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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

Plot of the number of distinct ISU or OTU classes in each sample as a function of the number of reads.The number of ISU classes increases with the number of reads, but the number of OTU classes becomes constant above 20000–30000 reads.
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pone-0015406-g012: Plot of the number of distinct ISU or OTU classes in each sample as a function of the number of reads.The number of ISU classes increases with the number of reads, but the number of OTU classes becomes constant above 20000–30000 reads.

Mentions: Finally, species richness can be examined as a function of the number of reads across all 272 samples. This is plotted in Figure 12 for ISU and OTU sequences. In this case the white-filled symbols represent populations derived from samples classified as ‘normal’, and are expected to be dominated by one or a few species, and the red or blue-filled symbols represent populations classified as bacterial vaginosis (BV), where there is expected to be a more even distribution of species [35], [36]. There are strongly diminishing returns when more than 20000–25000 reads are obtained regardless of the diversity of the population; sampling more than 50000 reads was sufficient to sample all the available OTU diversity in the samples. Interestingly, the number of distinct ISU sequences increases linearly with the number of reads, providing further evidence that increasing the number of reads increases the background number of ISUs that contain PCR-derived errors. Taken together with the rarefaction, Chao1, ACE data, we conclude that the number of reads obtained by this Illumina sequencing is adequate to sample nearly saturating numbers of species in this environment.


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)

Plot of the number of distinct ISU or OTU classes in each sample as a function of the number of reads.The number of ISU classes increases with the number of reads, but the number of OTU classes becomes constant above 20000–30000 reads.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015406-g012: Plot of the number of distinct ISU or OTU classes in each sample as a function of the number of reads.The number of ISU classes increases with the number of reads, but the number of OTU classes becomes constant above 20000–30000 reads.
Mentions: Finally, species richness can be examined as a function of the number of reads across all 272 samples. This is plotted in Figure 12 for ISU and OTU sequences. In this case the white-filled symbols represent populations derived from samples classified as ‘normal’, and are expected to be dominated by one or a few species, and the red or blue-filled symbols represent populations classified as bacterial vaginosis (BV), where there is expected to be a more even distribution of species [35], [36]. There are strongly diminishing returns when more than 20000–25000 reads are obtained regardless of the diversity of the population; sampling more than 50000 reads was sufficient to sample all the available OTU diversity in the samples. Interestingly, the number of distinct ISU sequences increases linearly with the number of reads, providing further evidence that increasing the number of reads increases the background number of ISUs that contain PCR-derived errors. Taken together with the rarefaction, Chao1, ACE data, we conclude that the number of reads obtained by this Illumina sequencing is adequate to sample nearly saturating numbers of species in this environment.

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