Description
QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 for Illumina
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 Kit for Illumina (Cat. No. 015) strategy, with compatible reagents and Read 1 linker sequence introduced by the second strand synthesis 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 in reverse transcription, random primed in second strand synthesis (QuantSeq 3’ mRNA-Seq)
2.) OligodT primed in reverse transcription, target-specifically primed in second strand synthesis (targeted 3’ mRNA-Seq)
3.) Target-specifically primed in reverse transcription, random primed in second strand synthesis (targeted RNA-Seq, allows for identification of novel fusions)
4.) Target-specifically primed in reverse transcription, target-specifically primed in second strand synthesis (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.
Direct Counting for Gene Expression Quantification
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.
Cost Saving Multiplexing
QuantSeq libraries are intended for a high degree of multiplexing. Up to 9,216 samples can be multiplexed/lane on an Illumina flow cell using the up to 96 i7 indices included in the kit together with the 96 i5 indices as part of the Lexogen i5 6 nt Dual Indexing Add-on Kit (Cat. No. 047).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 indices (barcode primers) please note our recommendations for the custom primer design (see Appendix D: Primer Design, QuantSeq-Flex User Guide, p.28)
Rapid Turnaround
An RNA-Seq Experiment Tailored to Your Needs
Workflow
QuantSeq-Flex 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. Although if using random priming for RT a depletion may be advisable.
For viewing the whole workflow on page please click here
Multiplexing
QuantSeq libraries are intended for a high degree of multiplexing. The kit already includes up to 96 i7 primer (7001-7096) which are introduced as standard external barcodes during the PCR amplification. For dual indexing allowing multiplexing of up to 9,216 libraries, an Lexogen i5 6 nt Dual Indexing Add-on Kit (Cat. No. 047) with 96 additional i5 indices (5001-5096) is available.
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FAQ
Frequently Asked Questions
Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.
SS1 of the basic kit contains a random primer and 10 µl of SS1 are added per reaction, TS from the module does not contain any primers and only 7 µl are added per reaction leaving 2 µl available for the addition of custom primers.
First Strand Synthesis
Any primer used for First Strand cDNA Synthesis has to be designed with a partial Illumina P7 adapter extension. Adapter sequences are kept short pre-PCR in order to allow for efficient removal of short fragments during the purification step (steps 12 and 16). The full Illumina P7 (Read 2) adapter sequence will only be introduced during PCR (step 28).
Partial Illumina P7 Adapter Sequence (Read 2) for First Strand Synthesis Primer:
5’ GTTCAGACGTGTGCTCTTCCGATCT- Target sequence 3’
Here the target sequence has to be the reverse complement to the mRNA-sequence in question (=cDNA). The chosen target sequence should be as specific as possible with a Tm that is as close as possible to the intended reaction temperature (50 °C). In most cases 20 nts are enough. Target-specific primer sequences should not exceed a length of 50 nts. The entire primer including the Illumina Adapter sequence should not exceed 75 nts. The optimal primer length is 45 – 50 nts (25 nt Illumina Sequence + 20 – 25 nt targeted sequence).
We highly recommend checking your targeted primers using the NCBI Primer Blast tool available at https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome. Use the Primer Pair Specificity Check and run your primers against the RefSeq database (not just RefSeq mRNA). Primer specificity stringency settings can be adjusted regarding the allowed mis¬matches and positions of the mismatch within the primer.
Second Strand Synthesis
Any primer used for Second Strand cDNA Synthesis has to be designed with a partial Illumina P5 adapter extension. Adapter sequences are kept short pre-PCR in order to allow for efficient removal of short fragments during the purification step (steps 12 and 16).The full Illumina P5 (Read 1) adapter sequence will only be introduced during PCR (step 28).
Partial Illumina P5 Adapter Sequence (Read 1) for Second Strand Synthesis Primer:
5’ CACGACGCTCTTCCGATCT – Target sequence (mRNA-sequence) 3’
Here the target sequence has to be the mRNA-sequence in question. The chosen target sequence should be as specific as possible with a Tm that is as close as possible to the intended reaction temperature. In most cases 20 nts are enough. Target-specific primer sequences should not exceed a length of 50 nts. The entire primer including the Illumina Adapter sequence should not exceed 75 nt. The optimal primer length is 39 – 50 nt (19 nt Illumina Sequence + 20 – 31 nt targeted sequence). The Tm of the targeted primers should be within the range of the potential annealing temperature i.e., between 45 °C and 72 °C. Please note that temperatures below 45°C should not be used. Introduction of 1 – 3 phosphorothioate linkages (PTOs) at the 3’ end of the target-specific second strand synthesis primer may increase specificity.
