Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products

Authors

Gregory B. Gloor, The University of Western Ontario
Ruben Hummelen, Lawson Health Research Institute, London, ON
Jean M. Macklaim, The University of Western Ontario
Russell J. Dickson, The University of Western Ontario
Andrew D. Fernandes, The University of Western Ontario
Roderick MacPhee, The University of Western Ontario
Gregor Reid, The University of Western Ontario

Document Type

Article

Publication Date

10-26-2010

Journal

PLoS ONE

Volume

5

Issue

10

First Page

e15406

Last Page

e15406

URL with Digital Object Identifier

http://dx.doi.org/10.1371/journal.pone.0015406

Abstract

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads allowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an observation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bacterial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.