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Comparison of Poly(A) capture versus Ribosomal RNA depletion methods for RNA-seq

ABSTRACT: Methods: RNA-sequencing was performed on matched samples obtained across several different gene expression measurement methods including: (a) fresh-frozen (FF) RNA samples by mRNA-seq, Ribo-zero and DSN and (b) FFPE samples by Ribo-zero and DSN. We also assayed the matched samples with Agilent microarray. RNA-seq data was compared on the rRNA-removal efficiency, genome profile, library complexity, coverage uniformity and quantitative cosinstency across protocols and with microarray data. Results: Compared to mRNA-seq, Ribo-zero provides equivalent percentage of rRNA component, genome-based mapped reads, and consistent quantification of transcripts. Moreover, Ribo-zero and DSN protocols achieve concordant transcript profiling in FFPE samples, and provide substantially more information on non-poly(A) RNA, which cannot be captured by mRNA-seq. Therefore, our study provides evidence that RNA-sequencing can generate accurate and reproducible transcript quantification using FFPE tissues.

ORGANISM(S): Homo sapiens

PROVIDER: GSE51783 | GEO | 2014/08/20

REPOSITORIES: GEO

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Project description:Background: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

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Comparison of Poly(A) capture versus Ribosomal RNA depletion methods for RNA-seq

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