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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex UDI Module - Set C" target="_blank"><span>C05030023</span> - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex UDI Module - Set D" target="_blank"><span>C05030024</span> - D-Plex Unique Dual Indexes for Illumina - Set D </a></li>
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
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'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li><span style="font-weight: 400;">An innovative technology with template switching and UMIs</span></li>
<li><span>Ultra-sensitive methodology with low input capability down to 10 ng</span></li>
<li><span>High efficiency for greater gene detection and biomarker identification</span></li>
<li><span>Consistent gene expression profiling, optimal for gene signature definition</span></li>
<li><span>Precise measurement of strand orientation for anti-sense transcription detection</span></li>
</ul>
</div>
<h4></h4>
<center><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/dplex-mRNA-manual.pdf" target="_blank" title="dplex total rnaseq user manual"><strong>Download the manual</strong></a></center>
<p></p>
<p>The D-Plex mRNA-seq Library Preparation Kit is a tool designed for gene expression analysis. <span>The kit utilizes the</span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank"><span> </span>D-Plex technology</a><span><span> </span>to generate directional libraries for Illumina sequencing directly from total RNAs.</span></p>
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<p><span></span><span>The D-Plex mRNA-seq Kit offers a time-saving protocol that can be completed within 6 hours and requires minimal hands-on time. <span>The library preparation takes place in a </span><span>single tube</span><span>, increasing the efficiency tremendously. This new solution has been developped to capture an increased number of genes for great discovery efficiency.</span></span></p>
<p><span><span>The D-Plex mRNA-seq Kit includes all buffers and enzymes necessary for mRNA capture and library preparation. Specific D-Plex Unique Dual Indexes were designed and validated to fit the D-Plex technology and are available separately:</span></span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
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<div class="extra-spaced" align="center"></div>
<div class="row">
<div class="carrousel" style="background-position: center;">
<div class="slick">
<div>
<h3>Greater gene discovery efficiency at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/UHR_mRNA.png" alt="total RNA kit" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit enables the detection of higher numbers of genes even for low RNA inputs. Increased number of genes detected at 10x coverage is an indicator of greater sensitivity.</p>
</div>
</div>
<div>
<h3>High mappability to the coding transcriptome</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_biotypes.png" alt="total RNA diversity" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq libraries capture efficiently coding exons and introns present in the sample of interest.</p>
</div>
</div>
<div>
<h3>Consistent, sensitive gene expression profiling</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_violin_tpm_10.png" alt="total RNA" height="200px" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>Gene expression profiling obtained with D-Plex mRNA-seq kit is conserved across a broad dynamic range of inputs, especially for the lowest RNA inputs.</p>
</div>
</div>
<div>
<h3>Exceptional data concordance at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/robustness_corr_HUR_mrna.png" alt="total RNA" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit produces quality data with high data concordance between low and high RNA inputs. This result demonstrates that D-Plex is an ideal solution for precious samples with limited starting material.</p>
</div>
</div>
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<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
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<h2>Product Features</h2>
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<li>Ultra-low input capability, down to 10 pg for small RNAs and 100 pg for total RNA samples</li>
<li>High library complexity providing novel and diverse transcript detection</li>
<li>Versatile integration into numerous transcriptomic analysis workflow: ribosome profiling, CLIP-seq, RIP-seq and other</li>
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<p></p>
<p></p>
<center><em><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/MA-D-Plex.pdf" target="_blank" title="D-Plex Small RNA library preparation user manual"><strong>Download the manual</strong></a></em></center>
<p>D-Plex Small RNA-seq Library Prep Kit is a tool designed for the study of the <strong>small non-coding transcriptome</strong>. The kit is using the<a href="https://www.diagenode.com/en/pages/dplex" target="_blank"> D-Plex technology</a> to generate small RNA libraries for Illumina sequencing directly from total RNAs or enriched small RNAs.</p>
<p>The D-Plex technology utilizes two innovative <strong>ligation-free mechanisms</strong> - <strong>poly(A) tailing</strong> and <strong>template switching</strong> - to produce sequencing libraries from ultra-low input amounts, down to 10 pg for small RNAs and 100 pg for total RNAs. Combined with <strong>unique molecular identifiers</strong> (UMI), this complete solution ensures a <strong>realistic</strong> and <strong>accurate representation of diverse small RNA species</strong> (such as microRNAs, piRNAs, tRNAs, and siRNAs) with minimized quantitative bias.</p>
<p>D-Plex Small RNA-seq Kit offers a time saving protocol that can be completed within <strong>5 hours</strong> and requires minimal hands-on time. <span>The library preparation takes place in a </span><strong>single tube</strong><span>, increasing the efficiency tremendously. </span></p>
<p>D-Plex Small RNA-seq Kit includes all buffers and enzymes necessary for the library preparation. Specific D-Plex Indexes were designed and validated to fit the D-Plex technology and are available separately:</p>
<p><strong>For D-Plex UDI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><strong>For D-Plex SI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-A" title="D-Plex SI Module - Set A" target="_blank">C05030010 - D-Plex Single Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-B" title="D-Plex SI Module - Set B" target="_blank">C05030011 - D-Plex Single Indexes for Illumina - Set B</a></li>
</ul>
<p><strong>D-Plex is also available for MGI sequencing, check <a href="https://www.diagenode.com/en/p/d-plex-small-RNA-dnbseq-kit-x24" target="_blank">here</a>!</strong></p>
<p></p>
<p></p>
<p></p>
<center><strong>The D-Plex smRNA-seq is a versatile tool for a plethora of transciptomic analysis</strong></center>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/versatile-circle.png" /></center>
<p></p>
<p>With low-input capability, accuracy and ability to incorporate different transcript classes independent of chemical moieties, the strong D-Plex library-preparation method can be included in various analysis workflows (i.e., ribosome profiling, genome-wide mapping of protein: RNA binding sites).</p>
<p></p>
<p></p>',
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'info1' => '<div class="row">
<div class="small-12 columns">
<p><strong>High library diversity for ultra-low inputs</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-diversity-fig1.png" /></center></div>
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig2-captures.png" /></center></div>
</div>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>D-Plex smRNA-seq captures the smRNA complexity of the sample in a sensitive manner. A large number of features is detected for each biotype at a CPM ≥5, as shown above for different species.</p>
</div>
</div>
<p></p>
<p><strong>Accurate representation of the smRNA content of a sample</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-ratplasma.png" /></center></div>
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-miRNAdistribution.png" /></center></div>
</div>
<div class="row">
<div class="small-6 columns">
<p><strong>Accurate identification of PCR duplicates in low-input samples using unique molecular identifiers (UMIs).</strong><br />The UMI read deduplication that is embedded in the D-Plex construct is used to avoid over-estimation of transcripts by identifying clones generated during the PCR amplification of the library. Despite limiting starting RNA input, the UMI correction removed technical copies of transcripts, maintaining the initial sample abundance.</p>
</div>
<div class="small-6 columns">
<p>D-Plex (template switching) technology enables recovery of the initial miRNA distribution of the miRXplore Universal Reference (Miltenyi Biotech Inc., Cat. No. 130-093-521). In contrast, ligation-based kits display a strong distortion of the miRNA equimolar distribution initially present in the pool, indicating sample incorporation bias.</p>
</div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-foldchange.jpg" /></center></div>
<div class="small-6 columns"><strong>D-Plex smRNA-seq in association with the MGcount analysis correlates strongly (R² > 0.8) with the RT-qPCR analysis, which is considered the gold-standard for accuracy. </strong><br />The figures shows a fold-change accuracy of a panel of 20 different small-RNA (figure taken from MGcount publication - Hita et al., BMC bioinformatics, 23-39(2022).</div>
</div>
<p></p>
<p></p>
<p><strong>Maximize information from the regulatory transcriptome using D-Plex small RNA-seq kit and MGcount*</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-multialignments.png" /></center></div>
<div class="small-6 columns">By analyzing D-Plex dataset with MGcount, more reads are kept during the analysis, which ultimately preserves biological complexity linked to ncRNA expression without decreasing the accuracy of detection. <br /><span style="line-height: 1em;"><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></span></div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>The D-Plex technology uses two innovative ligation-free mechanisms, <strong>poly(A) tailing and template switching</strong>, to produce sequencing libraries from ultra-low input amounts.</p>
</div>
</div>
<div class="row" style="background-color: #f5f5f5;">
<div class="small-12 columns"><br />
<p><strong>WORKFLOW</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/FL-Image-Workflow.jpg" width="50%" /></center></div>
<div class="small-12 columns">
<p style="text-align: justify; padding: 15px;"><br />RNA molecules are polyadenylated at the 3’-end and primed with an oligo(dT) primer containing part of the ILMN 3’-adapter sequence. The addition of a template-switching oligonucleotide during cDNA synthesis enables fusion of part of the ILMN 5’-adapter during reverse transcription. After PCR, the library comprises double stranded DNA constructs that are ready for sequencing.</p>
</div>
</div>
<p><br /><br /></p>
<p></p>
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'info2' => '<p>A specific bioinformatics pipeline has been developed to process the special sequences present in the D-Plex construct, namely the UMI, the A-tail, and the template switch motif. All guidelines and free softwares are shared in the user manual. Subject to the compatibility of D-Plex constructs, other specific pipelines can be used.</p>
<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
<p>Need help for your bioinformatics analysis of miRNA?</p>
<p></p>
<div class="small-12 medium-3 large-3 columns"><center><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"><img src="https://www.diagenode.com/img/product/kits/dplex/miRMaster_logo.png" /></a></center>
<p> </p>
<p> </p>
</div>
<div class="small-12 medium-9 large-9 columns">
<p>Diagenode has collaborated with <strong>Andreas Keller</strong> and <strong>Tobias Fehlmann</strong> to develop a new bioinformatics pipeline for <span>the <strong>prediction of novel miRNAs</strong>.</span><br /><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"></a></p>
<ul>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster2">miRMaster 2</a></li>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank">miRMaster</a></li>
</ul>
</div>
<p> </p>
<p> </p>',
'label3' => 'Data analysis - Counting using MGcount',
'info3' => '<p>The counting or expression level calculation is the last step of the processing to generate an expression level matrix. We recommend using MGcount*, a counting tool developed at Diagenode with a suitable annotations file that include the small RNA transcripts that are object of study. MGcount, built on top of featureCounts, employs a flexible quantification approach to deal with datasets containing multiple RNA biotypes such as D-plex libraries. Non-coding RNAs varying in length, biogenesis, and function, may overlap in a genomic region, and are sometimes present in the genome with a high copy number. Consequently, reads may align equally well to more than one position in the reference genome or/and align to a position where more than one annotated transcript is located. MGcount employs two strategies to quantify these reads. Firstly, it assigns reads in rounds prioritizing small-RNAs over long-RNAs ensuring the unbiased read attribution to intergenic and intragenic small RNAs while preventing host-genes transcription over-estimation. Secondly, MGcount collapses loci where reads consistently multi-map into communities of loci with a graph-based approach. The resultant communities are groups of biologically related loci with nearly identical sequence. Subsequently, quantification of reads is performed at the loci-community level, reducing multi-alignments ambiguity at individual locus. Ultimately, this strategy maximizes the transcriptome information analysed and improves the interrogation of non-coding RNAs. MGcount software repository provides annotation files for A. thaliana, H. sapiens, M. musculus and C. elegans. The required input files for MGcount are a .txt file listing the paths to the alignment input files (.bam format) and the annotations file (.gtf format). The output directory path has to be provided as an input as well.</p>
<p><strong>Example command:</strong></p>
<p style="background-color: #f0f0f0;">MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf --outdir outputs --bam_infiles input_bamfilenames.txt</p>
<p>MGcount provides the choice to enable/disable the quantification of all RNA biotypes included in the annotation file in the form of "communities" as optional parameters for small-RNA (--ml_flag_small) and long-RNA (--ml_flag_long). Both are enabled by default. The main output of MGcount is the count_matrix.csv file containing an expression matrix that can be imported to R or any other software for downstream analyses.<br />A full user guide for MGCount is available here: <a href="https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html">https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html</a>.</p>
<p>Other standard counting tools such as featureCounts or HTSeq-counts can also be used alternatively. Given the high complexity of D-Plex libraries, we recommend having a clear understanding of the scientific question and the goal of the project before proceeding to the choice of the counting method as this will strongly impact downstream analyses. Diagenode provides bioinformatics support as a service (please, contact us if you need help with data analysis).</p>
<p>Notice that D-Plex produces forward-stranded data. Stranded libraries have the benefit that reads map to the genome strand where they were originated from. Therefore, when estimating transcript expression, reads aligned to the forward strand should be assigned only to transcript features in the forward strand whereas reads aligned to the reverse strand should be assigned only to transcript features in the reverse strand. For this, make sure you select “stranded mode” in any tool of choice. Stranded mode is selected by default in MGcount.</p>
<p><em><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></em></p>',
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'slug' => 'D-Plex-Small-RNA-seq-Library-Prep-x24',
'meta_title' => 'D-Plex Small RNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, small RNA kit, small RNA-seq kit, low input RNA-seq, UMI RNA-seq, RNA-seq library preparation, D-Plex, higher RNA diversity',
'meta_description' => 'Small RNA-seq library preparation for Illumina sequencing - Unique D-Plex technology - Optimized for ultra-low input (100 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - Compatibility with plasma samples - User-friendly and fast protocol',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set A includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li>C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex 24 Single Indexes for Illumina - Set #B">C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex 24 Single Indexes for Illumina - Set #C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex 24 Single Indexes for Illumina - Set #D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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<li>Compatible with<span> </span><a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24">D-Plex Small RNA-seq kit for Illumina</a> </li>
<li>Multiplexing up to <strong>96 samples</strong> when combining Set A, B, C and D</li>
<li>Allows for identification of index hopping</li>
</ul>',
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'info2' => '<p><span>D-Plex RNA-seq UDI library constructs bear the TruSeq (Illumina) HT adapters. In case of a multiplexing scenario, it is therefore recommended to submit the D-Plex libraries as TruSeq HT libraries to your sequencing provider. </span><span>Further details are provided in the D-Plex Unique Dual Indexes Module<span> </span><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf" target="_blank">manual</a>.</span></p>',
