Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products
Open accessAbstract
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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When DNA fragment size is shorter than single-end read length, current mergers generate erroneous results.
“When this does not hold, for example when sequencing the V6 region of 16S rRNA genes of bacterial samples [fragment sizes range between 110 and 130-bp (Gloor et al., 2010)] with read lengths of 150-bp (see case C in Fig. 1), current mergers will generate erroneous results.”
From PEAR: a fast and accurate Illumina Paired-End reAd mergeR (2014) · cites this paper as Gloor et al., 2010
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- Authors
- Gloor, G. B., Hummelen, R., Macklaim, J. M., Dickson, R. J., Fernandes, A. D., MacPhee, R., & Reid, G.
- Affiliations
- Western University (CA)
- Lawson Health Research Institute (CA)
- Corresponding author
- Gloor, Gregory B.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2010-10-26
- Volume
- 5
- Issue
- 10
- Pages
- e15406
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC2964327
- OpenAlex
- W2163295736
- Cited by
- 330
- References
- 39
- Citations by year
- 2026: 5 · 2025: 4 · 2024: 8 · 2023: 12 · 2022: 9 · 2021: 19 · 2020: 19 · 2019: 27 · 2018: 25 · 2017: 33 · 2016: 43 · 2015: 33 · 2014: 35 · 2013: 31 · 2012: 18
- FWCI
- 7.50
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- arXiv (Cornell University) (submittedVersion)
- RePub (Erasmus University Rotterdam) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Figshare (submittedVersion, cc-by)
- Scholarship@Western (Western University) (submittedVersion)
- RePub (Erasmus University Rotterdam) (submittedVersion)
- arXiv (Cornell University)
- Funders
- University of Toronto
- Natural Sciences and Engineering Research Council of Canada
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Keywords
- microbiome profiling, PCR primers, vaginal microbiota, sequence clustering, short-read sequencing, next-generation sequencing, protist diversity
- MeSH terms
- Base Sequence, Microbiology, Species Specificity, Polymerase Chain Reaction, DNA Primers, Gene Expression Profiling
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