A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples. Homo sapiens

RNA-sequencing (RNA-seq) has emerged as the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A)+ enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives. In this study, we compare for the first time three Illumina library preparation kits (TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access) as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels (intact, degraded, and highly degraded). We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. In particular, we show that the exome-capture protocol RNA Access performs well on samples with low amount and highly degraded RNA.