Abstract：
:RNA-sequencing has become the gold standard for whole-transcriptome gene expression quantification. Multiple algorithms have been developed to derive gene counts from sequencing reads. While a number of benchmarking studies have been conducted, the question remains how individual methods perform at accurately quantifying gene expression levels from RNA-sequencing reads. We performed an independent benchmarking study using RNA-sequencing data from the well established MAQCA and MAQCB reference samples. RNA-sequencing reads were processed using five workflows (Tophat-HTSeq, Tophat-Cufflinks, STAR-HTSeq, Kallisto and Salmon) and resulting gene expression measurements were compared to expression data generated by wet-lab validated qPCR assays for all protein coding genes. All methods showed high gene expression correlations with qPCR data. When comparing gene expression fold changes between MAQCA and MAQCB samples, about 85% of the genes showed consistent results between RNA-sequencing and qPCR data. Of note, each method revealed a small but specific gene set with inconsistent expression measurements. A significant proportion of these method-specific inconsistent genes were reproducibly identified in independent datasets. These genes were typically smaller, had fewer exons, and were lower expressed compared to genes with consistent expression measurements. We propose that careful validation is warranted when evaluating RNA-seq based expression profiles for this specific gene set.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Everaert C,Luypaert M,Maag JLV,Cheng QX,Dinger ME,Hellemans J,Mestdagh Pdoi
10.1038/s41598-017-01617-3subject
Has Abstractpub_date
2017-05-08 00:00:00pages
1559issue
1issn
2045-2322pii
10.1038/s41598-017-01617-3journal_volume
7pub_type
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