Abstract
To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.
Highlights
RNA sequencing (RNA-seq) has revolutionized the way biologists examine transcriptomes and has been successfully applied in biological research, drug discovery, and clinical development[1,2,3]
Our analyses showed that the Ribosomal RNA (rRNA) depletion method captured a wider diversity of unique transcriptome features, whereas the polyadenylated RNA (polyA)+ enrichment method outperformed the rRNA depletion method in exonic coverage and accuracy of gene quantification
For the blood and colon samples, 220% and 50% more reads, respectively, had to be sequenced in the rRNA depletion method to achieve the same level of exonic coverage as the polyA+ selection approach
Summary
RNA sequencing (RNA-seq) has revolutionized the way biologists examine transcriptomes and has been successfully applied in biological research, drug discovery, and clinical development[1,2,3]. Several recent studies have noted that rRNA-depleted RNA libraries cost more than polyA+ selection libraries to achieve comparable coverage of protein-coding reads in a transcriptome, it provides more information on polyA- transcripts. Another technical advantage that favours RNA-seq of rRNA-depleted libraries compared with polyA-selected libraries is that its performance is better for degraded RNAs8–12. For the blood and colon samples, 220% and 50% more reads, respectively, had to be sequenced in the rRNA depletion method to achieve the same level of exonic coverage as the polyA+ selection approach. Our results show that selection of the library preparation protocol in clinical research should be guided by the study objectives, and polyA+ selection is recommended for RNA-seq projects where the main goal is quantification of protein-coding genes
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