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

Date Published: October 26, 2010

Publisher: Public Library of Science

Author(s): Gregory B. Gloor, Ruben Hummelen, Jean M. Macklaim, Russell J. Dickson, Andrew D. Fernandes, Roderick MacPhee, Gregor Reid, Frank R. DeLeo. http://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.

Partial Text: Microbiome profiling is used to identify and enumerate the organisms in samples from diverse sources such as soil, clinical samples and oceanic environments [1]–[3]. This profiling is an important first step in determining the important bacterial and protist organisms in a biome and how they interact with and influence their environment.

We present and characterize a low-cost, high throughput method for microbiome profiling. The method uses combinatorial sequence tags attached to the 5′ end of PCR primers that amplify the rRNA V6 region, but may be easily adapted for use in other bacterial and eukaryotic sequences. Illumina paired-end sequencing of the amplified sequences generates millions of overlapping reads. The combinatorial sequence tags allows the investigator to examine hundreds of samples with far fewer primers than is required for single-end bar-code sequencing. We propose that this method will be useful for the deep sequencing of any short sequence that is informative; these include the V3 and V5 regions of the bacterial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity. The use of the V3 and V5 regions is currently being attempted by our group.

Source:

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