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'meta_title' => 'D-Plex Unique Dual Indexes for Illumina - RNA Library Prep Kit - Set A | Diagenode',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set B includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A">C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li>C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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<li>Multiplexing up to <strong>96 samples</strong> when combining Set A, B, C and D</li>
<li>Allows for identification of index hopping</li>
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'meta_description' => 'Compatible with D-Plex RNA-seq kits - Unique Dual Indexes for Illumina - Index hopping mitigation - Suitable for ultra-low input - Contains UMI - Multiplexing up to 48 samples',
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<div class="small-12 medium-12 large-12 columns">
<h2 style="font-size: 22px;">DNA断片化、ライブラリー調製、自動化：NGSのワンストップショップ</h2>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-12 medium-12 large-12 columns">
<h4>1. 断片化装置を選択してください：150 bp〜75 kbの範囲でDNAを断片化します。</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-pico-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/bioruptor_pico.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/megaruptor2-1-unit"><img src="https://www.diagenode.com/img/product/shearing_technologies/B06010001_megaruptor2.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-one-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/br-one-profil.png" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns">5μlまで断片化：150 bp〜2 kb<br />NGS DNAライブラリー調製およびFFPE核酸抽出に最適で、</td>
<td class="small-4 medium-4 large-4 columns">2 kb〜75 kbの範囲をできます。<br />メイトペアライブラリー調製および長いフラグメントDNAシーケンシングに最適で、この軽量デスクトップデバイスで</td>
<td class="small-4 medium-4 large-4 columns">20または50μlの断片化が可能です。</td>
</tr>
</tbody>
</table>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-8 medium-8 large-8 columns">
<h4>2. 最適化されたライブラリー調整キットを選択してください。</h4>
</th>
<th class="small-4 medium-4 large-4 columns">
<h4>3. ライブラリー前処理自動化を選択して、比類のないデータ再現性を実感</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><img src="https://www.diagenode.com/img/product/kits/microPlex_library_preparation.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/ideal-library-preparation-kit-x24-incl-index-primer-set-1-24-rxns"><img src="https://www.diagenode.com/img/product/kits/box_kit.jpg" style="display: block; margin-left: auto; margin-right: auto;" height="173" width="250" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/sx-8g-ip-star-compact-automated-system-1-unit"><img src="https://www.diagenode.com/img/product/automation/B03000002%20_ipstar_compact.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">50pgの低入力：MicroPlex Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">5ng以上：iDeal Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">Achieve great NGS data easily</td>
</tr>
</tbody>
</table>
</div>
</div>
<blockquote>
<div class="row">
<div class="small-12 medium-12 large-12 columns"><span class="label" style="margin-bottom: 16px; margin-left: -22px; font-size: 15px;">DiagenodeがNGS研究にぴったりなプロバイダーである理由</span>
<p>Diagenodeは15年以上もエピジェネティクス研究に専念、専門としています。 ChIP研究クロマチン用のユニークな断片化システムの開発から始まり、 専門知識を活かし、5μlのせん断体積まで可能で、NGS DNAライブラリーの調製に最適な最先端DNA断片化装置の開発にたどり着きました。 我々は以来、ChIP-seq、Methyl-seq、NGSライブラリー調製用キットを研究開発し、業界をリードする免疫沈降研究と同様に、ライブラリー調製を自動化および完結させる独自の自動化システムを開発にも成功しました。</p>
<ul>
<li>信頼されるせん断装置</li>
<li>様々なインプットからのライブラリ作成キット</li>
<li>独自の自動化デバイス</li>
</ul>
</div>
</div>
</blockquote>
<div class="row">
<div class="small-12 columns">
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#panel1a">次世代シーケンシングへの理解とその専門知識</a>
<div id="panel1a" class="content">
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>次世代シーケンシング (NGS)</strong> ）は、著しいスケールとハイスループットでシーケンシングを行い、1日に数十億もの塩基生成を可能にします。 NGSのハイスループットは迅速でありながら正確で、再現性のあるデータセットを実現し、さらにシーケンシング費用を削減します。 NGSは、ゲノムシーケンシング、ゲノム再シーケンシング、デノボシーケンシング、トランスクリプトームシーケンシング、その他にDNA-タンパク質相互作用の検出やエピゲノムなどを示します。 指数関数的に増加するシーケンシングデータの需要は、計算分析の障害や解釈、データストレージなどの課題を解決します。</p>
<p>アプリケーションおよび出発物質に応じて、数百万から数十億の鋳型DNA分子を大規模に並行してシーケンシングすることが可能です。その為に、異なる化学物質を使用するいくつかの市販のNGSプラットフォームを利用することができます。 NGSプラットフォームの種類によっては、事前準備とライブラリー作成が必要です。</p>
<p>NGSにとっても、特にデータ処理と分析に関した大きな課題はあります。第3世代技術はゲノミクス研究にさらに革命を起こすであろうと大きく期待されています。</p>
</div>
</div>
<div class="row">
<div class="small-6 medium-6 large-6 columns">
<p><strong>NGS アプリケーション</strong></p>
<ul>
<li>全ゲノム配列決定</li>
<li>デノボシーケンシング</li>
<li>標的配列</li>
<li>Exomeシーケンシング</li>
<li>トランスクリプトーム配列決定</li>
<li>ゲノム配列決定</li>
<li>ミトコンドリア配列決定</li>
<li>DNA-タンパク質相互作用（ChIP-seq</li>
<li>バリアント検出</li>
<li>ゲノム仕上げ</li>
</ul>
</div>
<div class="small-6 medium-6 large-6 columns">
<p><strong>研究分野におけるNGS:</strong></p>
<ul>
<li>腫瘍学</li>
<li>リプロダクティブ・ヘルス</li>
<li>法医学ゲノミクス</li>
<li>アグリゲノミックス</li>
<li>複雑な病気</li>
<li>微生物ゲノミクス</li>
<li>食品・環境ゲノミクス</li>
<li>創薬ゲノミクス - パーソナライズド・メディカル</li>
</ul>
</div>
<div class="small-12 medium-12 large-12 columns">
<p><strong>NGSの用語</strong></p>
<dl>
<dt>リード（読み取り）</dt>
<dd>この装置から得られた連続した単一のストレッチ</dd>
<dt>断片リード</dt>
<dd>フラグメントライブラリからの読み込み。 シーケンシングプラットフォームに応じて、読み取りは通常約100〜300bp。</dd>
<dt>断片ペアエンドリード</dt>
<dd>断片ライブラリーからDNA断片の各末端2つの読み取り。</dd>
<dt>メイトペアリード</dt>
<dd>大きなDNA断片（通常は予め定義されたサイズ範囲）の各末端から2つの読み取り。</dd>
<dt>カバレッジ（例）</dt>
<dd>30×適用範囲とは、参照ゲノム中の各塩基対が平均30回の読み取りを示す。</dd>
</dl>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2>NGSプラットフォーム</h2>
<h3><a href="http://www.illumina.com" target="_blank">イルミナ</a></h3>
<p>イルミナは、クローン的に増幅された鋳型DNA（クラスター）上に位置する、蛍光標識された可逆的鎖ターミネーターヌクレオチドを用いた配列別合成技術を使用。 DNAクラスターは、ガラスフローセルの表面上に固定化され、 ワークフローは、4つのヌクレオチド（それぞれ異なる蛍光色素で標識された）の組み込み、4色イメージング、色素や末端基の切断、取り込み、イメージングなどを繰り返します。フローセルは大規模な並列配列決定を受ける。 この方法により、単一蛍光標識されたヌクレオチドの制御添加によるモノヌクレオチドのエラーを回避する可能性があります。 読み取りの長さは、通常約100〜150 bpです。</p>
<h3><a href="http://www.lifetechnologies.com" target="_blank">イオン トレント</a></h3>
<p>イオントレントは、半導体技術チップを用いて、合成中にヌクレオチドを取り込む際に放出されたプロトンを検出します。 これは、イオン球粒子と呼ばれるビーズの表面にエマルションPCR（emPCR）を使用し、リンクされた特定のアダプターを用いてDNA断片を増幅します。 各ビーズは1種類のDNA断片で覆われていて、異なるDNA断片を有するビーズは次いで、チップの陽子感知ウェル内に配置されます。 チップには一度に4つのヌクレオチドのうちの1つが浸水し、このプロセスは異なるヌクレオチドで15秒ごとに繰り返されます。 配列決定の間に4つの塩基の各々が1つずつ導入されます、組み込みの場合はプロトンが放出され、電圧信号が取り込みに比例して検出されます。.</p>
<h3><a href="http://www.pacificbiosciences.com" target="_blank">パシフィック バイオサイエンス</a></h3>
<p>パシフィックバイオサイエンスでは、20kbを超える塩基対の読み取りも、単一分子リアルタイム（SMRT）シーケンシングによる構造および細胞タイプの変化を観察することができます。 このプラットフォームでは、超長鎖二本鎖DNA（dsDNA）断片が、Megaruptor（登録商標）のようなDiagenode装置を用いたランダムシアリングまたは目的の標的領域の増幅によって生成されます。 SMRTbellライブラリーは、ユニバーサルヘアピンアダプターをDNA断片の各末端に連結することによって生成します。 サイズ選択条件による洗浄ステップの後、配列決定プライマーをSMRTbellテンプレートにアニーリングし、鋳型DNAに結合したDNAポリメラーゼを含む配列決定を、蛍光標識ヌクレオチドの存在下で開始。 各塩基が取り込まれると、異なる蛍光のパルスをリアルタイムで検出します。</p>
<h3><a href="https://nanoporetech.com" target="_blank">オックスフォード ナノポア</a></h3>
<p>Oxford Nanoporeは、単一のDNA分子配列決定に基づく技術を開発します。その技術により生物学的分子、すなわちDNAが一群の電気抵抗性高分子膜として位置するナノスケールの孔（ナノ細孔）またはその近くを通過し、イオン電流が変化します。 この変化に関する情報は、例えば4つのヌクレオチド（AまたはG r CまたはT）ならびに修飾されたヌクレオチドすべてを区別することによって分子情報に訳されます。 シーケンシングミニオンデバイスのフローセルは、数百個のナノポアチャネルのセンサアレイを含みます。 DNAサンプルは、Diagenode社のMegaruptor（登録商標）を用いてランダムシアリングによって生成され得る超長鎖DNAフラグメントが必要です。</p>
<h3><a href="http://www.lifetechnologies.com/be/en/home/life-science/sequencing/next-generation-sequencing/solid-next-generation-sequencing.html" target="_blank">SOLiD</a></h3>
<p>SOLiDは、ユニークな化学作用により、何千という個々のDNA分子の同時配列決定を可能にします。 それは、アダプター対ライブラリーのフラグメントが適切で、せん断されたゲノムDNAへのアダプターのライゲーションによるライブラリー作製から始まります。 次のステップでは、エマルジョンPCR（emPCR）を実施して、ビーズの表面上の個々の鋳型DNA分子をクローン的に増幅。 emPCRでは、個々の鋳型DNAをPCR試薬と混合し、水中油型エマルジョン内の疎水性シェルで囲まれた水性液滴内のプライマーコートビーズを、配列決定のためにロードするスライドガラスの表面にランダムに付着。 この技術は、シークエンシングプライマーへのライゲーションで競合する4つの蛍光標識されたジ塩基プローブのセットを使用します。</p>
<h3><a href="http://454.com/products/technology.asp" target="_blank">454</a></h3>
<p>454は、大規模並列パイロシーケンシングを利用しています。 始めに全ゲノムDNAまたは標的遺伝子断片の300〜800bp断片のライブラリー調製します。 次に、DNAフラグメントへのアダプターの付着および単一のDNA鎖の分離。 その後アダプターに連結されたDNAフラグメントをエマルジョンベースのクローン増幅（emPCR）で処理し、DNAライブラリーフラグメントをミクロンサイズのビーズ上に配置します。 各DNA結合ビーズを光ファイバーチップ上のウェルに入れ、器具に挿入します。 4つのDNAヌクレオチドは、配列決定操作中に固定された順序で連続して加えられ、並行して配列決定されます。</p>
</div>
</div>
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<p><span style="font-weight: 400;">Most of the major next-generation sequencing platforms require ligation of specific adaptor oligos to </span><a href="../applications/dna-rna-shearing"><span style="font-weight: 400;">fragmented DNA or RNA</span></a><span style="font-weight: 400;"> prior to sequencing</span></p>
<p><span style="font-weight: 400;">After input DNA has been fragmented, it is end-repaired and blunt-ended</span><span style="font-weight: 400;">. The next step is a A-tailing in which dAMP is added to the 3´ end of the blunt phosphorylated DNA fragments to prevent concatemerization and to allow the ligation of adaptors with complementary dT overhangs. In addition, barcoded adapters can be incorporated to facilitate multiplexing prior to or during amplification.</span></p>
<center><img src="https://www.diagenode.com/img/categories/library-prep/flux.png" /></center>
<p><span style="font-weight: 400;">Diagenode offers a comprehensive product portfolio for library preparation:<br /></span></p>
<strong><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">D-Plex RNA-seq Library Preparation Kits</a></strong><br />
<p><span style="font-weight: 400;">Diagenode’s new RNA-sequencing solutions utilize the innovative c</span><span style="font-weight: 400;">apture and a</span><span style="font-weight: 400;">mplification by t</span><span style="font-weight: 400;">ailing and s</span><span style="font-weight: 400;">witching”</span><span style="font-weight: 400;">, a ligation-free method to produce DNA libraries for next generation sequencing from low input amounts of RNA. </span><span style="font-weight: 400;"></span><a href="../categories/Library-preparation-for-RNA-seq">Learn more</a></p>
<strong><a href="../categories/library-preparation-for-ChIP-seq">ChIP-seq and DNA sequencing library preparation solutions</a></strong><br />
<p><span style="font-weight: 400;">Our kits have been optimized for DNA library preparation used for next generation sequencing for a wide range of inputs. Using a simple three-step protocols, our</span><a href="http://www.diagenode.com/p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><span style="font-weight: 400;"> </span></a><span style="font-weight: 400;">kits are an optimal choice for library preparation from DNA inputs down to 50 pg. </span><a href="../categories/library-preparation-for-ChIP-seq">Learn more</a></p>
<a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span><strong>Bioruptor Pico - short fragments</strong></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">Our well-cited Bioruptor Pico is the shearing device of choice for chromatin and DNA fragmentation. Obtain uniform and tight fragment distributions between 150bp -2kb. </span><a href="../p/bioruptor-pico-sonication-device">Learn more</a></p>
<strong><a href="../p/megaruptor2-1-unit"><span href="../p/bioruptor-pico-sonication-device">Megaruptor</span>® - long fragments</a></strong><a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">The Megaruptor is designed to shear DNA from 3kb-75kb for long-read sequencing. <a href="../p/megaruptor2-1-unit">Learn more</a></span></p>
<span href="../p/bioruptor-pico-sonication-device"></span><span style="font-weight: 400;"></span></div>
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'name' => 'Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADPribosylation dependent manner to induce translational stalling.',
'authors' => 'Kejiou N. S. et al.',
'description' => '<p><span>In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.</span></p>',
'date' => '2023-05-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37224531',
'doi' => '10.1093/nar/gkad440',
'modified' => '2023-06-15 08:38:59',
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'name' => 'miR-210 expression is strongly hypoxia-induced in anaplastic thyroidcancer cell lines and is associated with extracellular vesicles \&Argonaute-2',
'authors' => 'Powell Bonita H. et al.',
'description' => '<p>Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2\% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.</p>',
'date' => '2022-12-01',
'pmid' => 'https://www.biorxiv.org/content/10.1101/2022.11.23.515840v1.full',
'doi' => '10.1101/2022.11.23.515840',
'modified' => '2023-03-13 10:48:28',
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'name' => 'Light affects behavioral despair involving the clock gene Period 1.',
'authors' => 'Olejniczak I. et al.',
'description' => '<p>Light at night has strong effects on physiology and behavior of mammals. It affects mood in humans, which is exploited as light therapy, and has been shown to reset the circadian clock in the suprachiasmatic nuclei (SCN). This resetting is paramount to align physiological and biochemical timing to the environmental light-dark cycle. Here we provide evidence that light at zeitgeber time (ZT) 22 affects mood-related behaviors also in mice by activating the clock gene Period1 (Per1) in the lateral habenula (LHb), a brain region known to modulate mood-related behaviors. We show that complete deletion of Per1 in mice led to depressive-like behavior and loss of the beneficial effects of light on this behavior. In contrast, specific deletion of Per1 in the region of the LHb did not affect mood-related behavior, but suppressed the beneficial effects of light. RNA sequence analysis in the mesolimbic dopaminergic system revealed profound changes of gene expression after a light pulse at ZT22. In the nucleus accumbens (NAc), sensory perception of smell and G-protein coupled receptor signaling were affected the most. Interestingly, most of these genes were not affected in Per1 knock-out animals, indicating that induction of Per1 by light serves as a filter for light-mediated gene expression in the brain. Taken together we show that light affects mood-related behavior in mice at least in part via induction of Per1 in the LHb with consequences on mood-related behavior and signaling mechanisms in the mesolimbic dopaminergic system.</p>',
'date' => '2021-07-01',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/34237069/',
'doi' => '10.1371/journal.pgen.1009625',
'modified' => '2022-11-18 12:13:01',
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'name' => 'Small-Medium Extracellular Vesicles and Their miRNA Cargo inRetinal Health and Degeneration: Mediators of Homeostasis, andVehicles for Targeted Gene Therapy.',
'authors' => 'Wooff Yvette et al.',
'description' => '<p>Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.</p>',
'date' => '2020-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32670023',
'doi' => '10.3389/fncel.2020.00160',
'modified' => '2022-09-28 08:54:11',
'created' => '2022-09-08 16:32:20',
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'name' => 'The ribosomal protein S1-dependent standby site in mRNA consists ofa single-stranded region and a 5' structure element.',
'authors' => 'Romilly Cédric et al.',
'description' => '<p>In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient mRNAs, we provide a thorough characterization of the standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.</p>',
'date' => '2019-08-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31320593',
'doi' => '10.1073/pnas.1904309116',
'modified' => '2022-09-28 08:52:57',
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'name' => 'mir-21 is associated with inactive low molecular weight Argonautecomplexes in thyroid cancer cell lines',
'authors' => 'Powell Bonita H. et al.',
'description' => '<p>Thyroid cancer is the most prevalent endocrine malignancy. We and others have shown that several microRNAs, which are post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues, as well as cell lines derived from these cancers. In the cell, miRNAs are bound to Argonaute (AGO) proteins as what could be termed low molecular weight RNA-Induced Silencing Complexes (LMW-RISCs) that can assemble with additional proteins, mRNA, and translation machinery into high molecular weight RISCs (HMW-RISCs) that exert regulatory function. In this study, we sought to analyze the association of miRNAs with RISC complexes in ATC and PTC. For ATC and PTC lines, miRNA species were enriched in both HMW-RISC and LMW-RISC cellular fractions, compared with intermediate molecular weight fractions and very low molecular weight (AGO-poor) fractions. Furthermore, 60\% of all miRNAs were slightly more abundant in LMW-RISC versus HMW-RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines and one of the most widely studied oncogenic miRNAs in many solid tumors, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings may suggest that miR-21 has a role or roles distinct from canonical post-transcriptional regulation in cancer. Furthermore, the methodology described here is a useful way to assess the distribution of miR-21 between HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.</p>',
'date' => '0000-00-00',
'pmid' => 'https://www.biorxiv.org/content/10.1101/2020.03.24.006072v3',
'doi' => '10.1101/2020.03.24.006072',
'modified' => '2023-02-17 10:16:49',
