How deep is deep enough for RNA-Seq profiling of bacterial transcriptomes?

BMC Genomics
Authors
Keywords
Abstract

BACKGROUND: High-throughput sequencing of cDNA libraries (RNA-Seq) has proven to be a highly effective approach for studying bacterial transcriptomes. A central challenge in designing RNA-Seq-based experiments is estimating a priori the number of reads per sample needed to detect and quantify thousands of individual transcripts with a large dynamic range of abundance.

RESULTS: We have conducted a systematic examination of how changes in the number of RNA-Seq reads per sample influences both profiling of a single bacterial transcriptome and the comparison of gene expression among samples. Our findings suggest that the number of reads typically produced in a single lane of the Illumina HiSeq sequencer far exceeds the number needed to saturate the annotated transcriptomes of diverse bacteria growing in monoculture. Moreover, as sequencing depth increases, so too does the detection of cDNAs that likely correspond to spurious transcripts or genomic DNA contamination. Finally, even when dozens of barcoded individual cDNA libraries are sequenced in a single lane, the vast majority of transcripts in each sample can be detected and numerous genes differentially expressed between samples can be identified.

CONCLUSIONS: Our analysis provides a guide for the many researchers seeking to determine the appropriate sequencing depth for RNA-Seq-based studies of diverse bacterial species.

Year of Publication
2012
Journal
BMC Genomics
Volume
13
Pages
734
Date Published
2012 Dec 27
ISSN
1471-2164
URL
DOI
10.1186/1471-2164-13-734
PubMed ID
23270466
PubMed Central ID
PMC3543199
Links
Grant list
AI-076608 / AI / NIAID NIH HHS / United States
HHSN272200900018C / PHS HHS / United States