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

Get the utmost quality out of our well-established QuantSeq 3’ mRNA-Seq gene expression kits, thanks to the improved V2 series! With the increasing reading capacity of next-generation sequencers, Lexogen’s patented 12 nt-long Unique Dual Indices will help you preserve most of your precious reads.

If you already have a library preparation kit without UDI (e.g., QuantSeq-Flex, Reverse, or Forward), our UDI Add-on V2 modules are available for purchase separately. They are delivered together with an improved PCR system, which can be seamlessly adapted to previous QuantSeq kits. For QuantSeq-Pool kits, you only need the UDI Sets (without purchasing additional PCR enzyme and buffer).

Our UDI Add-on V2 kits are also compatible with library preparation kits from other vendors.

For more information, please reach out to our specialists at support@lexogen.com

Performance

Within the QuantSeq family, QuantSeq V2 with UDI is ideally suited for high-throughput RNA sequencing experiments (high number of samples).

Whether your samples come from FFPE preparations, or fresh frozen (FF) material, you can be confident that data output will provide consistent gene counts and correlations.

Fig-1_QuantSeq-UDI V2-2

Figure 1 | Sequencing data obtained with 10 ng input from two different mouse spleen samples, 1M reads, data 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)

Overcycling may happen when working with low input samples. This results in shorter libraries and increasing amounts of amplification artefacts (such as linker-linker amplicons). With our new, improved V2 kits, even 6 additional cycles will not affect the quality of your library.

Fig-2_QuantSeq-UDI V2

Figure 2 | Agilent Bioanalyzer traces of four libraries amplified with QuantSeq V1 with UDI or V2 with UDI kits, 100 ng input. Red: V1blue: V2green: V1 + 6 cyclesviolet: V2 + 6 cycles. The V2 technology prevents library shortening, even with substantial overcycling.

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.

023_UDI-12nt_Workflow-Index-errors

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).

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 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.

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!

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Downloads

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.

Ordering Information

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

199.96Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A2, 96 preps
200.96Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A3, 96 preps
201.96Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set A4, 96 preps
202.96Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Set B1, 96 preps
203.384Lexogen UDI 12 nt Unique Dual Indexing V2 Add-on Kit Sets A1 to A4, 384 preps

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