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'description' => '<div class="row"><div class="small-12 medium-4 large-4 columns"><div class="panel"><h3><em>lncRNAs in cancer</em></h3><p class="text-left">Gastrointestinal cancer: Gastrointestinal cancers occur as a result of dysregulated signaling pathways and cellular processes, such as the cell cycle or apoptosis. LncRNAs can regulate proliferation of gastrointestinal cancer through interacting with RNA targets, localization to chromatin, or binding to proteins. Read more about Long non-coding RNAs: crucial regulators of gastrointestinal cancer cell proliferation</p><h3><em>microRNAs in cancer</em></h3><p class="text-left">miR-155 has been found overexpressed in many cancer types including hematopoietic cancers, breast, lung and colon cancer<br /><br /> <a href="https://www.cell.com/trends/molecular-medicine/pdf/S1471-4914(14)00101-4.pdf">https://www.cell.com/trends/molecular-medicine/pdf/S1471-4914(14)00101-4.pdf</a></p></div></div><div class="small-12 medium-8 large-8 columns"><center><img src="https://www.diagenode.com/img/cancer/long-non-coding-rna.jpg" width="550" height="345" /></center><p></p><p>Various non-coding RNAs (ncRNAs) have been found to function as key regulators for transcription, chromatin remodelling, and post-transcriptional modification, thus making them relevant in oncogenesis, both as tumor suppressors and as drivers. For example, a number of microRNAs (miRNAs), one of the more well-studied types of ncRNAs, have been identified as potential cancer biomarkers and clinical targets. Other ncRNAs, such as long noncoding RNAs are involved in cancer regulation and proliferation. Understanding the role of ncRNAs will be critical for cancer research and therapeutic development.</p></div></div>',
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex UDI Module - Set C" target="_blank"><span>C05030023</span> - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex UDI Module - Set D" target="_blank"><span>C05030024</span> - D-Plex Unique Dual Indexes for Illumina - Set D </a></li>
</ul>
<p></p>
<p>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</p>',
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<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="Small RNA seq Bioinformatics pipeline" width="925" height="196" /></p>',
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'meta_title' => 'D-Plex Total RNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, RNA-seq library preparation; low input RNA-seq, UMI RNA-seq, UDI RNA-seq, D-Plex, higher RNA sensitivity, biomarker discovery',
'meta_description' => ' RNA-seq library preparation for Illumina sequencing - Unique D-Plex technology - Optimized for ultra-low input (50 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - Compatibility with FFPE samples - User-friendly and fast protocol',
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'name' => 'D-Plex mRNA-seq Kit for Illumina',
'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li><span style="font-weight: 400;">An innovative technology with template switching and UMIs</span></li>
<li><span>Ultra-sensitive methodology with low input capability down to 10 ng</span></li>
<li><span>High efficiency for greater gene detection and biomarker identification</span></li>
<li><span>Consistent gene expression profiling, optimal for gene signature definition</span></li>
<li><span>Precise measurement of strand orientation for anti-sense transcription detection</span></li>
</ul>
</div>
<h4></h4>
<center><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/dplex-mRNA-manual.pdf" target="_blank" title="dplex total rnaseq user manual"><strong>Download the manual</strong></a></center>
<p></p>
<p>The D-Plex mRNA-seq Library Preparation Kit is a tool designed for gene expression analysis. <span>The kit utilizes the</span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank"><span> </span>D-Plex technology</a><span><span> </span>to generate directional libraries for Illumina sequencing directly from total RNAs.</span></p>
<p><span>The D-Plex technology utilizes two innovative ligation-free mechanisms - poly(A) tailing and template switching - to produce sequencing libraries from low input samples, down to 10 ng of total RNAs. Combined with unique molecular identifiers (UMI), this complete solution delivers a high-sensitivity detection method for gene expression analysis and discovery applications in the coding transcriptome.</span></p>
<p><span></span><span>The D-Plex mRNA-seq Kit offers a time-saving protocol that can be completed within 6 hours and requires minimal hands-on time. <span>The library preparation takes place in a </span><span>single tube</span><span>, increasing the efficiency tremendously. This new solution has been developped to capture an increased number of genes for great discovery efficiency.</span></span></p>
<p><span><span>The D-Plex mRNA-seq Kit includes all buffers and enzymes necessary for mRNA capture and library preparation. Specific D-Plex Unique Dual Indexes were designed and validated to fit the D-Plex technology and are available separately:</span></span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
</ul>
<div class="extra-spaced" align="center"></div>
<div class="row">
<div class="carrousel" style="background-position: center;">
<div class="slick">
<div>
<h3>Greater gene discovery efficiency at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/UHR_mRNA.png" alt="total RNA kit" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit enables the detection of higher numbers of genes even for low RNA inputs. Increased number of genes detected at 10x coverage is an indicator of greater sensitivity.</p>
</div>
</div>
<div>
<h3>High mappability to the coding transcriptome</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_biotypes.png" alt="total RNA diversity" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq libraries capture efficiently coding exons and introns present in the sample of interest.</p>
</div>
</div>
<div>
<h3>Consistent, sensitive gene expression profiling</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_violin_tpm_10.png" alt="total RNA" height="200px" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>Gene expression profiling obtained with D-Plex mRNA-seq kit is conserved across a broad dynamic range of inputs, especially for the lowest RNA inputs.</p>
</div>
</div>
<div>
<h3>Exceptional data concordance at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/robustness_corr_HUR_mrna.png" alt="total RNA" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit produces quality data with high data concordance between low and high RNA inputs. This result demonstrates that D-Plex is an ideal solution for precious samples with limited starting material.</p>
</div>
</div>
</div>
</div>
</div>
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'info2' => '<p><span>Specific D-Plex indexes </span><span>were designed and validated to fit the D-Plex technology for Illumina sequencing and </span><span>are not included in the kit. They can be bought separately according to your needs. Please choose the format that suits you best among the compatible references to:</span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
</ul>
<p><span></span></p>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<script src="chrome-extension://hhojmcideegachlhfgfdhailpfhgknjm/web_accessible_resources/index.js"></script>',
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'info3' => '<p><span>A specific bioinformatics pipeline has been developed to process the special sequences present in the D-Plex construct, namely the UMI, the A-tail, and the template switch motif. All guidelines and free softwares are shared in the user manual. Subject to the compatibility of D-Plex constructs, other specific pipelines can be used.</span></p>
<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
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'meta_title' => 'D-Plex mRNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, RNA-seq library preparation; low input RNA-seq, UMI RNA-seq, UDI RNA-seq, D-Plex, higher RNA sensitivity, Gene expression analysis',
'meta_description' => 'Gene expression analysis - Unique D-Plex technology for Illumina sequencing - Optimized for ultra-low input (50 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - User-friendly and fast protocol',
'modified' => '2021-06-18 07:27:36',
'created' => '2021-04-13 14:20:32',
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'name' => 'D-Plex Small RNA-seq Kit for Illumina',
'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li>Ultra-low input capability, down to 10 pg for small RNAs and 100 pg for total RNA samples</li>
<li>High library complexity providing novel and diverse transcript detection</li>
<li>Versatile integration into numerous transcriptomic analysis workflow: ribosome profiling, CLIP-seq, RIP-seq and other</li>
<li>Improved quantification accuracy through the use of UMIs</li>
<li>Easy to use with minimal hands-on time: one day, one tube protocol</li>
</ul>
</div>
<p></p>
<p></p>
<center><em><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/MA-D-Plex.pdf" target="_blank" title="D-Plex Small RNA library preparation user manual"><strong>Download the manual</strong></a></em></center>
<p>D-Plex Small RNA-seq Library Prep Kit is a tool designed for the study of the <strong>small non-coding transcriptome</strong>. The kit is using the<a href="https://www.diagenode.com/en/pages/dplex" target="_blank"> D-Plex technology</a> to generate small RNA libraries for Illumina sequencing directly from total RNAs or enriched small RNAs.</p>
<p>The D-Plex technology utilizes two innovative <strong>ligation-free mechanisms</strong> - <strong>poly(A) tailing</strong> and <strong>template switching</strong> - to produce sequencing libraries from ultra-low input amounts, down to 10 pg for small RNAs and 100 pg for total RNAs. Combined with <strong>unique molecular identifiers</strong> (UMI), this complete solution ensures a <strong>realistic</strong> and <strong>accurate representation of diverse small RNA species</strong> (such as microRNAs, piRNAs, tRNAs, and siRNAs) with minimized quantitative bias.</p>
<p>D-Plex Small RNA-seq Kit offers a time saving protocol that can be completed within <strong>5 hours</strong> and requires minimal hands-on time. <span>The library preparation takes place in a </span><strong>single tube</strong><span>, increasing the efficiency tremendously. </span></p>
<p>D-Plex Small RNA-seq Kit includes all buffers and enzymes necessary for the library preparation. Specific D-Plex Indexes were designed and validated to fit the D-Plex technology and are available separately:</p>
<p><strong>For D-Plex UDI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><strong>For D-Plex SI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-A" title="D-Plex SI Module - Set A" target="_blank">C05030010 - D-Plex Single Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-B" title="D-Plex SI Module - Set B" target="_blank">C05030011 - D-Plex Single Indexes for Illumina - Set B</a></li>
</ul>
<p><strong>D-Plex is also available for MGI sequencing, check <a href="https://www.diagenode.com/en/p/d-plex-small-RNA-dnbseq-kit-x24" target="_blank">here</a>!</strong></p>
<p></p>
<p></p>
<p></p>
<center><strong>The D-Plex smRNA-seq is a versatile tool for a plethora of transciptomic analysis</strong></center>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/versatile-circle.png" /></center>
<p></p>
<p>With low-input capability, accuracy and ability to incorporate different transcript classes independent of chemical moieties, the strong D-Plex library-preparation method can be included in various analysis workflows (i.e., ribosome profiling, genome-wide mapping of protein: RNA binding sites).</p>
<p></p>
<p></p>',
'label1' => 'Characteristics and library prep. workflow ',
'info1' => '<div class="row">
<div class="small-12 columns">
<p><strong>High library diversity for ultra-low inputs</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-diversity-fig1.png" /></center></div>
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig2-captures.png" /></center></div>
</div>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>D-Plex smRNA-seq captures the smRNA complexity of the sample in a sensitive manner. A large number of features is detected for each biotype at a CPM ≥5, as shown above for different species.</p>
</div>
</div>
<p></p>
<p><strong>Accurate representation of the smRNA content of a sample</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-ratplasma.png" /></center></div>
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-miRNAdistribution.png" /></center></div>
</div>
<div class="row">
<div class="small-6 columns">
<p><strong>Accurate identification of PCR duplicates in low-input samples using unique molecular identifiers (UMIs).</strong><br />The UMI read deduplication that is embedded in the D-Plex construct is used to avoid over-estimation of transcripts by identifying clones generated during the PCR amplification of the library. Despite limiting starting RNA input, the UMI correction removed technical copies of transcripts, maintaining the initial sample abundance.</p>
</div>
<div class="small-6 columns">
<p>D-Plex (template switching) technology enables recovery of the initial miRNA distribution of the miRXplore Universal Reference (Miltenyi Biotech Inc., Cat. No. 130-093-521). In contrast, ligation-based kits display a strong distortion of the miRNA equimolar distribution initially present in the pool, indicating sample incorporation bias.</p>
</div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-foldchange.jpg" /></center></div>
<div class="small-6 columns"><strong>D-Plex smRNA-seq in association with the MGcount analysis correlates strongly (R² > 0.8) with the RT-qPCR analysis, which is considered the gold-standard for accuracy. </strong><br />The figures shows a fold-change accuracy of a panel of 20 different small-RNA (figure taken from MGcount publication - Hita et al., BMC bioinformatics, 23-39(2022).</div>
</div>
<p></p>
<p></p>
<p><strong>Maximize information from the regulatory transcriptome using D-Plex small RNA-seq kit and MGcount*</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-multialignments.png" /></center></div>
<div class="small-6 columns">By analyzing D-Plex dataset with MGcount, more reads are kept during the analysis, which ultimately preserves biological complexity linked to ncRNA expression without decreasing the accuracy of detection. <br /><span style="line-height: 1em;"><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></span></div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>The D-Plex technology uses two innovative ligation-free mechanisms, <strong>poly(A) tailing and template switching</strong>, to produce sequencing libraries from ultra-low input amounts.</p>
</div>
</div>
<div class="row" style="background-color: #f5f5f5;">
<div class="small-12 columns"><br />
<p><strong>WORKFLOW</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/FL-Image-Workflow.jpg" width="50%" /></center></div>
<div class="small-12 columns">
<p style="text-align: justify; padding: 15px;"><br />RNA molecules are polyadenylated at the 3’-end and primed with an oligo(dT) primer containing part of the ILMN 3’-adapter sequence. The addition of a template-switching oligonucleotide during cDNA synthesis enables fusion of part of the ILMN 5’-adapter during reverse transcription. After PCR, the library comprises double stranded DNA constructs that are ready for sequencing.</p>
</div>
</div>
<p><br /><br /></p>
<p></p>
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'info2' => '<p>A specific bioinformatics pipeline has been developed to process the special sequences present in the D-Plex construct, namely the UMI, the A-tail, and the template switch motif. All guidelines and free softwares are shared in the user manual. Subject to the compatibility of D-Plex constructs, other specific pipelines can be used.</p>
<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
<p>Need help for your bioinformatics analysis of miRNA?</p>
<p></p>
<div class="small-12 medium-3 large-3 columns"><center><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"><img src="https://www.diagenode.com/img/product/kits/dplex/miRMaster_logo.png" /></a></center>
<p> </p>
<p> </p>
</div>
<div class="small-12 medium-9 large-9 columns">
<p>Diagenode has collaborated with <strong>Andreas Keller</strong> and <strong>Tobias Fehlmann</strong> to develop a new bioinformatics pipeline for <span>the <strong>prediction of novel miRNAs</strong>.</span><br /><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"></a></p>
<ul>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster2">miRMaster 2</a></li>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank">miRMaster</a></li>
</ul>
</div>
<p> </p>
<p> </p>',
'label3' => 'Data analysis - Counting using MGcount',
'info3' => '<p>The counting or expression level calculation is the last step of the processing to generate an expression level matrix. We recommend using MGcount*, a counting tool developed at Diagenode with a suitable annotations file that include the small RNA transcripts that are object of study. MGcount, built on top of featureCounts, employs a flexible quantification approach to deal with datasets containing multiple RNA biotypes such as D-plex libraries. Non-coding RNAs varying in length, biogenesis, and function, may overlap in a genomic region, and are sometimes present in the genome with a high copy number. Consequently, reads may align equally well to more than one position in the reference genome or/and align to a position where more than one annotated transcript is located. MGcount employs two strategies to quantify these reads. Firstly, it assigns reads in rounds prioritizing small-RNAs over long-RNAs ensuring the unbiased read attribution to intergenic and intragenic small RNAs while preventing host-genes transcription over-estimation. Secondly, MGcount collapses loci where reads consistently multi-map into communities of loci with a graph-based approach. The resultant communities are groups of biologically related loci with nearly identical sequence. Subsequently, quantification of reads is performed at the loci-community level, reducing multi-alignments ambiguity at individual locus. Ultimately, this strategy maximizes the transcriptome information analysed and improves the interrogation of non-coding RNAs. MGcount software repository provides annotation files for A. thaliana, H. sapiens, M. musculus and C. elegans. The required input files for MGcount are a .txt file listing the paths to the alignment input files (.bam format) and the annotations file (.gtf format). The output directory path has to be provided as an input as well.</p>
