Abstract
BackgroundRNA-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.MethodsWe 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).ResultsWe 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 (R2 > 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.ConclusionsWe 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. We also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples.
Highlights
RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis
Poly(A) + enrichment using oligodT coated beads is the most common approach to quantify the polyadenylated RNA fraction of the transcriptome including coding mRNAs. This method fails at profiling other RNA populations and suffers from biases when applied to low quality or low quantity RNA samples [24,25,26]
To evaluate the performance of RNA-seq methods in profiling non-optimal samples, we conducted a technical assessment of the three different RNA library preparation protocols mentioned above, namely TruSeq, RiboZero and RNA Access, on two human reference RNA samples previously used in the MAQC/SEQC studies; these samples are the Universal Human Reference RNA (UHRR or SEQC-A) and the Human Brain Reference RNA (HBRR or SEQC-B) [34]
Summary
RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Several generation sequencing protocols are currently available for the profiling of RNA samples, each with its own strengths and weaknesses These methods use different strategies to reduce the representation of abundant ribosomal RNAs (rRNA) in RNA-seq libraries prior to sequencing. RNA capture is a novel approach used to profile poor quality RNA samples like those extracted from formalin-fixed, paraffinembedded (FFPE) tissue samples [28]; this method uses capture probes targeting known exons to enrich for coding RNAs. The “TruSeq” Stranded mRNA Kit, the “RiboZero” rRNA Removal Kit, and the “RNA Access” Library Prep Kit represent respectively implementations of the poly(A) + enrichment, ribosome depletion and exomecapture approaches. These kits are commercial products (Illumina) with standardized, reproducible and easy to implement protocol steps and suitable for any research laboratories conducting gene expression studies
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