Comparison of RNA-Seq by poly A capture, ribosomal RNA depletion, and DNA microarray for expression profilingReport as inadecuate




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BMC Genomics

, 15:419

Human and rodent genomics

Abstract

BackgroundRNA sequencing RNA-Seq is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using polyA enrichment of the mRNA mRNA-Seq to remove ribosomal RNA rRNA, however, this method fails to capture non-polyA transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded FFPE samples.

ResultsTo address the desire to perform RNA-Seq on FFPE materials, we evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. We obtained paired Fresh Frozen FF and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. We tested 11 human breast tumor samples using: a FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq Duplex-Specific Nuclease and b FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. We also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set.

The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3′ ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome 20-30%; in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45–65 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray.

ConclusionsOur results demonstrate that compared to mRNA-Seq and microarrays, Ribo-Zero-Seq provides equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and consistently high quality quantification of transcripts. Moreover, Ribo-Zero-Seq and DSN-Seq have consistent transcript quantification using FFPE RNAs, suggesting that RNA-Seq can be used with FFPE-derived RNAs for gene expression profiling.

KeywordsRNA sequencing FFPE RNA depletion Ribo-zero Gene expression Microarray Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-15-419 contains supplementary material, which is available to authorized users.

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Author: Wei Zhao - Xiaping He - Katherine A Hoadley - Joel S Parker - David Neil Hayes - Charles M Perou

Source: https://link.springer.com/







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