<p><strong>Example command:</strong></p>
<p style="background-color: #f0f0f0;">MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf --outdir outputs --bam_infiles input_bamfilenames.txt</p>
<p>MGcount provides the choice to enable/disable the quantification of all RNA biotypes included in the annotation file in the form of "communities" as optional parameters for small-RNA (--ml_flag_small) and long-RNA (--ml_flag_long). Both are enabled by default. The main output of MGcount is the count_matrix.csv file containing an expression matrix that can be imported to R or any other software for downstream analyses.<br />A full user guide for MGCount is available here: <a href="https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html">https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html</a>.</p>
<p>Other standard counting tools such as featureCounts or HTSeq-counts can also be used alternatively. Given the high complexity of D-Plex libraries, we recommend having a clear understanding of the scientific question and the goal of the project before proceeding to the choice of the counting method as this will strongly impact downstream analyses. Diagenode provides bioinformatics support as a service (please, contact us if you need help with data analysis).</p>
<p>Notice that D-Plex produces forward-stranded data. Stranded libraries have the benefit that reads map to the genome strand where they were originated from. Therefore, when estimating transcript expression, reads aligned to the forward strand should be assigned only to transcript features in the forward strand whereas reads aligned to the reverse strand should be assigned only to transcript features in the reverse strand. For this, make sure you select “stranded mode” in any tool of choice. Stranded mode is selected by default in MGcount.</p>
<p><em><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></em></p>',
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<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set A includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
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<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set B includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A">C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li>C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
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<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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'description' => '<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2 style="font-size: 22px;">DNA断片化、ライブラリー調製、自動化：NGSのワンストップショップ</h2>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-12 medium-12 large-12 columns">
<h4>1. 断片化装置を選択してください：150 bp〜75 kbの範囲でDNAを断片化します。</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-pico-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/bioruptor_pico.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/megaruptor2-1-unit"><img src="https://www.diagenode.com/img/product/shearing_technologies/B06010001_megaruptor2.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-one-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/br-one-profil.png" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns">5μlまで断片化：150 bp〜2 kb<br />NGS DNAライブラリー調製およびFFPE核酸抽出に最適で、</td>
<td class="small-4 medium-4 large-4 columns">2 kb〜75 kbの範囲をできます。<br />メイトペアライブラリー調製および長いフラグメントDNAシーケンシングに最適で、この軽量デスクトップデバイスで</td>
<td class="small-4 medium-4 large-4 columns">20または50μlの断片化が可能です。</td>
</tr>
</tbody>
</table>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-8 medium-8 large-8 columns">
<h4>2. 最適化されたライブラリー調整キットを選択してください。</h4>
</th>
<th class="small-4 medium-4 large-4 columns">
<h4>3. ライブラリー前処理自動化を選択して、比類のないデータ再現性を実感</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><img src="https://www.diagenode.com/img/product/kits/microPlex_library_preparation.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/ideal-library-preparation-kit-x24-incl-index-primer-set-1-24-rxns"><img src="https://www.diagenode.com/img/product/kits/box_kit.jpg" style="display: block; margin-left: auto; margin-right: auto;" height="173" width="250" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/sx-8g-ip-star-compact-automated-system-1-unit"><img src="https://www.diagenode.com/img/product/automation/B03000002%20_ipstar_compact.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">50pgの低入力：MicroPlex Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">5ng以上：iDeal Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">Achieve great NGS data easily</td>
</tr>
</tbody>
</table>
</div>
</div>
<blockquote>
<div class="row">
<div class="small-12 medium-12 large-12 columns"><span class="label" style="margin-bottom: 16px; margin-left: -22px; font-size: 15px;">DiagenodeがNGS研究にぴったりなプロバイダーである理由</span>
<p>Diagenodeは15年以上もエピジェネティクス研究に専念、専門としています。 ChIP研究クロマチン用のユニークな断片化システムの開発から始まり、 専門知識を活かし、5μlのせん断体積まで可能で、NGS DNAライブラリーの調製に最適な最先端DNA断片化装置の開発にたどり着きました。 我々は以来、ChIP-seq、Methyl-seq、NGSライブラリー調製用キットを研究開発し、業界をリードする免疫沈降研究と同様に、ライブラリー調製を自動化および完結させる独自の自動化システムを開発にも成功しました。</p>
<ul>
<li>信頼されるせん断装置</li>
<li>様々なインプットからのライブラリ作成キット</li>
<li>独自の自動化デバイス</li>
</ul>
</div>
</div>
</blockquote>
<div class="row">
<div class="small-12 columns">
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#panel1a">次世代シーケンシングへの理解とその専門知識</a>
<div id="panel1a" class="content">
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>次世代シーケンシング (NGS)</strong> ）は、著しいスケールとハイスループットでシーケンシングを行い、1日に数十億もの塩基生成を可能にします。 NGSのハイスループットは迅速でありながら正確で、再現性のあるデータセットを実現し、さらにシーケンシング費用を削減します。 NGSは、ゲノムシーケンシング、ゲノム再シーケンシング、デノボシーケンシング、トランスクリプトームシーケンシング、その他にDNA-タンパク質相互作用の検出やエピゲノムなどを示します。 指数関数的に増加するシーケンシングデータの需要は、計算分析の障害や解釈、データストレージなどの課題を解決します。</p>
<p>アプリケーションおよび出発物質に応じて、数百万から数十億の鋳型DNA分子を大規模に並行してシーケンシングすることが可能です。その為に、異なる化学物質を使用するいくつかの市販のNGSプラットフォームを利用することができます。 NGSプラットフォームの種類によっては、事前準備とライブラリー作成が必要です。</p>
<p>NGSにとっても、特にデータ処理と分析に関した大きな課題はあります。第3世代技術はゲノミクス研究にさらに革命を起こすであろうと大きく期待されています。</p>
</div>
</div>
<div class="row">
<div class="small-6 medium-6 large-6 columns">
<p><strong>NGS アプリケーション</strong></p>
<ul>
<li>全ゲノム配列決定</li>
<li>デノボシーケンシング</li>
<li>標的配列</li>
<li>Exomeシーケンシング</li>
<li>トランスクリプトーム配列決定</li>
<li>ゲノム配列決定</li>
<li>ミトコンドリア配列決定</li>
<li>DNA-タンパク質相互作用（ChIP-seq</li>
<li>バリアント検出</li>
<li>ゲノム仕上げ</li>
</ul>
</div>
<div class="small-6 medium-6 large-6 columns">
<p><strong>研究分野におけるNGS:</strong></p>
<ul>
<li>腫瘍学</li>
<li>リプロダクティブ・ヘルス</li>
<li>法医学ゲノミクス</li>
<li>アグリゲノミックス</li>
<li>複雑な病気</li>
<li>微生物ゲノミクス</li>
<li>食品・環境ゲノミクス</li>
<li>創薬ゲノミクス - パーソナライズド・メディカル</li>
</ul>
</div>
<div class="small-12 medium-12 large-12 columns">
<p><strong>NGSの用語</strong></p>
<dl>
<dt>リード（読み取り）</dt>
<dd>この装置から得られた連続した単一のストレッチ</dd>
<dt>断片リード</dt>
<dd>フラグメントライブラリからの読み込み。 シーケンシングプラットフォームに応じて、読み取りは通常約100〜300bp。</dd>
<dt>断片ペアエンドリード</dt>
<dd>断片ライブラリーからDNA断片の各末端2つの読み取り。</dd>
<dt>メイトペアリード</dt>
<dd>大きなDNA断片（通常は予め定義されたサイズ範囲）の各末端から2つの読み取り。</dd>
<dt>カバレッジ（例）</dt>
<dd>30×適用範囲とは、参照ゲノム中の各塩基対が平均30回の読み取りを示す。</dd>
</dl>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2>NGSプラットフォーム</h2>
<h3><a href="http://www.illumina.com" target="_blank">イルミナ</a></h3>
<p>イルミナは、クローン的に増幅された鋳型DNA（クラスター）上に位置する、蛍光標識された可逆的鎖ターミネーターヌクレオチドを用いた配列別合成技術を使用。 DNAクラスターは、ガラスフローセルの表面上に固定化され、 ワークフローは、4つのヌクレオチド（それぞれ異なる蛍光色素で標識された）の組み込み、4色イメージング、色素や末端基の切断、取り込み、イメージングなどを繰り返します。フローセルは大規模な並列配列決定を受ける。 この方法により、単一蛍光標識されたヌクレオチドの制御添加によるモノヌクレオチドのエラーを回避する可能性があります。 読み取りの長さは、通常約100〜150 bpです。</p>
<h3><a href="http://www.lifetechnologies.com" target="_blank">イオン トレント</a></h3>
<p>イオントレントは、半導体技術チップを用いて、合成中にヌクレオチドを取り込む際に放出されたプロトンを検出します。 これは、イオン球粒子と呼ばれるビーズの表面にエマルションPCR（emPCR）を使用し、リンクされた特定のアダプターを用いてDNA断片を増幅します。 各ビーズは1種類のDNA断片で覆われていて、異なるDNA断片を有するビーズは次いで、チップの陽子感知ウェル内に配置されます。 チップには一度に4つのヌクレオチドのうちの1つが浸水し、このプロセスは異なるヌクレオチドで15秒ごとに繰り返されます。 配列決定の間に4つの塩基の各々が1つずつ導入されます、組み込みの場合はプロトンが放出され、電圧信号が取り込みに比例して検出されます。.</p>
<h3><a href="http://www.pacificbiosciences.com" target="_blank">パシフィック バイオサイエンス</a></h3>
<p>パシフィックバイオサイエンスでは、20kbを超える塩基対の読み取りも、単一分子リアルタイム（SMRT）シーケンシングによる構造および細胞タイプの変化を観察することができます。 このプラットフォームでは、超長鎖二本鎖DNA（dsDNA）断片が、Megaruptor（登録商標）のようなDiagenode装置を用いたランダムシアリングまたは目的の標的領域の増幅によって生成されます。 SMRTbellライブラリーは、ユニバーサルヘアピンアダプターをDNA断片の各末端に連結することによって生成します。 サイズ選択条件による洗浄ステップの後、配列決定プライマーをSMRTbellテンプレートにアニーリングし、鋳型DNAに結合したDNAポリメラーゼを含む配列決定を、蛍光標識ヌクレオチドの存在下で開始。 各塩基が取り込まれると、異なる蛍光のパルスをリアルタイムで検出します。</p>
<h3><a href="https://nanoporetech.com" target="_blank">オックスフォード ナノポア</a></h3>
<p>Oxford Nanoporeは、単一のDNA分子配列決定に基づく技術を開発します。その技術により生物学的分子、すなわちDNAが一群の電気抵抗性高分子膜として位置するナノスケールの孔（ナノ細孔）またはその近くを通過し、イオン電流が変化します。 この変化に関する情報は、例えば4つのヌクレオチド（AまたはG r CまたはT）ならびに修飾されたヌクレオチドすべてを区別することによって分子情報に訳されます。 シーケンシングミニオンデバイスのフローセルは、数百個のナノポアチャネルのセンサアレイを含みます。 DNAサンプルは、Diagenode社のMegaruptor（登録商標）を用いてランダムシアリングによって生成され得る超長鎖DNAフラグメントが必要です。</p>
<h3><a href="http://www.lifetechnologies.com/be/en/home/life-science/sequencing/next-generation-sequencing/solid-next-generation-sequencing.html" target="_blank">SOLiD</a></h3>
<p>SOLiDは、ユニークな化学作用により、何千という個々のDNA分子の同時配列決定を可能にします。 それは、アダプター対ライブラリーのフラグメントが適切で、せん断されたゲノムDNAへのアダプターのライゲーションによるライブラリー作製から始まります。 次のステップでは、エマルジョンPCR（emPCR）を実施して、ビーズの表面上の個々の鋳型DNA分子をクローン的に増幅。 emPCRでは、個々の鋳型DNAをPCR試薬と混合し、水中油型エマルジョン内の疎水性シェルで囲まれた水性液滴内のプライマーコートビーズを、配列決定のためにロードするスライドガラスの表面にランダムに付着。 この技術は、シークエンシングプライマーへのライゲーションで競合する4つの蛍光標識されたジ塩基プローブのセットを使用します。</p>
<h3><a href="http://454.com/products/technology.asp" target="_blank">454</a></h3>
<p>454は、大規模並列パイロシーケンシングを利用しています。 始めに全ゲノムDNAまたは標的遺伝子断片の300〜800bp断片のライブラリー調製します。 次に、DNAフラグメントへのアダプターの付着および単一のDNA鎖の分離。 その後アダプターに連結されたDNAフラグメントをエマルジョンベースのクローン増幅（emPCR）で処理し、DNAライブラリーフラグメントをミクロンサイズのビーズ上に配置します。 各DNA結合ビーズを光ファイバーチップ上のウェルに入れ、器具に挿入します。 4つのDNAヌクレオチドは、配列決定操作中に固定された順序で連続して加えられ、並行して配列決定されます。</p>
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<div class="small-12 medium-12 large-12 columns">
<p><span style="font-weight: 400;">Most of the major next-generation sequencing platforms require ligation of specific adaptor oligos to </span><a href="../applications/dna-rna-shearing"><span style="font-weight: 400;">fragmented DNA or RNA</span></a><span style="font-weight: 400;"> prior to sequencing</span></p>
<p><span style="font-weight: 400;">After input DNA has been fragmented, it is end-repaired and blunt-ended</span><span style="font-weight: 400;">. The next step is a A-tailing in which dAMP is added to the 3´ end of the blunt phosphorylated DNA fragments to prevent concatemerization and to allow the ligation of adaptors with complementary dT overhangs. In addition, barcoded adapters can be incorporated to facilitate multiplexing prior to or during amplification.</span></p>
<center><img src="https://www.diagenode.com/img/categories/library-prep/flux.png" /></center>
<p><span style="font-weight: 400;">Diagenode offers a comprehensive product portfolio for library preparation:<br /></span></p>
<strong><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">D-Plex RNA-seq Library Preparation Kits</a></strong><br />
<p><span style="font-weight: 400;">Diagenode’s new RNA-sequencing solutions utilize the innovative c</span><span style="font-weight: 400;">apture and a</span><span style="font-weight: 400;">mplification by t</span><span style="font-weight: 400;">ailing and s</span><span style="font-weight: 400;">witching”</span><span style="font-weight: 400;">, a ligation-free method to produce DNA libraries for next generation sequencing from low input amounts of RNA. </span><span style="font-weight: 400;"></span><a href="../categories/Library-preparation-for-RNA-seq">Learn more</a></p>
<strong><a href="../categories/library-preparation-for-ChIP-seq">ChIP-seq and DNA sequencing library preparation solutions</a></strong><br />
<p><span style="font-weight: 400;">Our kits have been optimized for DNA library preparation used for next generation sequencing for a wide range of inputs. Using a simple three-step protocols, our</span><a href="http://www.diagenode.com/p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><span style="font-weight: 400;"> </span></a><span style="font-weight: 400;">kits are an optimal choice for library preparation from DNA inputs down to 50 pg. </span><a href="../categories/library-preparation-for-ChIP-seq">Learn more</a></p>
<a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span><strong>Bioruptor Pico - short fragments</strong></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">Our well-cited Bioruptor Pico is the shearing device of choice for chromatin and DNA fragmentation. Obtain uniform and tight fragment distributions between 150bp -2kb. </span><a href="../p/bioruptor-pico-sonication-device">Learn more</a></p>
<strong><a href="../p/megaruptor2-1-unit"><span href="../p/bioruptor-pico-sonication-device">Megaruptor</span>® - long fragments</a></strong><a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">The Megaruptor is designed to shear DNA from 3kb-75kb for long-read sequencing. <a href="../p/megaruptor2-1-unit">Learn more</a></span></p>
<span href="../p/bioruptor-pico-sonication-device"></span><span style="font-weight: 400;"></span></div>
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'name' => 'Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADPribosylation dependent manner to induce translational stalling.',
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'description' => '<p><span>In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.</span></p>',
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'description' => '<p>Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2\% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.</p>',
'date' => '2022-12-01',
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'name' => 'Light affects behavioral despair involving the clock gene Period 1.',
'authors' => 'Olejniczak I. et al.',
'description' => '<p>Light at night has strong effects on physiology and behavior of mammals. It affects mood in humans, which is exploited as light therapy, and has been shown to reset the circadian clock in the suprachiasmatic nuclei (SCN). This resetting is paramount to align physiological and biochemical timing to the environmental light-dark cycle. Here we provide evidence that light at zeitgeber time (ZT) 22 affects mood-related behaviors also in mice by activating the clock gene Period1 (Per1) in the lateral habenula (LHb), a brain region known to modulate mood-related behaviors. We show that complete deletion of Per1 in mice led to depressive-like behavior and loss of the beneficial effects of light on this behavior. In contrast, specific deletion of Per1 in the region of the LHb did not affect mood-related behavior, but suppressed the beneficial effects of light. RNA sequence analysis in the mesolimbic dopaminergic system revealed profound changes of gene expression after a light pulse at ZT22. In the nucleus accumbens (NAc), sensory perception of smell and G-protein coupled receptor signaling were affected the most. Interestingly, most of these genes were not affected in Per1 knock-out animals, indicating that induction of Per1 by light serves as a filter for light-mediated gene expression in the brain. Taken together we show that light affects mood-related behavior in mice at least in part via induction of Per1 in the LHb with consequences on mood-related behavior and signaling mechanisms in the mesolimbic dopaminergic system.</p>',
'date' => '2021-07-01',
'pmid' => 'https://pubmed.ncbi.nlm.nih.gov/34237069/',
'doi' => '10.1371/journal.pgen.1009625',
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'name' => 'Small-Medium Extracellular Vesicles and Their miRNA Cargo inRetinal Health and Degeneration: Mediators of Homeostasis, andVehicles for Targeted Gene Therapy.',
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'description' => '<p>Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.</p>',
'date' => '2020-01-01',
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'name' => 'The ribosomal protein S1-dependent standby site in mRNA consists ofa single-stranded region and a 5' structure element.',
'authors' => 'Romilly Cédric et al.',
'description' => '<p>In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient mRNAs, we provide a thorough characterization of the standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.</p>',
'date' => '2019-08-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31320593',
'doi' => '10.1073/pnas.1904309116',
'modified' => '2022-09-28 08:52:57',
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(int) 5 => array(
'id' => '4457',
'name' => 'mir-21 is associated with inactive low molecular weight Argonautecomplexes in thyroid cancer cell lines',
'authors' => 'Powell Bonita H. et al.',
'description' => '<p>Thyroid cancer is the most prevalent endocrine malignancy. We and others have shown that several microRNAs, which are post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues, as well as cell lines derived from these cancers. In the cell, miRNAs are bound to Argonaute (AGO) proteins as what could be termed low molecular weight RNA-Induced Silencing Complexes (LMW-RISCs) that can assemble with additional proteins, mRNA, and translation machinery into high molecular weight RISCs (HMW-RISCs) that exert regulatory function. In this study, we sought to analyze the association of miRNAs with RISC complexes in ATC and PTC. For ATC and PTC lines, miRNA species were enriched in both HMW-RISC and LMW-RISC cellular fractions, compared with intermediate molecular weight fractions and very low molecular weight (AGO-poor) fractions. Furthermore, 60\% of all miRNAs were slightly more abundant in LMW-RISC versus HMW-RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines and one of the most widely studied oncogenic miRNAs in many solid tumors, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings may suggest that miR-21 has a role or roles distinct from canonical post-transcriptional regulation in cancer. Furthermore, the methodology described here is a useful way to assess the distribution of miR-21 between HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.</p>',
