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Polyadenylation with ease

– QuantSeq REV V2!

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QuantSeq 3’ mRNA-Seq V2 Library Prep Kit REV

The QuantSeq REV Kit generates Illumina-compatible 3’ mRNA-Seq libraries directly targeting the 3’ end of polyadenylated transcripts. The first nucleotide of the read- out corresponds to the genuine transcription end site. The combination of cost-efficient 3’ mRNA sequencing and authentic end site representation makes QuantSeq REV the perfect tool to investigate alternative polyadenylation sites for projects at any scale.

Alternative Polyadenylation (APA)

(noun) / ȯl-ˈtər-nə-tiv päl-ē- -ə-ˈden-ə- lā shən

Alternative polyadenylation (APA) is a fundamental mechanism in the regulation of eukaryotic gene expression. Unlike traditional polyadenylation at the canonical 3’ end of a transcript, APA generates distinct mRNA isoforms through selection of alternate polyadenylation sites located at a different position within the 3’ end of the transcript or even in another exon. Alternative 3’ ends impact transcript properties and expression levels and are of utmost importance for diverse biological processes, e.g., development and disease. By investigating the dynamics of APA, we deepen our comprehension of cellular regulation, which opens new avenues for exploring potential therapeutic interventions.

Applications

Performance

QuantSeq REV has a broad input range of 1 ng – 500 ng total RNA without the need for any prior poly(A) selection or ribodepletion and reliably delivers high-quality data and excellent gene detection rates (Tab. 1).

Table 1 | Mapping stats for QuantSeq REV libraries across different input ranges of Universal Human Reference RNA (UHRR). Libraries were sequenced in paired end mode at 500 K reads and Read 2 was analyzed, averages over at least two replicates per experiment are presented.

Input RNA amount
500 ng10 ng1 ng
% Reads Mapped98.9 %98.6 %98.0 %
% Uniquely Mapping Reads91.9 %90.0 %88.8 %
% Protein Coding95.5 %94.6 %93.4 %
No. of Genes Detected14,21514,2749,922

The required input amount of total RNA is as low as 1 ng. QuantSeq REV is suitable to reproducibly generate libraries from low quality RNA, including FFPE samples.

QuantSeq REV allows to exactly pinpoint the 3’ end of poly(A) RNA and to obtain accurate information about a transcripts’ 3’ UTR, including alternative polyadenylation events (Fig. 1. Tab. 2).

Alternative polyadenylation in human blood
Figure 1 | Alternative polyadenylation of OSBP2 in human blood detected with QuantSeq REV V2. 10 ng RNA derived from human cell lines and 50 ng human blood was used to prepare QuantSeq REV libraries. The Globin Block Module (RS-GBHs) was used in combination with QuantSeq REV to effectively block library generation from globin transcripts in human whole blood RNA, focusing reads on RNAs of interest. Alternative polyadenylation site detection revealed two distinct transcript end sites for OSBP2 (human Oxysterol Binding Protein 2).

Table 2 | Alternative poly(A) site detection in samples derived from human cell lines vs. whole blood.
AllSignificant*
No. of Genes with alternative poly(A) sites1896118
Known alternative poly(A) sites detected1894118
Novel alternative poly(A) sites detected941

*Significance filtering using DEXSeq v1.44.0 at padj <0.1

Libraries were generated with QuantSeq REV V2 and sequenced with a read depth of 2 – 2.5 M reads / sample. Alternative poly(A) site analysis was conducted by peak calling after filtering internally poly(A)-priming reads, removal of low abundance peaks (<5 reads) and removal of intergenic, antisense peaks, and peaks associated with overlapping genes.

Need help with your data analysis? Lexogen offers alternative poly(A) site analysis as part of our Lexogen Bioinformatics Services portfolio. Find out more!

Globin Block Modules for QuantSeq enable the generation of globin-depleted, ready-to-sequence 3’ mRNA-Seq libraries from as little as 50 ng of total RNA from whole blood without additional processing steps. Globin Block reduces QuantSeq reads mapping to globin mRNAs from 80 % down to 5 % (Fig. 2) thereby dramatically increasing gene detection (Fig. 3).

Sequencing reads mapping to human globin mRNAs, library preparation with QuantSeq.
Figure 2 | Percentage of sequencing reads mapping to globin mRNAs from human (Hs) and pig (Ss) blood QuantSeq libraries. Input RNA was extracted with 1 SPLIT RNA Extraction Kit, 2 other kit including red blood cell lysis, or 3 other kit without red blood cell lysis.
Gene detection in human blood. Globin block. QuantSeq.
Figure 3 | Increased gene detection in human blood QuantSeq libraries using Globin Block. CPM = Counts Per Million. Input RNA was extracted with 1 SPLIT RNA Extraction Kit, or 2 other kit including red blood cell lysis.

