Our group has come up with a method to multiplex hundreds of microbiota samples in a single Illumina lane run (300-500 sample per run) resulting in a drastic cost reduction for microbial community profiling. The method paper was recently published in PLoS ONE:
Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, et al. 2010 Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products. PLoS ONE 5(10): e15406. doi: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.
Since there are several individuals and groups who might be interested in trying this method on their microbiota samples but may not have 400 samples to make the sequencing cost-effective, we're offering a cost-recovery academic service aimed to pool samples together from different sources for a single run. All you need to send is your isolated DNA!
The service is linked off of Dr. Greg Gloor's faculty page here:
Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, et al. 2010 Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products. PLoS ONE 5(10): e15406. doi: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.
Since there are several individuals and groups who might be interested in trying this method on their microbiota samples but may not have 400 samples to make the sequencing cost-effective, we're offering a cost-recovery academic service aimed to pool samples together from different sources for a single run. All you need to send is your isolated DNA!
The service is linked off of Dr. Greg Gloor's faculty page here:
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