D-Plex Small RNA-seq Kit x24 for Illumina

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D-Plex Small RNA-seq library prep kit for Illumina

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Diagenode’s latest innovation in RNA-seq, D-Plex, is based on a template-switching technology that delivers a ligation-free method for RNA library preparation, ensuring realistic representation of diverse small RNA species (such as miRNA, piRNA, tRNA, and siRNA) with minimized bias. This solution allows you to produce Illumina®-compatible DNA libraries for NGS from low input amounts of RNA with minimized PhiX spike-in quantity for the most representative results.

The D-Plex small RNA solution was optimized for 10 pg - 10 ng enriched small RNA (or 100 pg - 100 ng total RNA) and contains our latest TSO (Template-Switch Oligonucleotides) technology including UMIs. The kit is provided with our new bioinformatics tool especially developed in parallel to get the best of your data.

  • Read more
    • Truest representation - UMIs with ligation-free protocol ensures the most representative and unbiased data
    • Easy - One-tube, one-day protocol Lowest inputs - Down to 10 pg enriched small RNA or 100 pg total RNA starting amount
    • Clinical sample compatibility - Tested with RNA from biofluids
    • Highest sequencing quality - Minimized PhiX quantity
    • Enhanced data - Our new bioinformatics pipeline provided in the manual was optimized and developed specifically for getting the best out of your D-Plex data including UMI processing

    miRMaster : your bioinformatics tool for miRNA

    Diagenode has collaborated with Andreas Keller and Tobias Fehlmann to develop a new bioinformatics pipeline in order to allow novel miRNA prediction.
    miRMaster is a comprehensive analysis online free tool for NGS miRNA samples.

Focused on your target

small RNA sequencing kit

D-Plex: the highest yield technology to detect every kind of small RNA simultaneously (miRNA, snRNA, piRNA, …)

Get rich content

small RNA diversity

One day to enrich your sample with a large variety of small RNAs in one tube. Identify your reads accurately with UMIs.

Robust technology

small non coding RNA

Independent of your input amount -- go down to 10 pg small RNA as a starting amount.

Raise your quality output

small RNA library preparation for Illumina

Get the best of your data -- use our designed bioinformatics pipeline and guidelines. We provide everything you need to greatly simplify your analysis.

[ To check figures in details, click on "Figure" in the menu below ]

  • Indexes

    Specific D-Plex indexes were designed and validated to fit the D-Plex technology for Illumina sequencing and 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:

    For D-Plex UDI library construction:

    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.

    For D-Plex SI library construction:

  • Data analysis

    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.

    small RNA sequencing bioinformatics pipeline

  • Figures

    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.

    Example command:

    MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf --outdir outputs --bam_infiles input_bamfilenames.txt

    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.
    A full user guide for MGCount is available here: https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html.

    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).

    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.

    *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). https://doi.org/10.1186/s12859-021-04544-3

  •  文档
    D-Plex Small RNA-seq Kit for Illumina MANUAL
    Note: The D-Plex Small RNA-seq Kit manual describes the protocol for both UDI and SI li...
    True and accurate small RNA-seq: Use the power of UMIs without ligation FLYER
    D-Plex RNA-seq library preparation solutions FLYER
    D-Plex Small RNA-seq Library Prep Kit FLYER
    Go deeper with your RNA research & Biomarker discovery
  •  Safety sheets
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  •  出版物

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    DIS3 ribonuclease prevents the cytoplasmic accumulation of lncRNAs carrying non-canonical ORFs, which represent a source of cancer immunopeptides.
    Foretek D. et al.
    Around 12% of multiple myeloma (MM) cases harbour mutations in DIS3, which encodes an RNA decay enzyme that controls the turnover of some long noncoding RNAs (lncRNAs). Although lncRNAs, by definition, do not encode proteins, some can be a source of (poly)peptides with biological importance, such as antigens. T...

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    The piRNA-pathway factor FKBP6 is essential for spermatogenesis butdispensable for control of meiotic LINE-1 expression in humans.
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    Infertility affects around 7\% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with ...

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    Neutral sphingomyelinase 2 inhibition attenuates extracellular vesiclerelease and improves neurobehavioral deficits in murine HIV.
    Zhu X. et al.
    People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PL...

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    Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5' untranslated ...

    Interspecies effectors of a transgenerational memory of bacterial infection in Caenorhabditis elegans .
    Legüe Marcela et al.
    The inheritance of memory is an adaptive trait. Microbes challenge the immunity of organisms and trigger behavioral adaptations that can be inherited, but how bacteria produce inheritance of a trait is unknown. We use and its bacteria to study the transgenerational RNA dynamics of interspecies crosstalk leading to a...