'date' => '0000-00-00',
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'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li><span style="font-weight: 400;">An innovative technology with template switching and UMIs</span></li>
<li><span>Ultra-sensitive methodology with low input capability down to 10 ng</span></li>
<li><span>High efficiency for greater gene detection and biomarker identification</span></li>
<li><span>Consistent gene expression profiling, optimal for gene signature definition</span></li>
<li><span>Precise measurement of strand orientation for anti-sense transcription detection</span></li>
</ul>
</div>
<h4></h4>
<center><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/dplex-mRNA-manual.pdf" target="_blank" title="dplex total rnaseq user manual"><strong>Download the manual</strong></a></center>
<p></p>
<p>The D-Plex mRNA-seq Library Preparation Kit is a tool designed for gene expression analysis. <span>The kit utilizes the</span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank"><span> </span>D-Plex technology</a><span><span> </span>to generate directional libraries for Illumina sequencing directly from total RNAs.</span></p>
<p><span>The D-Plex technology utilizes two innovative ligation-free mechanisms - poly(A) tailing and template switching - to produce sequencing libraries from low input samples, down to 10 ng of total RNAs. Combined with unique molecular identifiers (UMI), this complete solution delivers a high-sensitivity detection method for gene expression analysis and discovery applications in the coding transcriptome.</span></p>
<p><span></span><span>The D-Plex mRNA-seq Kit offers a time-saving protocol that can be completed within 6 hours and requires minimal hands-on time. <span>The library preparation takes place in a </span><span>single tube</span><span>, increasing the efficiency tremendously. This new solution has been developped to capture an increased number of genes for great discovery efficiency.</span></span></p>
<p><span><span>The D-Plex mRNA-seq Kit includes all buffers and enzymes necessary for mRNA capture and library preparation. Specific D-Plex Unique Dual Indexes were designed and validated to fit the D-Plex technology and are available separately:</span></span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
</ul>
<div class="extra-spaced" align="center"></div>
<div class="row">
<div class="carrousel" style="background-position: center;">
<div class="slick">
<div>
<h3>Greater gene discovery efficiency at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/UHR_mRNA.png" alt="total RNA kit" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit enables the detection of higher numbers of genes even for low RNA inputs. Increased number of genes detected at 10x coverage is an indicator of greater sensitivity.</p>
</div>
</div>
<div>
<h3>High mappability to the coding transcriptome</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_biotypes.png" alt="total RNA diversity" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq libraries capture efficiently coding exons and introns present in the sample of interest.</p>
</div>
</div>
<div>
<h3>Consistent, sensitive gene expression profiling</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_violin_tpm_10.png" alt="total RNA" height="200px" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>Gene expression profiling obtained with D-Plex mRNA-seq kit is conserved across a broad dynamic range of inputs, especially for the lowest RNA inputs.</p>
</div>
</div>
<div>
<h3>Exceptional data concordance at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/robustness_corr_HUR_mrna.png" alt="total RNA" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit produces quality data with high data concordance between low and high RNA inputs. This result demonstrates that D-Plex is an ideal solution for precious samples with limited starting material.</p>
</div>
</div>
</div>
</div>
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'info2' => '<p><span>Specific D-Plex indexes </span><span>were designed and validated to fit the D-Plex technology for Illumina sequencing and </span><span>are not included in the kit. They can be bought separately according to your needs. Please choose the format that suits you best among the compatible references to:</span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
</ul>
<p><span></span></p>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
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'info3' => '<p><span>A specific bioinformatics pipeline has been developed to process the special sequences present in the D-Plex construct, namely the UMI, the A-tail, and the template switch motif. All guidelines and free softwares are shared in the user manual. Subject to the compatibility of D-Plex constructs, other specific pipelines can be used.</span></p>
<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
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'slug' => 'D-Plex-mRNA-seq-Library-Prep-x24',
'meta_title' => 'D-Plex mRNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, RNA-seq library preparation; low input RNA-seq, UMI RNA-seq, UDI RNA-seq, D-Plex, higher RNA sensitivity, Gene expression analysis',
'meta_description' => 'Gene expression analysis - Unique D-Plex technology for Illumina sequencing - Optimized for ultra-low input (50 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - User-friendly and fast protocol',
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'name' => 'D-Plex Small RNA-seq Kit for Illumina',
'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li>Ultra-low input capability, down to 10 pg for small RNAs and 100 pg for total RNA samples</li>
<li>High library complexity providing novel and diverse transcript detection</li>
<li>Versatile integration into numerous transcriptomic analysis workflow: ribosome profiling, CLIP-seq, RIP-seq and other</li>
<li>Improved quantification accuracy through the use of UMIs</li>
<li>Easy to use with minimal hands-on time: one day, one tube protocol</li>
</ul>
</div>
<p></p>
<p></p>
<center><em><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/MA-D-Plex.pdf" target="_blank" title="D-Plex Small RNA library preparation user manual"><strong>Download the manual</strong></a></em></center>
<p>D-Plex Small RNA-seq Library Prep Kit is a tool designed for the study of the <strong>small non-coding transcriptome</strong>. The kit is using the<a href="https://www.diagenode.com/en/pages/dplex" target="_blank"> D-Plex technology</a> to generate small RNA libraries for Illumina sequencing directly from total RNAs or enriched small RNAs.</p>
<p>The D-Plex technology utilizes two innovative <strong>ligation-free mechanisms</strong> - <strong>poly(A) tailing</strong> and <strong>template switching</strong> - to produce sequencing libraries from ultra-low input amounts, down to 10 pg for small RNAs and 100 pg for total RNAs. Combined with <strong>unique molecular identifiers</strong> (UMI), this complete solution ensures a <strong>realistic</strong> and <strong>accurate representation of diverse small RNA species</strong> (such as microRNAs, piRNAs, tRNAs, and siRNAs) with minimized quantitative bias.</p>
<p>D-Plex Small RNA-seq Kit offers a time saving protocol that can be completed within <strong>5 hours</strong> and requires minimal hands-on time. <span>The library preparation takes place in a </span><strong>single tube</strong><span>, increasing the efficiency tremendously. </span></p>
<p>D-Plex Small RNA-seq Kit includes all buffers and enzymes necessary for the library preparation. Specific D-Plex Indexes were designed and validated to fit the D-Plex technology and are available separately:</p>
<p><strong>For D-Plex UDI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><strong>For D-Plex SI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-A" title="D-Plex SI Module - Set A" target="_blank">C05030010 - D-Plex Single Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-B" title="D-Plex SI Module - Set B" target="_blank">C05030011 - D-Plex Single Indexes for Illumina - Set B</a></li>
</ul>
<p><strong>D-Plex is also available for MGI sequencing, check <a href="https://www.diagenode.com/en/p/d-plex-small-RNA-dnbseq-kit-x24" target="_blank">here</a>!</strong></p>
<p></p>
<p></p>
<p></p>
<center><strong>The D-Plex smRNA-seq is a versatile tool for a plethora of transciptomic analysis</strong></center>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/versatile-circle.png" /></center>
<p></p>
<p>With low-input capability, accuracy and ability to incorporate different transcript classes independent of chemical moieties, the strong D-Plex library-preparation method can be included in various analysis workflows (i.e., ribosome profiling, genome-wide mapping of protein: RNA binding sites).</p>
<p></p>
<p></p>',
'label1' => 'Characteristics and library prep. workflow ',
'info1' => '<div class="row">
<div class="small-12 columns">
<p><strong>High library diversity for ultra-low inputs</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-diversity-fig1.png" /></center></div>
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig2-captures.png" /></center></div>
</div>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>D-Plex smRNA-seq captures the smRNA complexity of the sample in a sensitive manner. A large number of features is detected for each biotype at a CPM ≥5, as shown above for different species.</p>
</div>
</div>
<p></p>
<p><strong>Accurate representation of the smRNA content of a sample</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-ratplasma.png" /></center></div>
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-miRNAdistribution.png" /></center></div>
</div>
<div class="row">
<div class="small-6 columns">
<p><strong>Accurate identification of PCR duplicates in low-input samples using unique molecular identifiers (UMIs).</strong><br />The UMI read deduplication that is embedded in the D-Plex construct is used to avoid over-estimation of transcripts by identifying clones generated during the PCR amplification of the library. Despite limiting starting RNA input, the UMI correction removed technical copies of transcripts, maintaining the initial sample abundance.</p>
</div>
<div class="small-6 columns">
<p>D-Plex (template switching) technology enables recovery of the initial miRNA distribution of the miRXplore Universal Reference (Miltenyi Biotech Inc., Cat. No. 130-093-521). In contrast, ligation-based kits display a strong distortion of the miRNA equimolar distribution initially present in the pool, indicating sample incorporation bias.</p>
</div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-foldchange.jpg" /></center></div>
<div class="small-6 columns"><strong>D-Plex smRNA-seq in association with the MGcount analysis correlates strongly (R² > 0.8) with the RT-qPCR analysis, which is considered the gold-standard for accuracy. </strong><br />The figures shows a fold-change accuracy of a panel of 20 different small-RNA (figure taken from MGcount publication - Hita et al., BMC bioinformatics, 23-39(2022).</div>
</div>
<p></p>
<p></p>
<p><strong>Maximize information from the regulatory transcriptome using D-Plex small RNA-seq kit and MGcount*</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-multialignments.png" /></center></div>
<div class="small-6 columns">By analyzing D-Plex dataset with MGcount, more reads are kept during the analysis, which ultimately preserves biological complexity linked to ncRNA expression without decreasing the accuracy of detection. <br /><span style="line-height: 1em;"><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></span></div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>The D-Plex technology uses two innovative ligation-free mechanisms, <strong>poly(A) tailing and template switching</strong>, to produce sequencing libraries from ultra-low input amounts.</p>
</div>
</div>
<div class="row" style="background-color: #f5f5f5;">
<div class="small-12 columns"><br />
<p><strong>WORKFLOW</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/FL-Image-Workflow.jpg" width="50%" /></center></div>
<div class="small-12 columns">
<p style="text-align: justify; padding: 15px;"><br />RNA molecules are polyadenylated at the 3’-end and primed with an oligo(dT) primer containing part of the ILMN 3’-adapter sequence. The addition of a template-switching oligonucleotide during cDNA synthesis enables fusion of part of the ILMN 5’-adapter during reverse transcription. After PCR, the library comprises double stranded DNA constructs that are ready for sequencing.</p>
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<p><br /><br /></p>
<p></p>
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<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
<p>Need help for your bioinformatics analysis of miRNA?</p>
<p></p>
<div class="small-12 medium-3 large-3 columns"><center><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"><img src="https://www.diagenode.com/img/product/kits/dplex/miRMaster_logo.png" /></a></center>
<p> </p>
<p> </p>
</div>
<div class="small-12 medium-9 large-9 columns">
<p>Diagenode has collaborated with <strong>Andreas Keller</strong> and <strong>Tobias Fehlmann</strong> to develop a new bioinformatics pipeline for <span>the <strong>prediction of novel miRNAs</strong>.</span><br /><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"></a></p>
<ul>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster2">miRMaster 2</a></li>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank">miRMaster</a></li>
</ul>
</div>
<p> </p>
<p> </p>',
'label3' => 'Data analysis - Counting using MGcount',
'info3' => '<p>The counting or expression level calculation is the last step of the processing to generate an expression level matrix. We recommend using MGcount*, a counting tool developed at Diagenode with a suitable annotations file that include the small RNA transcripts that are object of study. MGcount, built on top of featureCounts, employs a flexible quantification approach to deal with datasets containing multiple RNA biotypes such as D-plex libraries. Non-coding RNAs varying in length, biogenesis, and function, may overlap in a genomic region, and are sometimes present in the genome with a high copy number. Consequently, reads may align equally well to more than one position in the reference genome or/and align to a position where more than one annotated transcript is located. MGcount employs two strategies to quantify these reads. Firstly, it assigns reads in rounds prioritizing small-RNAs over long-RNAs ensuring the unbiased read attribution to intergenic and intragenic small RNAs while preventing host-genes transcription over-estimation. Secondly, MGcount collapses loci where reads consistently multi-map into communities of loci with a graph-based approach. The resultant communities are groups of biologically related loci with nearly identical sequence. Subsequently, quantification of reads is performed at the loci-community level, reducing multi-alignments ambiguity at individual locus. Ultimately, this strategy maximizes the transcriptome information analysed and improves the interrogation of non-coding RNAs. MGcount software repository provides annotation files for A. thaliana, H. sapiens, M. musculus and C. elegans. The required input files for MGcount are a .txt file listing the paths to the alignment input files (.bam format) and the annotations file (.gtf format). The output directory path has to be provided as an input as well.</p>
<p><strong>Example command:</strong></p>
<p style="background-color: #f0f0f0;">MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf --outdir outputs --bam_infiles input_bamfilenames.txt</p>
<p>MGcount provides the choice to enable/disable the quantification of all RNA biotypes included in the annotation file in the form of "communities" as optional parameters for small-RNA (--ml_flag_small) and long-RNA (--ml_flag_long). Both are enabled by default. The main output of MGcount is the count_matrix.csv file containing an expression matrix that can be imported to R or any other software for downstream analyses.<br />A full user guide for MGCount is available here: <a href="https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html">https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html</a>.</p>
<p>Other standard counting tools such as featureCounts or HTSeq-counts can also be used alternatively. Given the high complexity of D-Plex libraries, we recommend having a clear understanding of the scientific question and the goal of the project before proceeding to the choice of the counting method as this will strongly impact downstream analyses. Diagenode provides bioinformatics support as a service (please, contact us if you need help with data analysis).</p>
<p>Notice that D-Plex produces forward-stranded data. Stranded libraries have the benefit that reads map to the genome strand where they were originated from. Therefore, when estimating transcript expression, reads aligned to the forward strand should be assigned only to transcript features in the forward strand whereas reads aligned to the reverse strand should be assigned only to transcript features in the reverse strand. For this, make sure you select “stranded mode” in any tool of choice. Stranded mode is selected by default in MGcount.</p>
<p><em><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></em></p>',
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'slug' => 'D-Plex-Small-RNA-seq-Library-Prep-x24',
'meta_title' => 'D-Plex Small RNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, small RNA kit, small RNA-seq kit, low input RNA-seq, UMI RNA-seq, RNA-seq library preparation, D-Plex, higher RNA diversity',
