QuantSeq 3' mRNA-Seq Library Prep Kits
The QuantSeq Kit is a library preparation protocol designed to generate Illumina and Ion Torrent compatible libraries of the sequences close to the 3' end of the polyadenylated RNA.
The kits are available in two versions. In the first version NGS reads are generated 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. Using this kit it is possible to exactly pinpoint the 3' end (3' UTR) during Read 1 as the NGS reads are directly started from the poly(A) tail. However, to achieve cluster calling the Multiplex Read 1 Sequencing Primer needs to be replaced by a Custom Sequencing Primer (CSP, included in the kit).The reads generated during Read 1 reflect the cDNA sequence.
Analysis of Low Quality Samples
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.
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.
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 so speed up using e.g. Bowtie2 instead of TopHat2.
Simple workflow allows generating libraries ready for sequencing within 4.5 hours. 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 3'UTR.
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.
Cost Saving 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).
For the detailed information about barcodes and instructions how to use them please consult Appendix D: Multiplexing, QuantSeq for Ion Torrent User Guide (page 23) and Appendix E: Multiplexing, QuantSeq for Illumina User Guide (page 25).