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Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequenci...

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Autores principales: Sui, Hui-yu, Weil, Ana A., Nuwagira, Edwin, Qadri, Firdausi, Ryan, Edward T., Mezzari, Melissa P., Phipatanakul, Wanda, Lai, Peggy S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262970/
https://www.ncbi.nlm.nih.gov/pubmed/32528434
http://dx.doi.org/10.3389/fmicb.2020.00953
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author Sui, Hui-yu
Weil, Ana A.
Nuwagira, Edwin
Qadri, Firdausi
Ryan, Edward T.
Mezzari, Melissa P.
Phipatanakul, Wanda
Lai, Peggy S.
author_facet Sui, Hui-yu
Weil, Ana A.
Nuwagira, Edwin
Qadri, Firdausi
Ryan, Edward T.
Mezzari, Melissa P.
Phipatanakul, Wanda
Lai, Peggy S.
author_sort Sui, Hui-yu
collection PubMed
description Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0–3.9% of the variability), the most impact in vacuumed dust (12–16% of the variability) and intermediate values for sputum (9.2–12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.
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spelling pubmed-72629702020-06-10 Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing Sui, Hui-yu Weil, Ana A. Nuwagira, Edwin Qadri, Firdausi Ryan, Edward T. Mezzari, Melissa P. Phipatanakul, Wanda Lai, Peggy S. Front Microbiol Microbiology Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0–3.9% of the variability), the most impact in vacuumed dust (12–16% of the variability) and intermediate values for sputum (9.2–12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method. Frontiers Media S.A. 2020-05-25 /pmc/articles/PMC7262970/ /pubmed/32528434 http://dx.doi.org/10.3389/fmicb.2020.00953 Text en Copyright © 2020 Sui, Weil, Nuwagira, Qadri, Ryan, Mezzari, Phipatanakul and Lai. http://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) and the copyright owner(s) 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
Sui, Hui-yu
Weil, Ana A.
Nuwagira, Edwin
Qadri, Firdausi
Ryan, Edward T.
Mezzari, Melissa P.
Phipatanakul, Wanda
Lai, Peggy S.
Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title_full Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title_fullStr Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title_full_unstemmed Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title_short Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing
title_sort impact of dna extraction method on variation in human and built environment microbial community and functional profiles assessed by shotgun metagenomics sequencing
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262970/
https://www.ncbi.nlm.nih.gov/pubmed/32528434
http://dx.doi.org/10.3389/fmicb.2020.00953
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