Reliable and most cost-efficient solution for gene expression analysis
– QuantSeq FWD

Straightforward expression profiling with QuantSeq 3’ mRNA-Seq gene expression kits: the cost-saving 3’ sequencing approach enables mRNA-focused readout and exceptional scalability.

QuantSeq FWD V2 with UDI generates sequencing-ready libraries from total RNA in <4.5 hours without the need for any pre-processing steps and presents the ideal choice for cost-efficient gene expression profiling studies of eukaryotic samples.

QuantSeq kits contain all reagents to complete RNA-Seq library preparation, including library generation reagents, library amplification reagents, Unique Dual Indices (UDI), and purification beads and solutions.

Add-on kits:

PCR Add-on Kit for Illumina (Cat. No. 208)
UMI Second Strand Synthesis Module for QuantSeq FWD (Cat. No. 081)
Globin Block Modules for QuantSeq (homo sapiens Cat. No. 070, sus scrofa Cat. No. 071) for blocking of globin mRNA
BC1 Block Module for QuantSeq (Cat. No. 167) for blocking of abundant BC1 transcripts in rodent brains

System compatibility:

Illumina (directly compatible)
Element AVITI (directly compatable with Cloudbreak Freestyle, or ADEPT)
MGI (compatible after conversion)

Within the QuantSeq family, QuantSeq V2 with UDI is ideally suited for cost-efficient high-throughput RNA sequencing experiments for expression profiling.

Excellent Gene Detection Across a Wide Range of Input Amounts

The majority of reads generated by QuantSeq library preparation from 500 ng or 1 ng RNA present uniquely mapping reads (Table 1). QuantSeq 3′ mRNA-Seq V2 also provides excellent gene detection across a wide range of input RNA amounts and is thus ideal for gene expression studies (Fig. 1).

Table 1 | Basic Mapping Stats for QuantSeq 3′ mRNA-Seq FWD V2 from 500 ng and 1 ng Input RNA.


QuantSeq 3' mRNA-Seq FWD V2
Input RNA	500 ng	1 ng
Mapped Reads	99.45%	99.12%
Uniquely Mapped Reads	89.77%	88.70%
Multimapping Reads	9.67%	10.42%
Reads Mapped to rRNA*	0.29%	1.45%
Reads Mapped to Protein-coding Genes	93.71%	88.72%
*Reads mapping to cytoplasmic rRNA excluding polyadenylated mitochondrial rRNA transcripts.

Figure 1 | Number of detected genes for different RNA input amounts. QuantSeq libraries were prepared from 500 ng and 1 ng Universal Human Reference RNA (UHRR). Gene detection (CPM > 1) is shown for 1 M reads / sample (1×90 bp, NextSeq2000).

High Reproducibility Across Various Inputs

QuantSeq 3′ mRNA-Seq generates excellent gene count correlation between replicates (Fig. 2A) and across input amounts, i.e., between 1 ng and 500 ng input RNA (Fig. 2B).

Figure 2 | Correlation analyses of gene counts obtained from QuantSeq 3′ mRNA-Seq V2 library preps generated from 500 ng and 1 ng Universal Human Reference RNA (UHRR). A) Correlation between replicates. B) Correlation between 1 ng and 500 ng input RNA amount. Libraries were sequenced on NextSeq2000 (1×90 bp) and 1 M reads / sample were used for correlation analyses.

Transcript Coverage of 3' Ends for Cost-efficient Sequencing

QuantSeq focuses sequencing reads to the 3′ end of mRNAs covering the 3′ UTR and the ultimate exon (Fig. 3). Therefore, QuantSeq requires lower sequencing depth than whole transcriptome library preps: 3 – 5 M / reads per sample are sufficient for accurate gene expression quantification.

Figure 3 | Accumulated transcript body coverage for whole transcriptome libraries vs. QuantSeq. Coverage across transcripts was generated using the geneBody_coverage.py tool provided by RSeQC (transcript length was normalized to 100 %).

Performance on FFPE Samples

QuantSeq FWD V2 with UDI shows consistent correlation of gene detection between mouse spleen samples derived from fresh frozen (FF) and FFPE tissue at various gene detection thresholds (Fig. 4)

For optimized performance on FFPE samples, QuantSeq FFPE kits are recommended. QuantSeq FFPE is validated and QC’ed on FFPE material and seamlessly integrates with SPLIT One-step FFPE RNA Extraction kits for a fully validated workflow.

Figure 4 | Overlap of detected genes between fresh frozen (FF) and FFPE samples from 10 ng input (different mouse spleen samples) at 1M reads / sample. Data is shown for 1 CPM, 5 CPM, and 10 CPM (from left to right). RIN values: 3.7 (FF) and 2.9 (FFPE), DV₂₀₀ values: 90% (FF) and 9% (FFPE).

