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Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias

Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may b...

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Autores principales: Laursen, Martin F., Dalgaard, Marlene D., Bahl, Martin I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633598/
https://www.ncbi.nlm.nih.gov/pubmed/29051756
http://dx.doi.org/10.3389/fmicb.2017.01934
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author Laursen, Martin F.
Dalgaard, Marlene D.
Bahl, Martin I.
author_facet Laursen, Martin F.
Dalgaard, Marlene D.
Bahl, Martin I.
author_sort Laursen, Martin F.
collection PubMed
description Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5–73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy.
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spelling pubmed-56335982017-10-19 Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias Laursen, Martin F. Dalgaard, Marlene D. Bahl, Martin I. Front Microbiol Microbiology Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5–73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy. Frontiers Media S.A. 2017-10-05 /pmc/articles/PMC5633598/ /pubmed/29051756 http://dx.doi.org/10.3389/fmicb.2017.01934 Text en Copyright © 2017 Laursen, Dalgaard and Bahl. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Laursen, Martin F.
Dalgaard, Marlene D.
Bahl, Martin I.
Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title_full Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title_fullStr Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title_full_unstemmed Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title_short Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias
title_sort genomic gc-content affects the accuracy of 16s rrna gene sequencing based microbial profiling due to pcr bias
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633598/
https://www.ncbi.nlm.nih.gov/pubmed/29051756
http://dx.doi.org/10.3389/fmicb.2017.01934
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