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

RNA-sequencing (RNA-seq) has emerged as one of 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.

Researchers at the Novartis Institutes for Biomedical Research compared for the first time three commercial 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).

The researchers 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. We found that each protocol generates highly reproducible results (R 2 > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input.

Bargraph of the alignment statistics for the SEQC-A sample and all three protocols

Each bar represents the averaged values across the three technical replicates per condition. The percentage of total aligned reads is represented by the height of the bar, and the percentage of reads aligning to exons is in red, introns in blue, and intergenic regions in green.

The researchers found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. They also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples.