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Quant-MRNA-SeqKit

QuantSeq 3’mRNA-Seq Library Prep Kit

Company:
  • Enabling development of your own gene panels
  • for custom or targeted RNA-Seq
  • Cost-effective sequencing of up to 96 samples/lane
  • RNA-Seq libraries in 4.5 hours
  • A kit for advanced users of RNA-Seq

Product Description

The QuantSeq Kit is a library preparation protocol designed to generate Illumina and Ion Torrent compatible libraries of sequences close to the 3’ end of the polyadenylated RNA.
QuantSeq kits are available in two versions. QuantSeq FWD generates NGS reads towards the poly(A) tail, directly reflecting the mRNA sequence. It is offered for both Illumina and Ion Torrent platforms. QuantSeq Reverse (REV) is only available for Illumina platforms and requires a Custom Sequencing Primer (CSP Version 2, included in the kit) for Read 1. With QuantSeq REV and the CSP it is possible to exactly pinpoint the 3’ end in Read 1 as the first nucleotide of your NGS read corresponds the very last nucleotide of the mRNA. The reads generated during Read 1 reflect the cDNA sequence

Input Amount

The required input amount of total RNA is as low as 100 pg (QuantSeq for Illumina) and 5 ng (QuantSeq for Ion Torrent). QuantSeq is suitable to reproducibly generate libraries from low quality RNA, including FFPE samples

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 QuantSeq 3’ mRNA-Seq Library Prep Kits for Illumina. In-line barcodes allowing up to 48 samples to be sequenced in one sequencing run of Ion Torrent instruments are included in QuantSeq 3’ mRNA-Seq Library Prep Kit for Ion Torrent. This high level of multiplexing allows saving costs as the length restriction in QuantSeq saves sequencing space. QuantSeq is also designed to yield insert sizes for short sequencing reads (SR50, SR100).

Performance

High Strand-Specificity

QuantSeq maintains exceptional strand-specificity of >99.9% and allows to map reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.

Rapid Turnaround

QuantSeq’s simple workflow allows generating ready-to sequence-NGS libraries within only 4.5 hours, including less than 2 hours hands-on time. This already includes hands-on time, which is less than 2 hours.

Mapping of TES

QuantSeq allows to exactly pinpoint the 3’ end of poly(A) RNA and therefore obtain accurate information about the 3’UTR.

Simple Bioinformatics Analysis

Read mapping is simplified by skipping the junction detection. Reads are generated at the transcripts’ most 3′ end where nearly no junctions are located. Data processing can hence be accelerated by using e.g. Bowtie2 instead of TopHat2.

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 renders QuantSeq the best alternative to microarrays and conventional RNA-Seq in gene expression and eQTL studies.

Workflow

Step 1 : Reverse Transcription

01quantseq_workflow
The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed.
02quantseq_workflow
Library generation starts with oligodT priming containing sequencing platform-compatible linker sequences.

Step 2 : Removal of RNA

03quantseq_workflow
After first strand synthesis the RNA is removed.

Step 3 : Second-Strand Synthesis

04quantseq_workflow
Second strand synthesis is initiated by random priming and a DNA polymerase. The random primer also contains sequencing platform-compatible linker sequences.
05quantseq_workflow
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

06quantseq_workflow
During the library amplification step the sequences required for cluster generation on Illumina platforms or colony formation on Ion Torrent platforms are introduced
07quantseq_workflow
Multiplexing can be performed with up to 96 external barcodes for Illumina and 24 in-line barcodes for Ion Torrent sequencing platforms.

