QuantSeq-Flex Targeted RNA-Seq Library Prep Kit

Description
Performance
Workflow
FAQs
Ordering Information
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Product Description

The QuantSeq-Flex Kit is a library preparation protocol designed to make Illumina compatible libraries from any RNA sample using custom primers. It is open for development by advanced RNA-Seq users based on their custom needs.
The kit is based on the QuantSeq FWD (Cat. No. 015) strategy, with compatible reagents and Read 1 linker sequence introduced by the Second Strand Synthesis (SSS) primer. Hence the reads generated during the NGS run directly correspond to the RNA sequence.
QuantSeq-Flex Kit provides modules for Reverse Transcription (RT) and SSS, allowing users to choose their own primers for either one of these steps or both of them. This offers maximum flexibility in the choice of targets and input material.
With this highly flexible QuantSeq kit the following types of libraries can be generated:

1. OligodT primed during RT, random primed during SSS (QuantSeq 3’ mRNA-Seq)
2. OligodT primed during RT, target-specifically primed during SSS (targeted 3’ mRNA-Seq)
3. Target-specifically primed during RT, random primed during SSS (targeted RNA-Seq, allows for identification of novel fusions)
4. Target-specifically primed during RT, target-specifically primed during SSS (targeted RNA-Seq, known targets detectable only)

Whether it is gene expression analysis, targeted sequencing, adapter ligation-based RNA-Seq or any other experiment where a certain RNA region needs to be sequenced, QuantSeq-Flex can be used together with user-supplied primers.

Multiplexing

QuantSeq libraries are intended for a high degree of multiplexing. External barcodes allowing up to 96 samples to be sequenced per lane on an Illumina flow cell are included in the kit. This high level of multiplexing allows saving costs as the length restriction in QuantSeq saves sequencing space.
In order to be compatible with the supplied barcode primers please note our recommendations for the custom primer design (Refer User Guide: Appendix B- Primer design)
For the detailed information about barcodes and instructions how to use them please refer User Guide: Appendix F- Multiplexing, QuantSeq-Flex User Guide).

An RNA-Seq experiment tailored to your needs

QuantSeq-Flex modular kit gives maximum flexibility to the user encouraging implementation of RNA-Seq as a part of the experimental pipeline. One fragment per transcript produced and usage of custom primers makes this kit a perfect solution for cost-saving high-throughput gene expression analysis. QuantSeq-Flex is suitable for targeted sequencing of gene panels, enrichment, detection of fusion genes and various other types of transcripts.

Performance

Rapid Turnaround

Simple workflow allows generating libraries ready for sequencing within 4.5 hours. This already includes hands-on time, which is less than 2 hours.

Perfect For Gene Counting

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

Workflow

Step 1 : Reverse Transcription

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

Library generation starts either with oligodT (included in the kit) or a target-specific primer containing sequencing platform – compatible linker sequences.

Step 2 : Removal of RNA

After first strand synthesis the RNA is removed.

Step 3 : Second-Strand Synthesis

Second strand synthesis is initiated by random (included in the kit) or target-specific priming and a DNA polymerase. The primers also contain sequencing platform – compatible linker sequences.

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.

Step 4 : Library Amplification

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

Multiplexing can be performed with up to 96 external barcodes for Illumina platforms.

FAQs

1. What is different between the First and Second Strand Synthesis components of QuantSeq 015 and the QuantSeq-Flex Modules?

2. Can I use QuantSeq-Flex with the QuantSeq REV version?

3. What should I take into consideration when designing my custom primers for reverse transcription or 2nd strand synthesis?

4. Can I use molecular barcodes for targeted sequencing?

5. Which primers are included in the kit?

6. Can I purchase one of the QuantSeq-Flex modules separately?

7. Do I need to get both the First Strand Synthesis Flex Module and the Second Strand Synthesis Module?

8. Can I add blocking oligos to prevent the second strand of high abundant RNAs?

9. What concentration of targeted primers should I use?

10. What cycle number do I need to use to amplify my targeted sequencing library?

Ordering Information

Kit	Platform	Reactions	Cat.No.
with First Strand Synthesis Module	Illumina	24 preps	033.24
with First Strand Synthesis Module		96 preps	033.96
with Second Strand Synthesis Module		24 preps	034.24
with Second Strand Synthesis Module		96 preps	034.96
with First and Second Strand Synthesis Modules		24 preps	035.24
with First and Second Strand Synthesis Modules		96 preps	035.96

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