'meta_description' => 'Small RNA-seq library preparation for Illumina sequencing - Unique D-Plex technology - Optimized for ultra-low input (100 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - Compatibility with plasma samples - User-friendly and fast protocol',
'modified' => '2024-07-04 17:29:15',
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'name' => 'D-Plex Unique Dual Indexes for Illumina - Set A',
'description' => '<p><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set A includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li>C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex 24 Single Indexes for Illumina - Set #B">C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex 24 Single Indexes for Illumina - Set #C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex 24 Single Indexes for Illumina - Set #D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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<li>Multiplexing up to <strong>96 samples</strong> when combining Set A, B, C and D</li>
<li>Allows for identification of index hopping</li>
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'meta_title' => 'D-Plex Unique Dual Indexes for Illumina - RNA Library Prep Kit - Set A | Diagenode',
'meta_keywords' => 'RNA kit; small RNA kit; RNA-seq kit; low input RNA-seq kit; small RNA-seq, template switching kit, RNA-seq library preparation, D-Plex, higher RNA diversity',
'meta_description' => 'Compatible with D-Plex RNA-seq kits - Unique Dual Indexes for Illumina - Index hopping mitigation - Suitable for ultra-low input - Contains UMI - Multiplexing up to 48 samples',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set B includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A">C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li>C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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'description' => '<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2 style="font-size: 22px;">DNA断片化、ライブラリー調製、自動化：NGSのワンストップショップ</h2>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-12 medium-12 large-12 columns">
<h4>1. 断片化装置を選択してください：150 bp〜75 kbの範囲でDNAを断片化します。</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-pico-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/bioruptor_pico.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/megaruptor2-1-unit"><img src="https://www.diagenode.com/img/product/shearing_technologies/B06010001_megaruptor2.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-one-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/br-one-profil.png" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns">5μlまで断片化：150 bp〜2 kb<br />NGS DNAライブラリー調製およびFFPE核酸抽出に最適で、</td>
<td class="small-4 medium-4 large-4 columns">2 kb〜75 kbの範囲をできます。<br />メイトペアライブラリー調製および長いフラグメントDNAシーケンシングに最適で、この軽量デスクトップデバイスで</td>
<td class="small-4 medium-4 large-4 columns">20または50μlの断片化が可能です。</td>
</tr>
</tbody>
</table>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-8 medium-8 large-8 columns">
<h4>2. 最適化されたライブラリー調整キットを選択してください。</h4>
</th>
<th class="small-4 medium-4 large-4 columns">
<h4>3. ライブラリー前処理自動化を選択して、比類のないデータ再現性を実感</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><img src="https://www.diagenode.com/img/product/kits/microPlex_library_preparation.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/ideal-library-preparation-kit-x24-incl-index-primer-set-1-24-rxns"><img src="https://www.diagenode.com/img/product/kits/box_kit.jpg" style="display: block; margin-left: auto; margin-right: auto;" height="173" width="250" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/sx-8g-ip-star-compact-automated-system-1-unit"><img src="https://www.diagenode.com/img/product/automation/B03000002%20_ipstar_compact.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">50pgの低入力：MicroPlex Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">5ng以上：iDeal Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">Achieve great NGS data easily</td>
</tr>
</tbody>
</table>
</div>
</div>
<blockquote>
<div class="row">
<div class="small-12 medium-12 large-12 columns"><span class="label" style="margin-bottom: 16px; margin-left: -22px; font-size: 15px;">DiagenodeがNGS研究にぴったりなプロバイダーである理由</span>
<p>Diagenodeは15年以上もエピジェネティクス研究に専念、専門としています。 ChIP研究クロマチン用のユニークな断片化システムの開発から始まり、 専門知識を活かし、5μlのせん断体積まで可能で、NGS DNAライブラリーの調製に最適な最先端DNA断片化装置の開発にたどり着きました。 我々は以来、ChIP-seq、Methyl-seq、NGSライブラリー調製用キットを研究開発し、業界をリードする免疫沈降研究と同様に、ライブラリー調製を自動化および完結させる独自の自動化システムを開発にも成功しました。</p>
<ul>
<li>信頼されるせん断装置</li>
<li>様々なインプットからのライブラリ作成キット</li>
<li>独自の自動化デバイス</li>
</ul>
</div>
</div>
</blockquote>
<div class="row">
<div class="small-12 columns">
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#panel1a">次世代シーケンシングへの理解とその専門知識</a>
<div id="panel1a" class="content">
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>次世代シーケンシング (NGS)</strong> ）は、著しいスケールとハイスループットでシーケンシングを行い、1日に数十億もの塩基生成を可能にします。 NGSのハイスループットは迅速でありながら正確で、再現性のあるデータセットを実現し、さらにシーケンシング費用を削減します。 NGSは、ゲノムシーケンシング、ゲノム再シーケンシング、デノボシーケンシング、トランスクリプトームシーケンシング、その他にDNA-タンパク質相互作用の検出やエピゲノムなどを示します。 指数関数的に増加するシーケンシングデータの需要は、計算分析の障害や解釈、データストレージなどの課題を解決します。</p>
<p>アプリケーションおよび出発物質に応じて、数百万から数十億の鋳型DNA分子を大規模に並行してシーケンシングすることが可能です。その為に、異なる化学物質を使用するいくつかの市販のNGSプラットフォームを利用することができます。 NGSプラットフォームの種類によっては、事前準備とライブラリー作成が必要です。</p>
<p>NGSにとっても、特にデータ処理と分析に関した大きな課題はあります。第3世代技術はゲノミクス研究にさらに革命を起こすであろうと大きく期待されています。</p>
</div>
</div>
<div class="row">
<div class="small-6 medium-6 large-6 columns">
<p><strong>NGS アプリケーション</strong></p>
<ul>
<li>全ゲノム配列決定</li>
<li>デノボシーケンシング</li>
<li>標的配列</li>
<li>Exomeシーケンシング</li>
<li>トランスクリプトーム配列決定</li>
<li>ゲノム配列決定</li>
<li>ミトコンドリア配列決定</li>
<li>DNA-タンパク質相互作用（ChIP-seq</li>
<li>バリアント検出</li>
<li>ゲノム仕上げ</li>
</ul>
</div>
<div class="small-6 medium-6 large-6 columns">
<p><strong>研究分野におけるNGS:</strong></p>
<ul>
<li>腫瘍学</li>
<li>リプロダクティブ・ヘルス</li>
<li>法医学ゲノミクス</li>
<li>アグリゲノミックス</li>
<li>複雑な病気</li>
<li>微生物ゲノミクス</li>
<li>食品・環境ゲノミクス</li>
<li>創薬ゲノミクス - パーソナライズド・メディカル</li>
</ul>
</div>
<div class="small-12 medium-12 large-12 columns">
<p><strong>NGSの用語</strong></p>
<dl>
<dt>リード（読み取り）</dt>
<dd>この装置から得られた連続した単一のストレッチ</dd>
<dt>断片リード</dt>
<dd>フラグメントライブラリからの読み込み。 シーケンシングプラットフォームに応じて、読み取りは通常約100〜300bp。</dd>
<dt>断片ペアエンドリード</dt>
<dd>断片ライブラリーからDNA断片の各末端2つの読み取り。</dd>
<dt>メイトペアリード</dt>
<dd>大きなDNA断片（通常は予め定義されたサイズ範囲）の各末端から2つの読み取り。</dd>
<dt>カバレッジ（例）</dt>
<dd>30×適用範囲とは、参照ゲノム中の各塩基対が平均30回の読み取りを示す。</dd>
</dl>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2>NGSプラットフォーム</h2>
<h3><a href="http://www.illumina.com" target="_blank">イルミナ</a></h3>
<p>イルミナは、クローン的に増幅された鋳型DNA（クラスター）上に位置する、蛍光標識された可逆的鎖ターミネーターヌクレオチドを用いた配列別合成技術を使用。 DNAクラスターは、ガラスフローセルの表面上に固定化され、 ワークフローは、4つのヌクレオチド（それぞれ異なる蛍光色素で標識された）の組み込み、4色イメージング、色素や末端基の切断、取り込み、イメージングなどを繰り返します。フローセルは大規模な並列配列決定を受ける。 この方法により、単一蛍光標識されたヌクレオチドの制御添加によるモノヌクレオチドのエラーを回避する可能性があります。 読み取りの長さは、通常約100〜150 bpです。</p>
<h3><a href="http://www.lifetechnologies.com" target="_blank">イオン トレント</a></h3>
<p>イオントレントは、半導体技術チップを用いて、合成中にヌクレオチドを取り込む際に放出されたプロトンを検出します。 これは、イオン球粒子と呼ばれるビーズの表面にエマルションPCR（emPCR）を使用し、リンクされた特定のアダプターを用いてDNA断片を増幅します。 各ビーズは1種類のDNA断片で覆われていて、異なるDNA断片を有するビーズは次いで、チップの陽子感知ウェル内に配置されます。 チップには一度に4つのヌクレオチドのうちの1つが浸水し、このプロセスは異なるヌクレオチドで15秒ごとに繰り返されます。 配列決定の間に4つの塩基の各々が1つずつ導入されます、組み込みの場合はプロトンが放出され、電圧信号が取り込みに比例して検出されます。.</p>
<h3><a href="http://www.pacificbiosciences.com" target="_blank">パシフィック バイオサイエンス</a></h3>
<p>パシフィックバイオサイエンスでは、20kbを超える塩基対の読み取りも、単一分子リアルタイム（SMRT）シーケンシングによる構造および細胞タイプの変化を観察することができます。 このプラットフォームでは、超長鎖二本鎖DNA（dsDNA）断片が、Megaruptor（登録商標）のようなDiagenode装置を用いたランダムシアリングまたは目的の標的領域の増幅によって生成されます。 SMRTbellライブラリーは、ユニバーサルヘアピンアダプターをDNA断片の各末端に連結することによって生成します。 サイズ選択条件による洗浄ステップの後、配列決定プライマーをSMRTbellテンプレートにアニーリングし、鋳型DNAに結合したDNAポリメラーゼを含む配列決定を、蛍光標識ヌクレオチドの存在下で開始。 各塩基が取り込まれると、異なる蛍光のパルスをリアルタイムで検出します。</p>
<h3><a href="https://nanoporetech.com" target="_blank">オックスフォード ナノポア</a></h3>
<p>Oxford Nanoporeは、単一のDNA分子配列決定に基づく技術を開発します。その技術により生物学的分子、すなわちDNAが一群の電気抵抗性高分子膜として位置するナノスケールの孔（ナノ細孔）またはその近くを通過し、イオン電流が変化します。 この変化に関する情報は、例えば4つのヌクレオチド（AまたはG r CまたはT）ならびに修飾されたヌクレオチドすべてを区別することによって分子情報に訳されます。 シーケンシングミニオンデバイスのフローセルは、数百個のナノポアチャネルのセンサアレイを含みます。 DNAサンプルは、Diagenode社のMegaruptor（登録商標）を用いてランダムシアリングによって生成され得る超長鎖DNAフラグメントが必要です。</p>
<h3><a href="http://www.lifetechnologies.com/be/en/home/life-science/sequencing/next-generation-sequencing/solid-next-generation-sequencing.html" target="_blank">SOLiD</a></h3>
<p>SOLiDは、ユニークな化学作用により、何千という個々のDNA分子の同時配列決定を可能にします。 それは、アダプター対ライブラリーのフラグメントが適切で、せん断されたゲノムDNAへのアダプターのライゲーションによるライブラリー作製から始まります。 次のステップでは、エマルジョンPCR（emPCR）を実施して、ビーズの表面上の個々の鋳型DNA分子をクローン的に増幅。 emPCRでは、個々の鋳型DNAをPCR試薬と混合し、水中油型エマルジョン内の疎水性シェルで囲まれた水性液滴内のプライマーコートビーズを、配列決定のためにロードするスライドガラスの表面にランダムに付着。 この技術は、シークエンシングプライマーへのライゲーションで競合する4つの蛍光標識されたジ塩基プローブのセットを使用します。</p>
<h3><a href="http://454.com/products/technology.asp" target="_blank">454</a></h3>
<p>454は、大規模並列パイロシーケンシングを利用しています。 始めに全ゲノムDNAまたは標的遺伝子断片の300〜800bp断片のライブラリー調製します。 次に、DNAフラグメントへのアダプターの付着および単一のDNA鎖の分離。 その後アダプターに連結されたDNAフラグメントをエマルジョンベースのクローン増幅（emPCR）で処理し、DNAライブラリーフラグメントをミクロンサイズのビーズ上に配置します。 各DNA結合ビーズを光ファイバーチップ上のウェルに入れ、器具に挿入します。 4つのDNAヌクレオチドは、配列決定操作中に固定された順序で連続して加えられ、並行して配列決定されます。</p>
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<p><span style="font-weight: 400;">Most of the major next-generation sequencing platforms require ligation of specific adaptor oligos to </span><a href="../applications/dna-rna-shearing"><span style="font-weight: 400;">fragmented DNA or RNA</span></a><span style="font-weight: 400;"> prior to sequencing</span></p>
<p><span style="font-weight: 400;">After input DNA has been fragmented, it is end-repaired and blunt-ended</span><span style="font-weight: 400;">. The next step is a A-tailing in which dAMP is added to the 3´ end of the blunt phosphorylated DNA fragments to prevent concatemerization and to allow the ligation of adaptors with complementary dT overhangs. In addition, barcoded adapters can be incorporated to facilitate multiplexing prior to or during amplification.</span></p>
<center><img src="https://www.diagenode.com/img/categories/library-prep/flux.png" /></center>
<p><span style="font-weight: 400;">Diagenode offers a comprehensive product portfolio for library preparation:<br /></span></p>
<strong><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">D-Plex RNA-seq Library Preparation Kits</a></strong><br />
<p><span style="font-weight: 400;">Diagenode’s new RNA-sequencing solutions utilize the innovative c</span><span style="font-weight: 400;">apture and a</span><span style="font-weight: 400;">mplification by t</span><span style="font-weight: 400;">ailing and s</span><span style="font-weight: 400;">witching”</span><span style="font-weight: 400;">, a ligation-free method to produce DNA libraries for next generation sequencing from low input amounts of RNA. </span><span style="font-weight: 400;"></span><a href="../categories/Library-preparation-for-RNA-seq">Learn more</a></p>
<strong><a href="../categories/library-preparation-for-ChIP-seq">ChIP-seq and DNA sequencing library preparation solutions</a></strong><br />
<p><span style="font-weight: 400;">Our kits have been optimized for DNA library preparation used for next generation sequencing for a wide range of inputs. Using a simple three-step protocols, our</span><a href="http://www.diagenode.com/p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><span style="font-weight: 400;"> </span></a><span style="font-weight: 400;">kits are an optimal choice for library preparation from DNA inputs down to 50 pg. </span><a href="../categories/library-preparation-for-ChIP-seq">Learn more</a></p>
<a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span><strong>Bioruptor Pico - short fragments</strong></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">Our well-cited Bioruptor Pico is the shearing device of choice for chromatin and DNA fragmentation. Obtain uniform and tight fragment distributions between 150bp -2kb. </span><a href="../p/bioruptor-pico-sonication-device">Learn more</a></p>
<strong><a href="../p/megaruptor2-1-unit"><span href="../p/bioruptor-pico-sonication-device">Megaruptor</span>® - long fragments</a></strong><a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">The Megaruptor is designed to shear DNA from 3kb-75kb for long-read sequencing. <a href="../p/megaruptor2-1-unit">Learn more</a></span></p>
<span href="../p/bioruptor-pico-sonication-device"></span><span style="font-weight: 400;"></span></div>
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'description' => '<p><span>In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.</span></p>',
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'description' => '<p>Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2\% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.</p>',
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'description' => '<p>Light at night has strong effects on physiology and behavior of mammals. It affects mood in humans, which is exploited as light therapy, and has been shown to reset the circadian clock in the suprachiasmatic nuclei (SCN). This resetting is paramount to align physiological and biochemical timing to the environmental light-dark cycle. Here we provide evidence that light at zeitgeber time (ZT) 22 affects mood-related behaviors also in mice by activating the clock gene Period1 (Per1) in the lateral habenula (LHb), a brain region known to modulate mood-related behaviors. We show that complete deletion of Per1 in mice led to depressive-like behavior and loss of the beneficial effects of light on this behavior. In contrast, specific deletion of Per1 in the region of the LHb did not affect mood-related behavior, but suppressed the beneficial effects of light. RNA sequence analysis in the mesolimbic dopaminergic system revealed profound changes of gene expression after a light pulse at ZT22. In the nucleus accumbens (NAc), sensory perception of smell and G-protein coupled receptor signaling were affected the most. Interestingly, most of these genes were not affected in Per1 knock-out animals, indicating that induction of Per1 by light serves as a filter for light-mediated gene expression in the brain. Taken together we show that light affects mood-related behavior in mice at least in part via induction of Per1 in the LHb with consequences on mood-related behavior and signaling mechanisms in the mesolimbic dopaminergic system.</p>',
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'description' => '<p>Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.</p>',
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'name' => 'The ribosomal protein S1-dependent standby site in mRNA consists ofa single-stranded region and a 5' structure element.',
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'description' => '<p>In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient mRNAs, we provide a thorough characterization of the standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.</p>',
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'description' => '<p>Thyroid cancer is the most prevalent endocrine malignancy. We and others have shown that several microRNAs, which are post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues, as well as cell lines derived from these cancers. In the cell, miRNAs are bound to Argonaute (AGO) proteins as what could be termed low molecular weight RNA-Induced Silencing Complexes (LMW-RISCs) that can assemble with additional proteins, mRNA, and translation machinery into high molecular weight RISCs (HMW-RISCs) that exert regulatory function. In this study, we sought to analyze the association of miRNAs with RISC complexes in ATC and PTC. For ATC and PTC lines, miRNA species were enriched in both HMW-RISC and LMW-RISC cellular fractions, compared with intermediate molecular weight fractions and very low molecular weight (AGO-poor) fractions. Furthermore, 60\% of all miRNAs were slightly more abundant in LMW-RISC versus HMW-RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines and one of the most widely studied oncogenic miRNAs in many solid tumors, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings may suggest that miR-21 has a role or roles distinct from canonical post-transcriptional regulation in cancer. Furthermore, the methodology described here is a useful way to assess the distribution of miR-21 between HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.</p>',
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<p>将 <input name="data[Cart][quantity]" placeholder="1" value="1" min="1" style="width:60px;display:inline" type="number" id="CartQuantity" required="required"/> <strong> D-Plex Unique Dual Indexes for Illumina - Set A</strong> 添加至我的购物车。</p>
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
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<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex UDI Module - Set C" target="_blank"><span>C05030023</span> - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex UDI Module - Set D" target="_blank"><span>C05030024</span> - D-Plex Unique Dual Indexes for Illumina - Set D </a></li>
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<p>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</p>',
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<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li><span style="font-weight: 400;">An innovative technology with template switching and UMIs</span></li>
<li><span>Ultra-sensitive methodology with low input capability down to 10 ng</span></li>
<li><span>High efficiency for greater gene detection and biomarker identification</span></li>
<li><span>Consistent gene expression profiling, optimal for gene signature definition</span></li>
<li><span>Precise measurement of strand orientation for anti-sense transcription detection</span></li>
</ul>
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<h4></h4>
<center><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/dplex-mRNA-manual.pdf" target="_blank" title="dplex total rnaseq user manual"><strong>Download the manual</strong></a></center>
<p></p>