FAQs

Illumina SpecificIon Torrent SpecificautoQuantSeq Specific
1.1 What is the difference between the two versions (Cat. No. 015 and 016) of the QuantSeq kit?
1.2 What is the CSP and how do I use it?
1.3 What is the typical library fragment size?
1.4 What are the input RNA requirements?
1.5 What is the minimum input amount? Are there any recommendations for low RNA input?
1.6 How many PCR cycles are needed to amplify the libraries?
1.7 Is the kit suitable for preparation of libraries from degraded RNA or FFPE samples?
1.8 How long does it take to generate QuantSeq libraries?
1.9 What level of multiplexing can be provided with QuantSeq? What barcoding (indexing) system do you use?
1.10 What is the orientation of QuantSeq reads?
1.11 Which aligner should be used for data analysis?
1.12 What sequence should be trimmed?
1.13 Which sequencing platforms are suitable for QuantSeq libraries?
1.14 What sequencing read length is best suited for QuantSeq libraries?
1.15 I want to do a SR100 or PE100 run. Are there any special considerations for this?
1.16 I see poly(T) stretches in my QuantSeq REV (Cat. No. 016) data and low sequencing quality. What is the reason for this?
1.17 I see internal priming events. How can I prevent this?
1.18 What are the most critical steps in the QuantSeq library generation?
1.19 My Bioanalyzer trace shows a second high molecular weight peak between 1000-9000 bp. Where does it come from? Is it a problem?
1.20 There is a peak around and beyond the upper marker of the Bioanalyzer trace. Where does it come from?
1.21 What can I do if my libraries are undercycled?
1.22 What positive control do you recommend to use?
1.23 I want to do paired-end sequencing, which kit version should I use?
1.24 Do you have any protocol recommendations to get longer insert sizes?
1.25 How much of a QuantSeq library should be loaded for sequencing?
1.26 Which barcodes should be used for multiplexing?
2.1 What is the typical library fragment size?
2.2 What are the input RNA requirements?
2.3 Is the kit suitable for preparation of libraries from degraded RNA or FFPE samples?
2.4 How long does it take to generate QuantSeq libraries?
2.5 What level of multiplexing can be provided with QuantSeq for Ion Torrent? What barcoding (indexing) system do you use?
2.6 How do QuantSeq barcodes differ from IonXpress barcodes and can I multiplex QuantSeq libaries with other libraries?
2.7 Which barcodes are included in Barcode Set A and B?
2.8 What is the orientation of QuantSeq reads?
2.9 Which aligner should be used for data analysis?
2.10 Which sequences should be trimmed?
2.11 What sequencing read length is best suited for QuantSeq libraries? Can I pinpoint the exact 3’ end?
2.12 My Bioanalyzer trace shows a second high molecular weight peak between 1000-9000 bp. Where does it come from? Is it a problem?
2.13 There is a peak around and beyond the upper marker of the Bioanalyzer trace. Where does it come from?
2.14 My QuantSeq libraries worked fine previously but after switching to IonChef and the HiQ system we get low loading (~50%). What can I do to improve this?
2.15 Which sequencing platforms are suitable for QuantSeq for Ion Torrent libraries?
2.16 What are the most critical steps in the QuantSeq library generation?
2.17 What positive control do you recommend to use?
3.1. On which liquid handler is the autoQuantSeq protocol automated?
3.2 How many samples can I process in a single run?
3.3 We only have a Zephyr NGS workstation in our lab. Can we still use the protocol?
3.4 We have a ScicloneNGSx workstation (with sunken deck). Can we use the protocol?
3.5 We have a Hamilton Microlab STARlet workstation. Can we use the protocol?
3.6 When should I use the Master Plate on the PerkinElmer Sciclone?
3.7 Should I do the thermal treatment on- or off-deck?
3.8 I have a Hamilton Microlab STAR liquid handler. Can I install the protocol myself?
3.9 Is any manual intervention required during the run?
3.10 How can I know when the next intervention is due?
3.11 I do not have enough EB in the kit when running on the Hamilton STAR. Why is this and what shall I do?

Ordering Information

Kit Platform Reactions Cat.No.
with Barcode Set A for Ion Torrent 24 preps 012.24A
with Barcode Set B 24 preps 012.24B
Forward Illumina 24 preps 015.24
96 preps 015.96
2×96 preps 015.2×96
Reverse with custom sequencing primer Illumina 24 preps 016.24

Downloads

QuantSeq Application Note (Nature Methods, December 2014).
Application Note

QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina

User Guide, – update 22.02.2016 (New barcode sealing introduced; Recommendations for low RNA input given (Step 4 (RT) may be increased to 1 h @ 42°C; Step 6: reduce to 5 min 95 °C for
PCR Add-on Kit for Illumina Instruction Manual
Product Flyer
QuantSeq for Illumina External Barcodes Overview

QuantSeq 3′ mRNA-Seq Library Prep Kit for Ion Torrent

User Guide – update 15.03.2016 (Temperature of reverse transcription raised from 37 °C to 42 °C to get less unspecific hybridization; Protocol adjustments for or low input, FFPE, or low quality RNA in step 2, 6, 17, and 29.)
PCR Add-on Kit for Ion Torrent Instruction Manual
QuantSeq for Ion Torrent In-line Barcodes Overview