QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A3, (UDI12A_0193-0288), 1 rxn/UDI

Roll over image to zoom in

QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set A3, (UDI12A_0193-0288), 1 rxn/UDI

LEXOGENSKU: LEX-195.96

antSeq with UDI V2 kits are provided with four essential components:

a library generation module (same as the original QuantSeq FWD 015)
a library amplification module
a library purification module (magnetic beads)
a plate containing 24 or 96 Unique Dual Indices (UDI), packaged in single reactions
for the 384-reaction kit: four plates containing 384 different UDI (sets A1 to A4)

UDI Set B1 is designed for Illumina chemistries of iSeq 100, MiniSeq, NextSeq 500 – 2000, HiSeq 3000 and 4000, as well as NovaSeq 6000 (v1.5 reagent kits).

Sets A1 to A4 can also be used for the above instruments, but sequences are reversed (this needs to be specified during data analysis).

Features and Application:

Gene expression studies
Degraded RNA (low RIN, high DV₂₀₀) such as FFPE
Low inputs (down to 1ng)

System Compatibility:

iSeq 100
MiniSeq
NextSeq 500 – 2000
HiSeq 2000, 2500, 3000, 4000
NovaSeq 6000 (v1.0 and 1.5 reagent kits)

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.

Performance

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

Consistent correlation between fresh frozen and FFPE 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.

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)

Excellent resistance against overcycling

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.

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

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.

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.

Reverse Transcription

Step 1:

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.

Removal of RNA

Step 2:

After first strand synthesis the RNA is removed.

Second-Strand Synthesis

Step 3:

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).

Library Amplification

Step 4:

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.

Sequencing

Step 5:

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:

Revvity: 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

QuantSeq automation on other platforms may also be possible.

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 orders@dmarkbio.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:

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 (comparison with V1) – update 16.05.2023
Product Flyer for QuantSeq FWD V2 with UDI (protocol and benefits) – release 28.03.2023

Lexogen UDI 12 nt Unique Dual Index Sequences – update 26.03.2021

Recover password

Create my account