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Microbiome profiling by illumina sequencing of combinatorial sequence-tagged PCR products.

Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, MacPhee R, Reid G - PLoS ONE (2010)

Bottom Line: The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. ggloor@uwo.ca

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.

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Related in: MedlinePlus

Correspondence between Chao1, ACE and rarefaction curves for the 272 samples.The X and Y axes show the fraction of species that were found in each sample for the two estimates. Red-filled circles highlight those samples where the limit rarefaction value was less than 0.97.
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pone-0015406-g011: Correspondence between Chao1, ACE and rarefaction curves for the 272 samples.The X and Y axes show the fraction of species that were found in each sample for the two estimates. Red-filled circles highlight those samples where the limit rarefaction value was less than 0.97.

Mentions: Another method of examining species richness is to use the Chao1 or ACE methods to estimate the number of unseen species in the sample [32], [33]. We used both methods to determine the number of species expected in each of the 272 samples with the VEGAN package for biodiversity analysis [34]. There were 37 and 31 of 272 samples where the Chao1 and ACE estimates indicated that we observed of the real species. The correspondence between the Chao1 and ACE measures is plotted in Figure 11 and it is clear by these two measures that the vast majority of samples were expected to contain the majority of the available species. Included in this plot is the fraction of species found when the rarefaction analysis was performed with the number of reads in the sample. Rarefaction with a saturating number of reads again showed that the 206 of 272 samples identified all or almost all of the available species.


Microbiome profiling by illumina sequencing of combinatorial sequence-tagged PCR products.

Gloor GB, Hummelen R, Macklaim JM, Dickson RJ, Fernandes AD, MacPhee R, Reid G - PLoS ONE (2010)

Correspondence between Chao1, ACE and rarefaction curves for the 272 samples.The X and Y axes show the fraction of species that were found in each sample for the two estimates. Red-filled circles highlight those samples where the limit rarefaction value was less than 0.97.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2964327&req=5

pone-0015406-g011: Correspondence between Chao1, ACE and rarefaction curves for the 272 samples.The X and Y axes show the fraction of species that were found in each sample for the two estimates. Red-filled circles highlight those samples where the limit rarefaction value was less than 0.97.
Mentions: Another method of examining species richness is to use the Chao1 or ACE methods to estimate the number of unseen species in the sample [32], [33]. We used both methods to determine the number of species expected in each of the 272 samples with the VEGAN package for biodiversity analysis [34]. There were 37 and 31 of 272 samples where the Chao1 and ACE estimates indicated that we observed of the real species. The correspondence between the Chao1 and ACE measures is plotted in Figure 11 and it is clear by these two measures that the vast majority of samples were expected to contain the majority of the available species. Included in this plot is the fraction of species found when the rarefaction analysis was performed with the number of reads in the sample. Rarefaction with a saturating number of reads again showed that the 206 of 272 samples identified all or almost all of the available species.

Bottom Line: The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. ggloor@uwo.ca

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.

Show MeSH
Related in: MedlinePlus