<p>The D-Plex mRNA-seq Library Preparation Kit is a tool designed for gene expression analysis. <span>The kit utilizes the</span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank"><span> </span>D-Plex technology</a><span><span> </span>to generate directional libraries for Illumina sequencing directly from total RNAs.</span></p>
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<p><span></span><span>The D-Plex mRNA-seq Kit offers a time-saving protocol that can be completed within 6 hours and requires minimal hands-on time. <span>The library preparation takes place in a </span><span>single tube</span><span>, increasing the efficiency tremendously. This new solution has been developped to capture an increased number of genes for great discovery efficiency.</span></span></p>
<p><span><span>The D-Plex mRNA-seq Kit includes all buffers and enzymes necessary for mRNA capture and library preparation. Specific D-Plex Unique Dual Indexes were designed and validated to fit the D-Plex technology and are available separately:</span></span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
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<div class="slick">
<div>
<h3>Greater gene discovery efficiency at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/UHR_mRNA.png" alt="total RNA kit" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit enables the detection of higher numbers of genes even for low RNA inputs. Increased number of genes detected at 10x coverage is an indicator of greater sensitivity.</p>
</div>
</div>
<div>
<h3>High mappability to the coding transcriptome</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_biotypes.png" alt="total RNA diversity" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq libraries capture efficiently coding exons and introns present in the sample of interest.</p>
</div>
</div>
<div>
<h3>Consistent, sensitive gene expression profiling</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/HUR_mRNA_violin_tpm_10.png" alt="total RNA" height="200px" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>Gene expression profiling obtained with D-Plex mRNA-seq kit is conserved across a broad dynamic range of inputs, especially for the lowest RNA inputs.</p>
</div>
</div>
<div>
<h3>Exceptional data concordance at low inputs</h3>
<div class="large-10 small-12 medium-10 large-centered medium-centered small-centered columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/robustness_corr_HUR_mrna.png" alt="total RNA" /></center></div>
<div class="large-6 small-12 medium-6 large-centered medium-centered small-centered columns">
<p></p>
<p>D-Plex mRNA-seq kit produces quality data with high data concordance between low and high RNA inputs. This result demonstrates that D-Plex is an ideal solution for precious samples with limited starting material.</p>
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<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
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<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
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<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
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'meta_title' => 'D-Plex mRNA-seq Library Prep Kit | Diagenode ',
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'description' => '<div class="small-12 medium-12 large-12 columns" style="border: 3px solid #B02736; padding: 10px; margin: 10px;">
<h2>Product Features</h2>
<ul style="list-style-type: disc;">
<li>Ultra-low input capability, down to 10 pg for small RNAs and 100 pg for total RNA samples</li>
<li>High library complexity providing novel and diverse transcript detection</li>
<li>Versatile integration into numerous transcriptomic analysis workflow: ribosome profiling, CLIP-seq, RIP-seq and other</li>
<li>Improved quantification accuracy through the use of UMIs</li>
<li>Easy to use with minimal hands-on time: one day, one tube protocol</li>
</ul>
</div>
<p></p>
<p></p>
<center><em><a class="chip diahome button" href="https://www.diagenode.com/files/products/kits/MA-D-Plex.pdf" target="_blank" title="D-Plex Small RNA library preparation user manual"><strong>Download the manual</strong></a></em></center>
<p>D-Plex Small RNA-seq Library Prep Kit is a tool designed for the study of the <strong>small non-coding transcriptome</strong>. The kit is using the<a href="https://www.diagenode.com/en/pages/dplex" target="_blank"> D-Plex technology</a> to generate small RNA libraries for Illumina sequencing directly from total RNAs or enriched small RNAs.</p>
<p>The D-Plex technology utilizes two innovative <strong>ligation-free mechanisms</strong> - <strong>poly(A) tailing</strong> and <strong>template switching</strong> - to produce sequencing libraries from ultra-low input amounts, down to 10 pg for small RNAs and 100 pg for total RNAs. Combined with <strong>unique molecular identifiers</strong> (UMI), this complete solution ensures a <strong>realistic</strong> and <strong>accurate representation of diverse small RNA species</strong> (such as microRNAs, piRNAs, tRNAs, and siRNAs) with minimized quantitative bias.</p>
<p>D-Plex Small RNA-seq Kit offers a time saving protocol that can be completed within <strong>5 hours</strong> and requires minimal hands-on time. <span>The library preparation takes place in a </span><strong>single tube</strong><span>, increasing the efficiency tremendously. </span></p>
<p>D-Plex Small RNA-seq Kit includes all buffers and enzymes necessary for the library preparation. Specific D-Plex Indexes were designed and validated to fit the D-Plex technology and are available separately:</p>
<p><strong>For D-Plex UDI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A" title="D-Plex UDI Module - Set A" target="_blank"><span>C05030021</span> - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex UDI Module - Set B" target="_blank"><span>C05030022</span> - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><strong>For D-Plex SI library construction:</strong></p>
<ul style="list-style-type: disc;">
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-A" title="D-Plex SI Module - Set A" target="_blank">C05030010 - D-Plex Single Indexes for Illumina - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Single-Indexes-Set-B" title="D-Plex SI Module - Set B" target="_blank">C05030011 - D-Plex Single Indexes for Illumina - Set B</a></li>
</ul>
<p><strong>D-Plex is also available for MGI sequencing, check <a href="https://www.diagenode.com/en/p/d-plex-small-RNA-dnbseq-kit-x24" target="_blank">here</a>!</strong></p>
<p></p>
<p></p>
<p></p>
<center><strong>The D-Plex smRNA-seq is a versatile tool for a plethora of transciptomic analysis</strong></center>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/versatile-circle.png" /></center>
<p></p>
<p>With low-input capability, accuracy and ability to incorporate different transcript classes independent of chemical moieties, the strong D-Plex library-preparation method can be included in various analysis workflows (i.e., ribosome profiling, genome-wide mapping of protein: RNA binding sites).</p>
<p></p>
<p></p>',
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<div class="small-12 columns">
<p><strong>High library diversity for ultra-low inputs</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-diversity-fig1.png" /></center></div>
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig2-captures.png" /></center></div>
</div>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>D-Plex smRNA-seq captures the smRNA complexity of the sample in a sensitive manner. A large number of features is detected for each biotype at a CPM ≥5, as shown above for different species.</p>
</div>
</div>
<p></p>
<p><strong>Accurate representation of the smRNA content of a sample</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-ratplasma.png" /></center></div>
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-miRNAdistribution.png" /></center></div>
</div>
<div class="row">
<div class="small-6 columns">
<p><strong>Accurate identification of PCR duplicates in low-input samples using unique molecular identifiers (UMIs).</strong><br />The UMI read deduplication that is embedded in the D-Plex construct is used to avoid over-estimation of transcripts by identifying clones generated during the PCR amplification of the library. Despite limiting starting RNA input, the UMI correction removed technical copies of transcripts, maintaining the initial sample abundance.</p>
</div>
<div class="small-6 columns">
<p>D-Plex (template switching) technology enables recovery of the initial miRNA distribution of the miRXplore Universal Reference (Miltenyi Biotech Inc., Cat. No. 130-093-521). In contrast, ligation-based kits display a strong distortion of the miRNA equimolar distribution initially present in the pool, indicating sample incorporation bias.</p>
</div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-foldchange.jpg" /></center></div>
<div class="small-6 columns"><strong>D-Plex smRNA-seq in association with the MGcount analysis correlates strongly (R² > 0.8) with the RT-qPCR analysis, which is considered the gold-standard for accuracy. </strong><br />The figures shows a fold-change accuracy of a panel of 20 different small-RNA (figure taken from MGcount publication - Hita et al., BMC bioinformatics, 23-39(2022).</div>
</div>
<p></p>
<p></p>
<p><strong>Maximize information from the regulatory transcriptome using D-Plex small RNA-seq kit and MGcount*</strong></p>
<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/kits/dplex/dplex-fig-multialignments.png" /></center></div>
<div class="small-6 columns">By analyzing D-Plex dataset with MGcount, more reads are kept during the analysis, which ultimately preserves biological complexity linked to ncRNA expression without decreasing the accuracy of detection. <br /><span style="line-height: 1em;"><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></span></div>
</div>
<p></p>
<p></p>
<div class="row">
<div class="small-12 columns">
<p>The D-Plex technology uses two innovative ligation-free mechanisms, <strong>poly(A) tailing and template switching</strong>, to produce sequencing libraries from ultra-low input amounts.</p>
</div>
</div>
<div class="row" style="background-color: #f5f5f5;">
<div class="small-12 columns"><br />
<p><strong>WORKFLOW</strong></p>
<center><img src="https://www.diagenode.com/img/product/kits/dplex/FL-Image-Workflow.jpg" width="50%" /></center></div>
<div class="small-12 columns">
<p style="text-align: justify; padding: 15px;"><br />RNA molecules are polyadenylated at the 3’-end and primed with an oligo(dT) primer containing part of the ILMN 3’-adapter sequence. The addition of a template-switching oligonucleotide during cDNA synthesis enables fusion of part of the ILMN 5’-adapter during reverse transcription. After PCR, the library comprises double stranded DNA constructs that are ready for sequencing.</p>
</div>
</div>
<p><br /><br /></p>
<p></p>
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'info2' => '<p>A specific bioinformatics pipeline has been developed to process the special sequences present in the D-Plex construct, namely the UMI, the A-tail, and the template switch motif. All guidelines and free softwares are shared in the user manual. Subject to the compatibility of D-Plex constructs, other specific pipelines can be used.</p>
<p><img src="https://www.diagenode.com/img/product/kits/dplex/bioinfoPipe.png" alt="small RNA sequencing bioinformatics pipeline" caption="false" width="925" height="196" /></p>
<p>Need help for your bioinformatics analysis of miRNA?</p>
<p></p>
<div class="small-12 medium-3 large-3 columns"><center><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"><img src="https://www.diagenode.com/img/product/kits/dplex/miRMaster_logo.png" /></a></center>
<p> </p>
<p> </p>
</div>
<div class="small-12 medium-9 large-9 columns">
<p>Diagenode has collaborated with <strong>Andreas Keller</strong> and <strong>Tobias Fehlmann</strong> to develop a new bioinformatics pipeline for <span>the <strong>prediction of novel miRNAs</strong>.</span><br /><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank"></a></p>
<ul>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster2">miRMaster 2</a></li>
<li style="text-align: justify;"><a href="https://ccb-compute.cs.uni-saarland.de/mirmaster" target="_blank">miRMaster</a></li>
</ul>
</div>
<p> </p>
<p> </p>',
'label3' => 'Data analysis - Counting using MGcount',
'info3' => '<p>The counting or expression level calculation is the last step of the processing to generate an expression level matrix. We recommend using MGcount*, a counting tool developed at Diagenode with a suitable annotations file that include the small RNA transcripts that are object of study. MGcount, built on top of featureCounts, employs a flexible quantification approach to deal with datasets containing multiple RNA biotypes such as D-plex libraries. Non-coding RNAs varying in length, biogenesis, and function, may overlap in a genomic region, and are sometimes present in the genome with a high copy number. Consequently, reads may align equally well to more than one position in the reference genome or/and align to a position where more than one annotated transcript is located. MGcount employs two strategies to quantify these reads. Firstly, it assigns reads in rounds prioritizing small-RNAs over long-RNAs ensuring the unbiased read attribution to intergenic and intragenic small RNAs while preventing host-genes transcription over-estimation. Secondly, MGcount collapses loci where reads consistently multi-map into communities of loci with a graph-based approach. The resultant communities are groups of biologically related loci with nearly identical sequence. Subsequently, quantification of reads is performed at the loci-community level, reducing multi-alignments ambiguity at individual locus. Ultimately, this strategy maximizes the transcriptome information analysed and improves the interrogation of non-coding RNAs. MGcount software repository provides annotation files for A. thaliana, H. sapiens, M. musculus and C. elegans. The required input files for MGcount are a .txt file listing the paths to the alignment input files (.bam format) and the annotations file (.gtf format). The output directory path has to be provided as an input as well.</p>
<p><strong>Example command:</strong></p>
<p style="background-color: #f0f0f0;">MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf --outdir outputs --bam_infiles input_bamfilenames.txt</p>
<p>MGcount provides the choice to enable/disable the quantification of all RNA biotypes included in the annotation file in the form of "communities" as optional parameters for small-RNA (--ml_flag_small) and long-RNA (--ml_flag_long). Both are enabled by default. The main output of MGcount is the count_matrix.csv file containing an expression matrix that can be imported to R or any other software for downstream analyses.<br />A full user guide for MGCount is available here: <a href="https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html">https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html</a>.</p>
<p>Other standard counting tools such as featureCounts or HTSeq-counts can also be used alternatively. Given the high complexity of D-Plex libraries, we recommend having a clear understanding of the scientific question and the goal of the project before proceeding to the choice of the counting method as this will strongly impact downstream analyses. Diagenode provides bioinformatics support as a service (please, contact us if you need help with data analysis).</p>
<p>Notice that D-Plex produces forward-stranded data. Stranded libraries have the benefit that reads map to the genome strand where they were originated from. Therefore, when estimating transcript expression, reads aligned to the forward strand should be assigned only to transcript features in the forward strand whereas reads aligned to the reverse strand should be assigned only to transcript features in the reverse strand. For this, make sure you select “stranded mode” in any tool of choice. Stranded mode is selected by default in MGcount.</p>
<p><em><small>*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC Bioinformatics 23, 39 (2022). <a href="https://doi.org/10.1186/s12859-021-04544-3">https://doi.org/10.1186/s12859-021-04544-3</a></small></em></p>',
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'slug' => 'D-Plex-Small-RNA-seq-Library-Prep-x24',
'meta_title' => 'D-Plex Small RNA-seq Library Prep Kit | Diagenode ',
'meta_keywords' => 'RNA kit, RNA-seq kit, small RNA kit, small RNA-seq kit, low input RNA-seq, UMI RNA-seq, RNA-seq library preparation, D-Plex, higher RNA diversity',
'meta_description' => 'Small RNA-seq library preparation for Illumina sequencing - Unique D-Plex technology - Optimized for ultra-low input (100 pg total RNA) - UMI reduce PCR biases - UDI detect index hopping - Compatibility with plasma samples - User-friendly and fast protocol',
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<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set A includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li>C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-B" title="D-Plex 24 Single Indexes for Illumina - Set #B">C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C" title="D-Plex 24 Single Indexes for Illumina - Set #C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D" title="D-Plex 24 Single Indexes for Illumina - Set #D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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'meta_keywords' => 'RNA kit; small RNA kit; RNA-seq kit; low input RNA-seq kit; small RNA-seq, template switching kit, RNA-seq library preparation, D-Plex, higher RNA diversity',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set B includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-A">C05030021 - D-Plex Unique Dual Indexes for Illumina - Set A</a></li>
