Original Article

Subject Category: Integrated genomics and post-genomics approaches in microbial ecology

The ISME Journal (2010) 4, 896–907; doi:10.1038/ismej.2010.18; published online 11 March 2010

Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics

Frank J Stewart1,2, Elizabeth A Ottesen1,2 and Edward F DeLong1

1Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Parsons Laboratory, Cambridge, MA, USA

Correspondence: Edward F DeLong, Division of Biological Engineering, Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Parsons Laboratory 48-427, 15 Vassar Street, Cambridge, MA 02139, USA. E-mail: delong@mit.edu

2These authors contributed equally to this work.

Received 5 October 2009; Revised 4 January 2010; Accepted 26 January 2010; Published online 11 March 2010.



Metatranscriptomes generated by pyrosequencing hold significant potential for describing functional processes in complex microbial communities. Meeting this potential requires protocols that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as systematic comparisons to identify methodological artifacts and test for reproducibility across data sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S) that uses sample-specific probes and is applicable across diverse environmental samples. To test this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX technology) from bacterioplankton communities at two depths in the oligotrophic open ocean, yielding 10 data sets representing ~350Mbp. Subtractive hybridization reduced bacterial rRNA transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from 12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for detecting sequences replicated artificially via pyrosequencing errors and identified such replicates as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal, statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential transcript abundance (<0.2% of reference genes). However, gene overlap between data sets was remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing library template) sharing greater than 17% of unique reference genes. These results indicate that pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of reliable rRNA subtraction procedures to enhance sequencing coverage across the functional transcript pool.


functional genomics; gene expression; ribosomal RNA; Roche 454 pyrosequencing; RNA amplification; marine bacterioplankton