AEM Accepts, published online ahead of print on 11 January 2008

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Appl. Environ. Microbiol. doi:10.1128/AEM.02181-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Metagenomics: Read length matters

K. Eric Wommack, Jaysheel Bhavsar, and Jacques Ravel^*

Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711, USA.; Institute for Genome Sciences, Department of Microbiology & Immunology, University of Maryland School of Medicine, 20 Penn Street, Baltimore, MD 21201, USA

^* To whom correspondence should be addressed. Email: jravel{at}som.umaryland.edu.

Obtaining an unbiased view of the phylogenetic composition and functional diversity within a microbial community is one central objective of metagenomic analysis. New technologies, such as 454 pyrosequencing, have dramatically reduced sequencing costs to a level where metagenomic analysis may become a viable alternative to more focused assessments of the phylogenetic (eg. 16S rDNA) and functional diversity of microbial communities. To determine whether the short (100-200 bp) sequence reads obtained from pyrosequencing are appropriate for phylogenetic and functional characterization of microbial communities the results of BLAST and COG analyses were compared for long (750 bp) and randomly derived short reads from each of two microbial and one virioplankton metagenome libraries. Overall, BLASTX searches against GenBank nr found far fewer homologs within the short sequence library. This was especially pronounced for a Chesapeake Bay virioplankton metagenome library. Increasing the short read sampling depth or the length of derived short reads (up to 400 bp) did not completely resolve the discrepancy in BLASTX homolog detection. Only in cases where the long read sequence had a close homolog (low BLAST E-score) did the derived short read sequence also find a significant homolog. Thus, more distant homologs of microbial and viral genes are not detected by short read sequences. Among COG hits, derived short reads sampled at a depth of two short reads per long read missed up to 72% of the COGs found using long reads. Noting the current limitation in computational approaches for analysis of short sequences, use of short read length libraries does not appear to be an appropriate tool for metagenomic characterization of microbial communities.

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