Partial Illumina P7 Adapter Sequence (Read 2) for First Strand Synthesis Primer:
5’ GTTCAGACGTGTGCTCTTCCGATCT – NNNNNN(NN) – Target sequence (= cDNA sequence) 3’
If the molecular index is introduced in the First Strand Synthesis Primer a paired-end sequencing run is required for read-out.
Partial Illumina P5 Adapter Sequence (Read 1) for Second Strand Synthesis Primer:
5’ CACGACGCTCTTCCGATCT – NNNNNN(NN) – Target sequence (mRNA-sequence) 3’
The QuantSeq-Flex First Strand Synthesis Module can be used with the second strand synthesis components included in the QuantSeq 015 kit. Also, the QuantSeq-Flex Second Strand Synthesis Module V2 can be used with the first strand synthesis components included in the QuantSeq 015 kit, depending what kind of libraries are to be generated.
By getting only the QuantSeq-Flex Second Strand Synthesis Module V2 (Cat. No. 028) you can generate oligodT primed libraries during reverse transcription (using the First Strand Synthesis of QuantSeq 015) and target-specifically primed libraries during second strand synthesis, which means targeted 3’ mRNA-Seq libraries. Please note that the second strand synthesis reaction requires annealing temperatures of >45 °C. In case you need lower temperatures contact info@lexogen.com.
Getting both the QuantSeq-Flex First and Second Strand Synthesis Module V2 allows you to generate targeted libraries in both reverse transcription and second strand synthesis (targeted RNA-Seq) to identify known targets.
The concentration of a target-specific reverse transcription primer should be around 12.5 nM – 1.25 µM final concentration (in this case it would mean you would need 5 µl of a 50 nM – 5 µM target-specific primer). The total concentration of oligos in the first strand synthesis reaction should not exceed 2 µM. The higher the primer concentration the higher the likelihood of unspecific binding. However, the exact primer concentration and reaction temperature (up to 50 °C for first strand synthesis and second strand synthesis) strongly depends on the custom primer(s) used and has to be optimized accordingly.
Second Strand Synthesis
The concentration of a target-specific second strand synthesis primer should be 0.5 µM final concentration of the target primer (i.e., 2 µl of 7.5 pM Custom Target Primer). The total concentration of all oligos in the second strand synthesis reaction should not exceed 2 µM. The higher the primer concentration the higher the likelihood of unspecific binding. The annealing temperature should be chose according to the Tm of the targeted primers and can range from 45 °C to 72 °C. The extension temperature should be 72 °C.
- First Strand cDNA Synthesis (p.13-14): Do not cool the mastermix containing E1 (step 3) and have the RNA/Custom Targeted Primer or RNA/FS1/dT samples at 42-50 °C when adding the E1-containing mastermix (step 4) to avoid mishybridization. Mix properly by pipetting. Do not forget to shortly spin down the samples at room temperature before and after adding the E1-containing mastermix.
- Use higher input amounts of RNA where possible.
- Use 1 hour incubation time for reverse transcription (step 4).
- Use 48ul of PS at step 16.
- We recommend performing a qPCR assay to determine the optimal cycle number for library amplification. The PCR add-on kit for Illumina (Cat. No. 020.96) provides reagents for performing these assays. In addition SYBR Green I (Sigma-Aldrich, Cat. No. S9430; 10,000x in DMSO) is needed for the qPCR assay.
For further information about the i5 Dual Indexing Add-on Kit for QuantSeq/SENSE please see the online frequently asked questions on the QuantSeq FWD product page.
Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq or SENSE libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).
Downloads
QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2 for Illumina platforms
User Guide – update 08.08.2018
Send us your publication & get the RNA T-shirt!
Application Note for Automation of QuantSeq 3’ mRNA Kit on the Agilent NGS Workstation
PCR Add-on Kit for Illumina Instruction Manual – update 04.11.2020
Lexogen i5 6 nt Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019
Lexogen i7 and i5 Index Sequences – update 05.05.2020
Library Quantification Calculation File
autoQuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina
Agilent Bravo – Please inquire at info@lexogen.com for the automation scripts
Material Safety Datasheets
MSDS Information can be found in the Documents page.
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.
Want to try QuantSeq-Flex Targeted RNA-Seq V2 on your samples?
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If you are interested in discussing your QuantSeq Flex project with Lexogen scientist, please contact us at info@lexogen.com.
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