The BC1 Block Module (RS-BC1 Block) for QuantSeq prevents the generation of library fragments from the abundant BC1 transcripts that are present in rodent brain samples by blocking their extension during second strand synthesis.

QuantSeq maintains exceptional strand-specificity of >99.9 % and allows to map reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.

QuantSeq’s simple workflow allows generating ready-to sequence NGS libraries within only 4.5 hours, including less than 2 hours hands-on time.

Just one fragment per transcript is produced; therefore no length normalization is required. This allows more accurate determination of gene expression values and renders QuantSeq the best alternative to microarrays and conventional RNA-Seq in gene expression and eQTL (expression quantitative trait loci) studies.

QuantSeq REV libraries are intended for a high degree of multiplexing. The kit includes 24 or 96 Unique Dual Indices (UDIs) with an unprecedented error correction accuracy. Up to 384 UDIs are compatible with QuantSeq REV. For multiplexing needs beyond 384 samples, please consult with us at support@lexogen.com.

Lexogen offers an automated solution for QuantSeq data analysis based on state-of-the-art Lexogen’s proprietary pipeline. Thus, every user, even without bioinformatics experience, can analyze QuantSeq samples in a convenient and fast way. More information and details about Lexogen’s data analysis pipeline can be found here.

Workflow

QuantSeq has a short and simple workflow and can be completed within 4.5 hours. The required hands-on time is less than 2 hours. The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.

Step1:

The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.

Library generation starts with oligodT priming containing the Illumina-specific Read 1 linker sequence.

After first strand synthesis the RNA is removed.

Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer contains the Illumina specific Read 2 linker sequence.

No purification is required between first and second strand synthesis. Second strand synthesis is followed by a magnetic bead-based purification step rendering the protocol compatible with automation.

During the library amplification step sequences required for cluster generation are introduced.

Multiplexing can be performed with up to 9,216 barcode combinations using the 96 available i7 indices and 96 i5 indices.

NGS reads are started at the very last nucleotide of the mRNA using the Custom Sequencing Primer (CSP Version 2, included in the kit) for sequencing. The reads generated during Read 1 reflect the cDNA sequence. With QuantSeq REV also paired-end sequencing is possible.

Featured Publications

Automation

Automated QuantSeq protocol for 3’ mRNA-Seq Library Preparation

Automating the process of library preparation has the advantage of avoiding sample tracking errors, dramatically increasing throughput, and saving hands-on time.

QuantSeq library preparation and has been successfully implemented on several liquid handlers:

  • Perkin Elmer: Sciclone® / Zephyr®
  • Hamilton: Microlab STAR / STARlet
  • Agilent: NGS Workstation (NGS Bravo Option B)
  • Beckman Coulter: Biomek FXP, Biomek i5, Biomek i7
  • Eppendorf: EpMotion® 5075
  • Opentrons® OT-2


QuantSeq automation on other platforms may also be possible. Please contact support team for more information.

Two key parameters must always be considered when automating a protocol:

  • volume optimization
  • script compatibility


In some instances, our kits will provide enough reagents while, in other instances, you will need a higher reagent volume or script adjustments. At Lexogen, we will help you find the best solution, tailored to your needs.

Before starting any new project involving automation, please reach out to our experts at support@lexogen.com

Please, also consider liaising with the robotic platform support team – they will be able to share the most recent version of the script.

Lexogen gladly supports the implementation of Lexogen-manufactured kits on liquid handlers, but not hardware or software issues linked to the original liquid handling instrument supplier.

Related resources:

FAQ

Frequently Asked Questions

Access our frequently asked question (FAQ) resources via the buttons below.

Please also check our General Guidelines and FAQ resources!

How do you like the new online FAQ resource? Please share your feedback with us!

Downloads

QuantSeq 3′ mRNA-Seq Library Prep Kit REV for Illumina

autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

  • Perkin Elmer: Sciclone® / Zephyr®
  • Hamilton: Microlab STAR / STARlet
  • Agilent: NGS Workstation (NGS Bravo Option B)
  • Beckman Coulter: Biomek FXP, and Biomek i7
  • Eppendorf: EpMotion® 5075


Please inquire at info@lexogen.com for the automation scripts.

Safety Data Sheet

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

QuantSeq Bioinformatics Data Analysis

Find more about the QuantSeq Data Analysis here.

Ordering information

Cat. №Product Name
Full library preparation kits
225.24QuantSeq 3’ mRNA-Seq V2 Library Prep Kit (REV) with Custom Sequencing Primer and UDI 12 nt Set B1, 24 preps
225.96QuantSeq 3’ mRNA-Seq V2 Library Prep Kit (REV) with Custom Sequencing Primer and UDI 12 nt Set B1, 96 preps

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