Performance on Blood Samples

QuantSeq is ideal for gene expression studies from whole blood. Libraries prepared with QuantSeq and Globin Block Modules show significant reduction of reads mapped to globin with depletion rates >80 % (Fig. 5). Lexogen RNA-Seq workflows for blood samples are compatible with established blood collection systems, including PAXgene™ Blood RNA Tubes.

Figure 5 | Reads mapping to human globin mRNAs for QuantSeq libraries generated from 250 ng and 50 ng RNA input with and without Globin Block.

Find out more in Publications using QuantSeq with Globin Block.

Deplete Abundant Transcripts in Rodent Brain Samples with BC1 Block and QuantSeq

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 (Fig. 6). As a result, Reads mapping to BC1 are reduced and the number of uniquely mapping reads is increased (Table 2).

Figure 6 | Bioanalyzer traces for QuantSeq libraries from 10 ng mouse brain RNA prepared with (blue trace) and without (red trace) BC1 Block solution. RNA was isolated using the SPLIT RNA Extraction Kit (Lexogen). Dominant BC1 peaks that are visible in the libraries prepared without blockers are no longer visible after BC1 blocking.

Table 2 | Mapping stats for QuantSeq libraries generated from 10 ng Mus musculus brain RNA with and without BC1 Block.


	without BC1 Block	with BC1 Block
Mapped Reads	97.60%	98.00%
Uniquely Mapped Reads	62.00%	73.30%
Reads Mapped to BC1	12.70%	0.01%
Reads Mapped to BC1 Homologs	6.50%	0.01%

Performance on Plants

QuantSeq is compatible with plant samples. Lexogen offers end-to-end workflows for plant expression profiling (Fig. 7). No rRNA-depletion or poly(A) enrichment is needed prior to library generation.

Figure 7 | QuantSeq is ideal for expression profiling studies in plant. Lexogen offers end-to-end workflows including SPLIT RNA Extraction which efficiently removes secondary metabolites and plant-derived inhibitors. Data analysis is supported on the cloud-based Kangooroo Data Analysis platform.

Publications using QuantSeq for plant transcriptomics and crop science.

Optimal UDI deduplication capabilities

Lexogen’s patented 12 nt UDI allow for particularly high error correction rates, with optimally designed sequence-Levenshtein values. With Lexogen, you do not have to worry about lost reads or even misassigned reads! And you can benefit from our design even on libraries generated with other vendors’ kits since our UDI kits can adapt to most of the commercially available library generation solutions. For optimal demultiplexing and read rescue, we recommend Lexogen’s iDemux command line.

Please consult with us for more information: support@lexogen.com.

Figure 3 | Lexogen’s 12 nt design allows rescuing the majority of undetermined reads thanks to superior error correction features, thereby saving precious data (based on 96 pooled libraries; Illumina NextSeq500 run, demultiplexed with iDemux).

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 2 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 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).

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.

Lexogen’s 12nt UDI (i5 index and i7 index) are added during the library amplification step.

Multiplexing can be performed with up to 384 UDIs.

NGS reads are generated towards the poly(A) tail 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.

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 FX^P, Biomek i5, Biomek i7
Eppendorf: EpMotion^® 5075
Opentrons^® OT-2
Tecan: DreamPrep^® NGS

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

Considerations for Automation

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

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!

User Guide for QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set – update 12.07.2023

User Guide for Lexogen 12 nt Unique Dual Indexing Add-on Kit V2 – release 12.10.2022

Product Flyer for QuantSeq FWD V2 with UDI (protocol and benefits) – release 28.03.2023

Product Flyer for QuantSeq and CORALL – release 02.05.2023

Product Flyer for Blood Samples – release 13.12.2023

Lexogen UDI 12 nt Unique Dual Index Sequences – update 26.03.2021

Safety Data Sheet

Request 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.


Cat. №	Product Name
Full library preparation kits		Full library preparation kits	Full library preparation kits
191.24	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A1, 24 preps
192.24	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set B1, 24 preps
191.96	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A1, 96 preps
192.96	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set B1, 96 preps
193.384	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Sets A1 to A4, 384 preps
194.96	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A2, 96 preps
195.96	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A3, 96 preps
196.96	QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A4, 96 preps
UDI Add-on Kits (UDI plate and library amplification module)
198.96	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A1, 96 preps
199.96	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A2, 96 preps
200.96	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A3, 96 preps
201.96	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A4, 96 preps
202.96	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set B1, 96 preps
203.384	Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Sets A1 to A4, 384 preps

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QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI
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Reliable and most cost-efficient solution for gene expression analysis
– QuantSeq FWD

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QuantSeq 3’ mRNA-Seq V2 Library Prep Kit with UDI

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