    Diverse Monogenic Subforms of Human Spermatogenic Failure
    Nagirnaja L. et al.
    Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable with current medicine. Due to the biological complexity of sperm production, defining the genetic basis of NOA has proven challenging, and to date, the most advanced classification of NOA subforms is based on simple...

    Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development.
    Xiong Zhuqing et al.
    In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30-150 mouse oocy...

    Translation is a key determinant controlling the fate of cytoplasmic long non-coding RNAs
    Andjus Sara et al.
    Despite predicted to lack coding potential, cytoplasmic long non-coding (lnc)RNAs can associate with ribosomes, resulting in some cases into the production of functional peptides. However, the biological and mechanistic relevance of this pervasive lncRNAs translation remains poorly studied. In yeast, cytoplasmic Xrn...

    MGcount: a total RNA-seq quantification tool to address multi-mappingand multi-overlapping alignments ambiguity in non-coding transcripts
    Hita Andrea, Brocart Gilles, Fernandez Ana, Rehmsmeier Marc, Alemany Anna, Schvartzman Sol
    Background Total-RNA sequencing (total-RNA-seq) allows the simultaneous study of both the coding and the non-coding transcriptome. Yet, computational pipelines have traditionally focused on particular biotypes, making assumptions that are not fullfilled by total-RNA-seq datasets. Transcripts from distinct RNA biotyp...

    Single cell quantification of ribosome occupancy in early mouse development
    Tori Tonn et al.
    Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during ...

    Single cell quantification of ribosome occupancy in early mousedevelopment
    Tonn T. et al.
    Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during ...

    Functional microRNA targetome undergoes degeneration-induced shift in the retina
    Chu-Tan JA et al.
    Background: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex...

    Functional microRNA targetome undergoes degeneration-induced shift inthe retina.
    Chu-Tan Joshua A et al.
    BACKGROUND: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC)...

    Single-cell microRNA sequencing method comparison and application tocell lines and circulating lung tumor cells
    Hücker S. et al.
    Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1 pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best pro...

    Small RNAs in Seminal Plasma as Novel Biomarkers for Germ Cell Tumors
    Mørup N. et al.
    Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor le...

    Vesicle-bound regulatory RNAs are associated with tissue aging
    F. Kern, T. Kuhn, N. Ludwig, M. Simon, L. Gröger, N. Fabis, A. Salhab, T. Fehlmann, O. Hahn, A. Engel, M. Koch, J. Koehler, K. Winek, H. Soreq, G. Fuhrmann, T. Wyss-Coray, E. Meese, M. W. Laschke and A. Keller
    Previous work on murine models and human demonstrated global as well as tissue-specific molecular aging trajectories in solid tissues and body fluids1–8. Extracellular vesicles like exosomes play a crucial role in communication and information exchange in between such systemic factors and solid tissues9,10. We...

    Small RNAs in Seminal Plasma as Novel Biomarkers for GermCell Tumors.
    Mørup Nina et al.
    Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor le...

    miRMaster 2.0: multi-species non-coding RNA sequencing analyses at scale
    Tobias Fehlmann, Fabian Kern, Omar Laham, Christina Backes, Jeffrey Solomon, Pascal Hirsch, Carsten Volz, Rolf Müller, Andreas Keller
    Analyzing all features of small non-coding RNA sequencing data can be demanding and challenging. To facilitate this process, we developed miRMaster. After the analysis of over 125 000 human samples and 1.5 trillion human small RNA reads over 4 years, we present miRMaster 2 with a wide range of updates and new featur...

    Bacterial small RNAs and host epigenetic effectors of atransgenerational memory of pathogens in C. elegans
    Legüe M. et al.
    The inheritance of memories is adaptive for survival. Microbes interact with all organisms challenging their immunity and triggering behavioral adaptations. Some of these behaviors induced by bacteria can be inherited although the mechanisms of action are largely unexplored. In this work, we use C. elegans and its b...

    Interspecies RNA Interactome of Pathogen and Host in aHeritable Defensive Strategy.
    Legüe Marcela et al.
    Communication with bacteria deeply impacts the life history traits of their hosts. Through specific molecules and metabolites, bacteria can promote short- and long-term phenotypic and behavioral changes in the nematode . The chronic exposure of to pathogens promotes the adaptive behavior in the host's progeny called...

    Distinct Extracellular RNA Profiles in Different PlasmaComponents.
    Jia Jing et al.
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    Shilpa R. et al.
    Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, NSP1, for shutting down host translation. Despite this, it is currently unknown how viral pr...

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    Gylling HM, Gonzalez-Aguilera C, Smith MA, Kaczorowski DC, Groth A, Lund AH
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    The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expres...

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