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Open Access Highly Accessed Research article

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

Brian J Haas1, Melissa Chin1, Chad Nusbaum1, Bruce W Birren1 and Jonathan Livny12*

Author affiliations

1 Genome Sequencing and Analysis Program, The Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA

2 Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

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Citation and License

BMC Genomics 2012, 13:734  doi:10.1186/1471-2164-13-734

Published: 27 December 2012

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.