QuantSeq for Illumina for Ultra-High Throughput

QuantSeq 3’ mRNA-Seq Library Prep has already been widely used for high throughput gene expression studies. QuantSeq is a gene counting method and requires much less sequencing reads per sample compared to standard RNA-Seq.

“Using the QuantSeq 3´ mRNA-Seq library prep kits, we were able to multiplex >40 samples per sequencing lane and obtain between 2 to 5 million reads per sample. This enabled us to analyze numerous different strains with various exosome and roadblocking factors inactivated, showing that inactivating roadblocks shifted the window of NNS termination downstream.” – Kevin Roy, Postdoctoral Scholar, Lars Steinmetz Lab, Department of Genetics Stanford University School of Medicine

Increasing capabilities of the sequencing instruments give the opportunity to multiplex more and more samples in one run. To meet this need, Lexogen has released an ultra-high throughput version of QuantSeq. QuantSeq FWD HT Kit (Cat. No. 015.384) includes 384 preps and 4 x 96 i7 Index Plates. Four unique i5 indices have also been included in the kit, providing an opportunity for dual indexing and therefore allowing multiplexing of up to 384 unique samples in one lane of the sequencer. Learn more about QuantSeq FWD HT Kit.

Indices for QuantSeq and SENSE Kits for Illumina

Barcodes plates used for all versions of QuantSeq and SENSE kits have been updated for an even better nucleotide balance for sequencing when only few samples are multiplexed. The changes include:

  • The former barcode BC05 has been exchanged. It was the only index identical to one of the barcodes present in the Illumina barcodes set. Now you can multiplex SENSE or QuantSeq samples with any other library preps without concern of the indices overlap.
  • The barcodes have been renamed (i7 Index Plate (7001-7096), formerly Barcode Plate (BC01-96)).
  • BC00 primer without index, used for qPCR amplification has been renamed to P7 Primer 7000.
  • The online Index Balance Checker has been introduced to allow selection of the barcodes carefully for the most optimal balance on any Illumina sequencer.

QuantSeq Protocol for Illumina

In the updated User Guide of the upgraded QuantSeq protocol we have included recommendations for qPCR endpoint determination to be set to 50 % of the maximum fluorescence (used to be 33 %) for a higher yield but still avoiding overcycling.

Additionally, RS2 (RNA Removal Solution 2) and corresponding step 6 have been removed from the protocol, saving your consumables and making QuantSeq a more streamlined protocol. As a consequence, SS1 (Second Strand Synthesis Mix 1) is reduced to 10 µl (instead of 15 µl) and the volumes for the purification steps (16 µl PB in step 12 instead of 20 µl in previously step 13 and 56 µl PS in step 16 instead of 72 µl in previously step 17) were adjusted. RS1 (RNA Removal Solution 1) has been renamed to RS (RNA Removal Solution).

Visit QuantSeq and SENSE for Illumina web pages to learn more about these products:

QuantSeq 3’ mRNA-Seq Library Prep Kit (FWD) and QuantSeq 3’ mRNA-Seq Library Prep Kit (FWD) HT
QuantSeq 3’ mRNA-Seq Library Prep Kit (REV)
QuantSeq-Flex Targeted RNA-Seq Library Prep Kit V2

SENSE Total RNA-Seq Library Prep Kit
SENSE mRNA-Seq Library Prep Kit V2