QuantSeq 3' mRNA-Seq library prep: Efficient gene expression analysis by Next Generation Sequencing for low quality and FFPE samples

QuantSeq is an oligodT primed NGS library preparation protocol generating only one fragment per transcript. This enables accurate gene expression quantification independent of the RNA quality (including FFPE samples). Standard... [ view full abstract ]

QuantSeq is an oligodT primed NGS library preparation protocol generating only one fragment per transcript. This enables accurate gene expression quantification independent of the RNA quality (including FFPE samples). Standard mRNA-Seq protocols aim to cover the whole transcript, and result in a heavy 3’ bias when used on degraded RNA. Comparison between high quality and low quality RNA samples is therefore hampered and often biased. QuantSeq 3’ mRNA-Seq focuses on counting 3‘ ends and therefore delivers reliable and reproducible results independent of the RNA quality.

In this study a xenograft of the MOLP-8 human tumor cell line was split into two pieces, which were subsequently processed either as fresh frozen cryo-block (RIN 8.3) or embedded FFPE material (RIN 2.8, DV₂₀₀ of 87 %), leading to different RNA qualities from the same original sample. QuantSeq libraries were generated using 50 ng total RNA and sequenced on an Illumina HiSeq2500 with SR50.

Next Generation Sequencing data analysis revealed a high correlation of gene expression between libraries derived from FFPE and cryo-preserved RNA (R² 0.86).

At 26.5 M reads, 25 842 genes were detected in the high quality cryo RNA sample. At 2.5 M reads, 20 081 genes were detected in the cryo sample with at least 1 read, and 15 190 genes in the FFPE sample. With the detection threshold of 5 reads/gene, the difference in gene detection was reduced from 24 % to 3 %, and at 10 reads/gene to 1 %.

The high concordance of gene expression detected in cryo and FFPE RNA samples demonstrates that QuantSeq is the method of choice for gene expression quantification independent of the RNA input quality.