<li>C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
</ul>
<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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<li>Compatible with <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24">D-Plex Small RNA-seq kit for Illumina</a> </li>
<li>Multiplexing up to <strong>96 samples</strong> when combining Set A, B, C and D</li>
<li>Allows for identification of index hopping</li>
</ul>',
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'info2' => '<p><span>D-Plex RNA-seq UDI library constructs bear the TruSeq (Illumina) HT adapters. In case of a multiplexing scenario, it is therefore recommended to submit the D-Plex libraries as TruSeq HT libraries to your sequencing provider. </span><span>Further details are provided in the D-Plex Unique Dual Indexes Module<span> </span><a href="https://www.diagenode.com/files/products/kits/dplex-unique-dual-indexes-manual.pdf" target="_blank">manual</a>.</span></p>',
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'description' => '<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2 style="font-size: 22px;">DNA断片化、ライブラリー調製、自動化：NGSのワンストップショップ</h2>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-12 medium-12 large-12 columns">
<h4>1. 断片化装置を選択してください：150 bp〜75 kbの範囲でDNAを断片化します。</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-pico-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/bioruptor_pico.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/megaruptor2-1-unit"><img src="https://www.diagenode.com/img/product/shearing_technologies/B06010001_megaruptor2.jpg" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/bioruptor-one-sonication-device"><img src="https://www.diagenode.com/img/product/shearing_technologies/br-one-profil.png" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns">5μlまで断片化：150 bp〜2 kb<br />NGS DNAライブラリー調製およびFFPE核酸抽出に最適で、</td>
<td class="small-4 medium-4 large-4 columns">2 kb〜75 kbの範囲をできます。<br />メイトペアライブラリー調製および長いフラグメントDNAシーケンシングに最適で、この軽量デスクトップデバイスで</td>
<td class="small-4 medium-4 large-4 columns">20または50μlの断片化が可能です。</td>
</tr>
</tbody>
</table>
<table class="small-12 medium-12 large-12 columns">
<tbody>
<tr>
<th class="small-8 medium-8 large-8 columns">
<h4>2. 最適化されたライブラリー調整キットを選択してください。</h4>
</th>
<th class="small-4 medium-4 large-4 columns">
<h4>3. ライブラリー前処理自動化を選択して、比類のないデータ再現性を実感</h4>
</th>
</tr>
<tr style="background-color: #ffffff;">
<td class="small-12 medium-12 large-12 columns"></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns"><a href="../p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><img src="https://www.diagenode.com/img/product/kits/microPlex_library_preparation.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/ideal-library-preparation-kit-x24-incl-index-primer-set-1-24-rxns"><img src="https://www.diagenode.com/img/product/kits/box_kit.jpg" style="display: block; margin-left: auto; margin-right: auto;" height="173" width="250" /></a></td>
<td class="small-4 medium-4 large-4 columns"><a href="../p/sx-8g-ip-star-compact-automated-system-1-unit"><img src="https://www.diagenode.com/img/product/automation/B03000002%20_ipstar_compact.png" style="display: block; margin-left: auto; margin-right: auto;" /></a></td>
</tr>
<tr>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">50pgの低入力：MicroPlex Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">5ng以上：iDeal Library Preparation Kit</td>
<td class="small-4 medium-4 large-4 columns" style="text-align: center;">Achieve great NGS data easily</td>
</tr>
</tbody>
</table>
</div>
</div>
<blockquote>
<div class="row">
<div class="small-12 medium-12 large-12 columns"><span class="label" style="margin-bottom: 16px; margin-left: -22px; font-size: 15px;">DiagenodeがNGS研究にぴったりなプロバイダーである理由</span>
<p>Diagenodeは15年以上もエピジェネティクス研究に専念、専門としています。 ChIP研究クロマチン用のユニークな断片化システムの開発から始まり、 専門知識を活かし、5μlのせん断体積まで可能で、NGS DNAライブラリーの調製に最適な最先端DNA断片化装置の開発にたどり着きました。 我々は以来、ChIP-seq、Methyl-seq、NGSライブラリー調製用キットを研究開発し、業界をリードする免疫沈降研究と同様に、ライブラリー調製を自動化および完結させる独自の自動化システムを開発にも成功しました。</p>
<ul>
<li>信頼されるせん断装置</li>
<li>様々なインプットからのライブラリ作成キット</li>
<li>独自の自動化デバイス</li>
</ul>
</div>
</div>
</blockquote>
<div class="row">
<div class="small-12 columns">
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#panel1a">次世代シーケンシングへの理解とその専門知識</a>
<div id="panel1a" class="content">
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<p><strong>次世代シーケンシング (NGS)</strong> ）は、著しいスケールとハイスループットでシーケンシングを行い、1日に数十億もの塩基生成を可能にします。 NGSのハイスループットは迅速でありながら正確で、再現性のあるデータセットを実現し、さらにシーケンシング費用を削減します。 NGSは、ゲノムシーケンシング、ゲノム再シーケンシング、デノボシーケンシング、トランスクリプトームシーケンシング、その他にDNA-タンパク質相互作用の検出やエピゲノムなどを示します。 指数関数的に増加するシーケンシングデータの需要は、計算分析の障害や解釈、データストレージなどの課題を解決します。</p>
<p>アプリケーションおよび出発物質に応じて、数百万から数十億の鋳型DNA分子を大規模に並行してシーケンシングすることが可能です。その為に、異なる化学物質を使用するいくつかの市販のNGSプラットフォームを利用することができます。 NGSプラットフォームの種類によっては、事前準備とライブラリー作成が必要です。</p>
<p>NGSにとっても、特にデータ処理と分析に関した大きな課題はあります。第3世代技術はゲノミクス研究にさらに革命を起こすであろうと大きく期待されています。</p>
</div>
</div>
<div class="row">
<div class="small-6 medium-6 large-6 columns">
<p><strong>NGS アプリケーション</strong></p>
<ul>
<li>全ゲノム配列決定</li>
<li>デノボシーケンシング</li>
<li>標的配列</li>
<li>Exomeシーケンシング</li>
<li>トランスクリプトーム配列決定</li>
<li>ゲノム配列決定</li>
<li>ミトコンドリア配列決定</li>
<li>DNA-タンパク質相互作用（ChIP-seq</li>
<li>バリアント検出</li>
<li>ゲノム仕上げ</li>
</ul>
</div>
<div class="small-6 medium-6 large-6 columns">
<p><strong>研究分野におけるNGS:</strong></p>
<ul>
<li>腫瘍学</li>
<li>リプロダクティブ・ヘルス</li>
<li>法医学ゲノミクス</li>
<li>アグリゲノミックス</li>
<li>複雑な病気</li>
<li>微生物ゲノミクス</li>
<li>食品・環境ゲノミクス</li>
<li>創薬ゲノミクス - パーソナライズド・メディカル</li>
</ul>
</div>
<div class="small-12 medium-12 large-12 columns">
<p><strong>NGSの用語</strong></p>
<dl>
<dt>リード（読み取り）</dt>
<dd>この装置から得られた連続した単一のストレッチ</dd>
<dt>断片リード</dt>
<dd>フラグメントライブラリからの読み込み。 シーケンシングプラットフォームに応じて、読み取りは通常約100〜300bp。</dd>
<dt>断片ペアエンドリード</dt>
<dd>断片ライブラリーからDNA断片の各末端2つの読み取り。</dd>
<dt>メイトペアリード</dt>
<dd>大きなDNA断片（通常は予め定義されたサイズ範囲）の各末端から2つの読み取り。</dd>
<dt>カバレッジ（例）</dt>
<dd>30×適用範囲とは、参照ゲノム中の各塩基対が平均30回の読み取りを示す。</dd>
</dl>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2>NGSプラットフォーム</h2>
<h3><a href="http://www.illumina.com" target="_blank">イルミナ</a></h3>
<p>イルミナは、クローン的に増幅された鋳型DNA（クラスター）上に位置する、蛍光標識された可逆的鎖ターミネーターヌクレオチドを用いた配列別合成技術を使用。 DNAクラスターは、ガラスフローセルの表面上に固定化され、 ワークフローは、4つのヌクレオチド（それぞれ異なる蛍光色素で標識された）の組み込み、4色イメージング、色素や末端基の切断、取り込み、イメージングなどを繰り返します。フローセルは大規模な並列配列決定を受ける。 この方法により、単一蛍光標識されたヌクレオチドの制御添加によるモノヌクレオチドのエラーを回避する可能性があります。 読み取りの長さは、通常約100〜150 bpです。</p>
<h3><a href="http://www.lifetechnologies.com" target="_blank">イオン トレント</a></h3>
<p>イオントレントは、半導体技術チップを用いて、合成中にヌクレオチドを取り込む際に放出されたプロトンを検出します。 これは、イオン球粒子と呼ばれるビーズの表面にエマルションPCR（emPCR）を使用し、リンクされた特定のアダプターを用いてDNA断片を増幅します。 各ビーズは1種類のDNA断片で覆われていて、異なるDNA断片を有するビーズは次いで、チップの陽子感知ウェル内に配置されます。 チップには一度に4つのヌクレオチドのうちの1つが浸水し、このプロセスは異なるヌクレオチドで15秒ごとに繰り返されます。 配列決定の間に4つの塩基の各々が1つずつ導入されます、組み込みの場合はプロトンが放出され、電圧信号が取り込みに比例して検出されます。.</p>
<h3><a href="http://www.pacificbiosciences.com" target="_blank">パシフィック バイオサイエンス</a></h3>
<p>パシフィックバイオサイエンスでは、20kbを超える塩基対の読み取りも、単一分子リアルタイム（SMRT）シーケンシングによる構造および細胞タイプの変化を観察することができます。 このプラットフォームでは、超長鎖二本鎖DNA（dsDNA）断片が、Megaruptor（登録商標）のようなDiagenode装置を用いたランダムシアリングまたは目的の標的領域の増幅によって生成されます。 SMRTbellライブラリーは、ユニバーサルヘアピンアダプターをDNA断片の各末端に連結することによって生成します。 サイズ選択条件による洗浄ステップの後、配列決定プライマーをSMRTbellテンプレートにアニーリングし、鋳型DNAに結合したDNAポリメラーゼを含む配列決定を、蛍光標識ヌクレオチドの存在下で開始。 各塩基が取り込まれると、異なる蛍光のパルスをリアルタイムで検出します。</p>
<h3><a href="https://nanoporetech.com" target="_blank">オックスフォード ナノポア</a></h3>
<p>Oxford Nanoporeは、単一のDNA分子配列決定に基づく技術を開発します。その技術により生物学的分子、すなわちDNAが一群の電気抵抗性高分子膜として位置するナノスケールの孔（ナノ細孔）またはその近くを通過し、イオン電流が変化します。 この変化に関する情報は、例えば4つのヌクレオチド（AまたはG r CまたはT）ならびに修飾されたヌクレオチドすべてを区別することによって分子情報に訳されます。 シーケンシングミニオンデバイスのフローセルは、数百個のナノポアチャネルのセンサアレイを含みます。 DNAサンプルは、Diagenode社のMegaruptor（登録商標）を用いてランダムシアリングによって生成され得る超長鎖DNAフラグメントが必要です。</p>
<h3><a href="http://www.lifetechnologies.com/be/en/home/life-science/sequencing/next-generation-sequencing/solid-next-generation-sequencing.html" target="_blank">SOLiD</a></h3>
<p>SOLiDは、ユニークな化学作用により、何千という個々のDNA分子の同時配列決定を可能にします。 それは、アダプター対ライブラリーのフラグメントが適切で、せん断されたゲノムDNAへのアダプターのライゲーションによるライブラリー作製から始まります。 次のステップでは、エマルジョンPCR（emPCR）を実施して、ビーズの表面上の個々の鋳型DNA分子をクローン的に増幅。 emPCRでは、個々の鋳型DNAをPCR試薬と混合し、水中油型エマルジョン内の疎水性シェルで囲まれた水性液滴内のプライマーコートビーズを、配列決定のためにロードするスライドガラスの表面にランダムに付着。 この技術は、シークエンシングプライマーへのライゲーションで競合する4つの蛍光標識されたジ塩基プローブのセットを使用します。</p>
<h3><a href="http://454.com/products/technology.asp" target="_blank">454</a></h3>
<p>454は、大規模並列パイロシーケンシングを利用しています。 始めに全ゲノムDNAまたは標的遺伝子断片の300〜800bp断片のライブラリー調製します。 次に、DNAフラグメントへのアダプターの付着および単一のDNA鎖の分離。 その後アダプターに連結されたDNAフラグメントをエマルジョンベースのクローン増幅（emPCR）で処理し、DNAライブラリーフラグメントをミクロンサイズのビーズ上に配置します。 各DNA結合ビーズを光ファイバーチップ上のウェルに入れ、器具に挿入します。 4つのDNAヌクレオチドは、配列決定操作中に固定された順序で連続して加えられ、並行して配列決定されます。</p>
</div>
</div>
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</ul>
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<p><span style="font-weight: 400;">Most of the major next-generation sequencing platforms require ligation of specific adaptor oligos to </span><a href="../applications/dna-rna-shearing"><span style="font-weight: 400;">fragmented DNA or RNA</span></a><span style="font-weight: 400;"> prior to sequencing</span></p>
<p><span style="font-weight: 400;">After input DNA has been fragmented, it is end-repaired and blunt-ended</span><span style="font-weight: 400;">. The next step is a A-tailing in which dAMP is added to the 3´ end of the blunt phosphorylated DNA fragments to prevent concatemerization and to allow the ligation of adaptors with complementary dT overhangs. In addition, barcoded adapters can be incorporated to facilitate multiplexing prior to or during amplification.</span></p>
<center><img src="https://www.diagenode.com/img/categories/library-prep/flux.png" /></center>
<p><span style="font-weight: 400;">Diagenode offers a comprehensive product portfolio for library preparation:<br /></span></p>
<strong><a href="https://www.diagenode.com/en/categories/Library-preparation-for-RNA-seq">D-Plex RNA-seq Library Preparation Kits</a></strong><br />
<p><span style="font-weight: 400;">Diagenode’s new RNA-sequencing solutions utilize the innovative c</span><span style="font-weight: 400;">apture and a</span><span style="font-weight: 400;">mplification by t</span><span style="font-weight: 400;">ailing and s</span><span style="font-weight: 400;">witching”</span><span style="font-weight: 400;">, a ligation-free method to produce DNA libraries for next generation sequencing from low input amounts of RNA. </span><span style="font-weight: 400;"></span><a href="../categories/Library-preparation-for-RNA-seq">Learn more</a></p>
<strong><a href="../categories/library-preparation-for-ChIP-seq">ChIP-seq and DNA sequencing library preparation solutions</a></strong><br />
<p><span style="font-weight: 400;">Our kits have been optimized for DNA library preparation used for next generation sequencing for a wide range of inputs. Using a simple three-step protocols, our</span><a href="http://www.diagenode.com/p/microplex-library-preparation-kit-v2-x12-12-indices-12-rxns"><span style="font-weight: 400;"> </span></a><span style="font-weight: 400;">kits are an optimal choice for library preparation from DNA inputs down to 50 pg. </span><a href="../categories/library-preparation-for-ChIP-seq">Learn more</a></p>
<a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span><strong>Bioruptor Pico - short fragments</strong></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">Our well-cited Bioruptor Pico is the shearing device of choice for chromatin and DNA fragmentation. Obtain uniform and tight fragment distributions between 150bp -2kb. </span><a href="../p/bioruptor-pico-sonication-device">Learn more</a></p>
<strong><a href="../p/megaruptor2-1-unit"><span href="../p/bioruptor-pico-sonication-device">Megaruptor</span>® - long fragments</a></strong><a href="../p/bioruptor-pico-sonication-device"><span style="font-weight: 400;"></span></a><a href="../categories/library-preparation-for-ChIP-seq-and-DNA-sequencing"><span style="font-weight: 400;"></span></a><br />
<p><span style="font-weight: 400;"></span><span style="font-weight: 400;">The Megaruptor is designed to shear DNA from 3kb-75kb for long-read sequencing. <a href="../p/megaruptor2-1-unit">Learn more</a></span></p>
<span href="../p/bioruptor-pico-sonication-device"></span><span style="font-weight: 400;"></span></div>
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'description' => '<p>Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.</p>',
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'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32670023',
'doi' => '10.3389/fncel.2020.00160',
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'name' => 'The ribosomal protein S1-dependent standby site in mRNA consists ofa single-stranded region and a 5' structure element.',
'authors' => 'Romilly Cédric et al.',
'description' => '<p>In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient mRNAs, we provide a thorough characterization of the standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.</p>',
'date' => '2019-08-01',
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'name' => 'mir-21 is associated with inactive low molecular weight Argonautecomplexes in thyroid cancer cell lines',
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'description' => '<p>Thyroid cancer is the most prevalent endocrine malignancy. We and others have shown that several microRNAs, which are post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues, as well as cell lines derived from these cancers. In the cell, miRNAs are bound to Argonaute (AGO) proteins as what could be termed low molecular weight RNA-Induced Silencing Complexes (LMW-RISCs) that can assemble with additional proteins, mRNA, and translation machinery into high molecular weight RISCs (HMW-RISCs) that exert regulatory function. In this study, we sought to analyze the association of miRNAs with RISC complexes in ATC and PTC. For ATC and PTC lines, miRNA species were enriched in both HMW-RISC and LMW-RISC cellular fractions, compared with intermediate molecular weight fractions and very low molecular weight (AGO-poor) fractions. Furthermore, 60\% of all miRNAs were slightly more abundant in LMW-RISC versus HMW-RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines and one of the most widely studied oncogenic miRNAs in many solid tumors, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings may suggest that miR-21 has a role or roles distinct from canonical post-transcriptional regulation in cancer. Furthermore, the methodology described here is a useful way to assess the distribution of miR-21 between HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.</p>',
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'pmid' => 'https://www.biorxiv.org/content/10.1101/2020.03.24.006072v3',
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'description' => '<div class="row"><div class="small-12 medium-4 large-4 columns"><div class="panel"><h3><em>lncRNAs in cancer</em></h3><p class="text-left">Gastrointestinal cancer: Gastrointestinal cancers occur as a result of dysregulated signaling pathways and cellular processes, such as the cell cycle or apoptosis. LncRNAs can regulate proliferation of gastrointestinal cancer through interacting with RNA targets, localization to chromatin, or binding to proteins. Read more about Long non-coding RNAs: crucial regulators of gastrointestinal cancer cell proliferation</p><h3><em>microRNAs in cancer</em></h3><p class="text-left">miR-155 has been found overexpressed in many cancer types including hematopoietic cancers, breast, lung and colon cancer<br /><br /> <a href="https://www.cell.com/trends/molecular-medicine/pdf/S1471-4914(14)00101-4.pdf">https://www.cell.com/trends/molecular-medicine/pdf/S1471-4914(14)00101-4.pdf</a></p></div></div><div class="small-12 medium-8 large-8 columns"><center><img src="https://www.diagenode.com/img/cancer/long-non-coding-rna.jpg" width="550" height="345" /></center><p></p><p>Various non-coding RNAs (ncRNAs) have been found to function as key regulators for transcription, chromatin remodelling, and post-transcriptional modification, thus making them relevant in oncogenesis, both as tumor suppressors and as drivers. For example, a number of microRNAs (miRNAs), one of the more well-studied types of ncRNAs, have been identified as potential cancer biomarkers and clinical targets. Other ncRNAs, such as long noncoding RNAs are involved in cancer regulation and proliferation. Understanding the role of ncRNAs will be critical for cancer research and therapeutic development.</p></div></div>',
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<p style="text-align: left;"><span>D-Plex Unique Dual Indexes Module - Set B includes primer pairs with 24 unique dual barcodes (unique i5 and i7 indexes) for library multiplexing with the <a href="https://www.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24" target="_blank" title="D-Plex Small RNA-seq Kit">D-Plex Small RNA-seq Kit</a>. </span></p>
<p style="text-align: left;"><span>The use of UDI is highly recommended to mitigate errors introduced by read misassignment, including index hopping frequently observed with patterned flow cells such as Illumina’s NovaSeq system.</span></p>
<p><span>Four sets are available separately: </span></p>
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<li>C05030022 - D-Plex Unique Dual Indexes for Illumina - Set B</li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-C">C05030023 - D-Plex Unique Dual Indexes for Illumina - Set C</a></li>
<li><a href="https://www.diagenode.com/en/p/D-Plex-24-Unique-Dual-Indexes-Set-D">C05030024 - D-Plex Unique Dual Indexes for Illumina - Set D</a></li>
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<p><span>Each set can be used for library multiplexing up to 24. <span>Set A, B, C and D can be used simultaneously for library multiplexing up to 96.</span></span></p>
<p><span>Read more about the </span><a href="https://www.diagenode.com/en/pages/dplex" target="_blank">D-Plex technology</a><span>.</span><span> </span></p>',
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