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

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 sequ...

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Detalles Bibliográficos
Autores principales: Gloor, Gregory B., Hummelen, Ruben, Macklaim, Jean M., Dickson, Russell J., Fernandes, Andrew D., MacPhee, Roderick, Reid, Gregor
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964327/
https://www.ncbi.nlm.nih.gov/pubmed/21048977
http://dx.doi.org/10.1371/journal.pone.0015406
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author Gloor, Gregory B.
Hummelen, Ruben
Macklaim, Jean M.
Dickson, Russell J.
Fernandes, Andrew D.
MacPhee, Roderick
Reid, Gregor
author_facet Gloor, Gregory B.
Hummelen, Ruben
Macklaim, Jean M.
Dickson, Russell J.
Fernandes, Andrew D.
MacPhee, Roderick
Reid, Gregor
author_sort Gloor, Gregory B.
collection PubMed
description 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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spelling pubmed-29643272010-11-03 Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products Gloor, Gregory B. Hummelen, Ruben Macklaim, Jean M. Dickson, Russell J. Fernandes, Andrew D. MacPhee, Roderick Reid, Gregor PLoS One Research Article 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. Public Library of Science 2010-10-26 /pmc/articles/PMC2964327/ /pubmed/21048977 http://dx.doi.org/10.1371/journal.pone.0015406 Text en Gloor, et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Gloor, Gregory B.
Hummelen, Ruben
Macklaim, Jean M.
Dickson, Russell J.
Fernandes, Andrew D.
MacPhee, Roderick
Reid, Gregor
Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title_full Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title_fullStr Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title_full_unstemmed Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title_short Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
title_sort microbiome profiling by illumina sequencing of combinatorial sequence-tagged pcr products
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964327/
https://www.ncbi.nlm.nih.gov/pubmed/21048977
http://dx.doi.org/10.1371/journal.pone.0015406
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