QuantSeq 3’ mRNA-Seq Library Prep Kit FWD
The QuantSeq FWD Kit is a library preparation protocol designed to generate Illumina compatible libraries of sequences close to the 3’ end of polyadenylated RNA.
Dear Customer, QuantSeq V1 kits will be phased out soon. The last order date is October 31, 2023.
For more information, please get in touch with us at info@lexogen.com or sales@lexogen.com. Thank you!
QuantSeq FWD contains the Illumina Read 1 linker sequence in the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail, directly reflecting the mRNA sequence (see workflow). This version is the recommended standard for gene expression analysis.
Performance
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Figure 1 | Correlation of gene counts of FFPE and cryo samples.
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Figure 2 | Venn diagrams of genes detected by QuantSeq at a uniform read depth of 2.5 M reads in FFPE and cryo samples with 1, 5, and 10 reads/gene thresholds.
Figure 3 | QuantSeq read coverage versus normalized transcript length of NGS libraries derived from FFPE-RNA (blue) and cryo-preserved RNA (red).
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. No additional protocol steps are required. The RS-Globin Block solution from the module simply replaces the standard RNA Removal Solution from the QuantSeq Kit. Using Globin Block for QuantSeq, reads mapping to globin mRNAs can be reduced from 50 – 80 %, to as low as 5% (Fig. 4) and gene detection is dramatically increased (Fig.5).
* For Figures: Input RNA (50 ng, or 250 ng) 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.
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Figure 4 | Percentage of sequencing reads mapping to globin mRNAs from human (Hs) and pig (Ss) blood QuantSeq libraries.*
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Figure 5 | Increased gene detection in human blood QuantSeq libraries using Globin Block. CPM = Counts Per Million.*
The BC1 Block Module for QuantSeq prevents the generation of library fragments from the abundant BC1 transcripts that are present in mouse brain samples (Mus musculus, Mm), by blocking their extension during second strand synthesis. The provided RS-BC1 Block solution from the module simply replaces the standard RNA Removal Solution from the QuantSeq Kits (FWD and REV).
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 studies.
QuantSeq libraries are intended for a high degree of multiplexing. Up to 96 i7 indices are included in the single indexing kits (Cat. No. 015). Additional i5 indices can also be introduced using the Lexogen i5 6 nt Dual Indexing Add-on Kits (Cat. No. 047 or 015.384). Used together, Lexogen’s 96 i7 and 96 i5 6 nt indices enable up to 9,216 different index combinations for sequencing.
In order to maximise the potential (demultiplexing, read rescuing) of dual indexing, we recommend our QuantSeq 3’ mRNA-Seq V2 Library Prep Kit with UDI. If you already own a QuantSeq-FWD kit 015, you can also upgrade it by purchasing only the amplification module with Lexogen’s 12 nt UDI. For further questions, please contact support@lexogen.com.
This high level of multiplexing allows saving costs as the length restriction in QuantSeq saves sequencing space. QuantSeq is also designed to yield insert sizes for short sequencing reads (SR50, SR100).
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.
The UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Cat. No. 081.96) contains the UMI Second Strand Synthesis Mix (USS). This mix simply replaces the Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD Kit. The module allows unique tagging of individual transcripts with 6 nt long Unique Molecular Identifiers (UMI) located between the partial P5 adapter and the random priming sequence. Use this module to identify PCR duplicates and eliminate amplification bias.
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.
enrichment or rRNA depletion is needed.
the Illumina-specific Read 2 linker sequence.
and a DNA polymerase. The random primer contains
the Illumina-specific Read 1 linker sequence. At this step
Unique Molecular Identifiers (UMIs) can be introduced
by exchanging the Second Strand Synthesis Mix 1 (SS1)
from the standard QuantSeq FWD Kit with UMI
Second Strand Synthesis Mix (USS).
strand synthesis. Second strand synthesis is followed
by a magnetic bead-based purification step rendering
the protocol compatible with automation.
required for cluster generation are introduced.
barcode combinations using the 96 available
i7 indices and 96 i5 indices.
and directly correspond to the mRNA sequence.
To pinpoint the exact 3’ end, longer reads may be
required (SR50, SR100, SR150). Although paired-end
sequencing is possible, we do not recommend it
for QuantSeq FWD. Read 2 would start with
the poly(T) stretch, and as a result of sequencing
through the homopolymer stretch, the quality
of Read 2 would be very low.
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:
Application Note for Automation of QuantSeq 3’ mRNA Kit on the Agilent NGS Workstation
Application Flyer for Automation of QuantSeq 3’ mRNA Kit on Beckman Biomek NGS Workstation
Webinar: Automation of RNA-seq: from model organisms to the clinic
FAQ
Frequently Asked Questions
Access our frequently asked question (FAQ) resources via the buttons below.
Please also check our General Guidelines and FAQ resources!
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Downloads
QuantSeq Product Flyer – update 04.01.2023
QuantSeq Application Note (Nature Methods, December 2014) – external link
Application Note for QuantSeq – update 04.01.2023
Application Note for QuantSeq Data Analysis with PARTEK® FLOW®
Application Note for Globin Block Modules
QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina
User Guide – update 24.01.2023
User Guide HT – update 25.01.2023
User Guide for BC1 Block Module for QuantSeq – update 25.01.2023
For QuantSeq Add-on Module User Guides click here
User Guide for QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set (version 2) – update 12.07.2023
User Guide for QuantSeq 3’ mRNA-Seq Library Prep Kit FWD with UDI 12 nt Set (version 1) – update 25.01.2023
User Guide for Lexogen 12 nt Unique Dual Indexing Add-on Kit V2 (version 2) – release 12.10.2022
User Guide for Lexogen 12 nt Unique Dual Indexing Add-on Kit (version 1) – update 25.01.2023
PCR Add-on Kit for Illumina User Guide – update 03.01.2023
Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) User Guide – update 03.01.2023
Lexogen UDI 12 nt Unique Dual Index Sequences – update 26.03.2021
Lexogen i7 and i5 Index Sequences – update 05.05.2020
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.
Use this command line tool for demultiplexing QuantSeq-Pool data – Demultiplexing and Error Correction Tool – iDemux
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