Benchmarking of RNA-sequencing analysis workflows using whole-transcriptome RT-qPCR expression data
| Autor: | Jesper L.V. Maag, Quek Xiu Cheng, Pieter Mestdagh, Jan Hellemans, Marcel E. Dinger, Manuel Luypaert, Celine Everaert |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
RNA-Sequencing quantification TOPHAT Sequence analysis Science Biology Real-Time Polymerase Chain Reaction Article Workflow Transcriptome 03 medical and health sciences Exon Gene expression Databases Genetic Humans Gene Genetics Regulation of gene expression Multidisciplinary Sequence Analysis RNA RNA Biology and Life Sciences Gold standard (test) QUANTIFICATION GENE Benchmarking 030104 developmental biology SEQ Gene Expression Regulation Medicine TRANSCRIPT EXPRESSION |
| Zdroj: | Scientific Reports SCIENTIFIC REPORTS Scientific Reports, Vol 7, Iss 1, Pp 1-11 (2017) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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