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Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing
BACKGROUND AND OBJECTIVE: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Co-Action Publishing
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000428/ https://www.ncbi.nlm.nih.gov/pubmed/24778776 http://dx.doi.org/10.3402/jom.v6.23990 |
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author | Abusleme, Loreto Hong, Bo-Young Dupuy, Amanda K. Strausbaugh, Linda D. Diaz, Patricia I. |
author_facet | Abusleme, Loreto Hong, Bo-Young Dupuy, Amanda K. Strausbaugh, Linda D. Diaz, Patricia I. |
author_sort | Abusleme, Loreto |
collection | PubMed |
description | BACKGROUND AND OBJECTIVE: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. DESIGN: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. RESULTS: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. CONCLUSION: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies. |
format | Online Article Text |
id | pubmed-4000428 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Co-Action Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-40004282014-04-28 Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing Abusleme, Loreto Hong, Bo-Young Dupuy, Amanda K. Strausbaugh, Linda D. Diaz, Patricia I. J Oral Microbiol Original Article BACKGROUND AND OBJECTIVE: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. DESIGN: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. RESULTS: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. CONCLUSION: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies. Co-Action Publishing 2014-04-23 /pmc/articles/PMC4000428/ /pubmed/24778776 http://dx.doi.org/10.3402/jom.v6.23990 Text en © 2014 Loreto Abusleme et al. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Abusleme, Loreto Hong, Bo-Young Dupuy, Amanda K. Strausbaugh, Linda D. Diaz, Patricia I. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title | Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title_full | Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title_fullStr | Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title_full_unstemmed | Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title_short | Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing |
title_sort | influence of dna extraction on oral microbial profiles obtained via 16s rrna gene sequencing |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000428/ https://www.ncbi.nlm.nih.gov/pubmed/24778776 http://dx.doi.org/10.3402/jom.v6.23990 |
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