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Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples
OBJECTIVE: Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated D...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10422837/ https://www.ncbi.nlm.nih.gov/pubmed/37568179 http://dx.doi.org/10.1186/s13104-023-06451-7 |
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author | Sinclair, James West, Nicholas P Cox, Amanda J |
author_facet | Sinclair, James West, Nicholas P Cox, Amanda J |
author_sort | Sinclair, James |
collection | PubMed |
description | OBJECTIVE: Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34–69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods. RESULTS: Diversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13104-023-06451-7. |
format | Online Article Text |
id | pubmed-10422837 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-104228372023-08-13 Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples Sinclair, James West, Nicholas P Cox, Amanda J BMC Res Notes Research Note OBJECTIVE: Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34–69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods. RESULTS: Diversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13104-023-06451-7. BioMed Central 2023-08-11 /pmc/articles/PMC10422837/ /pubmed/37568179 http://dx.doi.org/10.1186/s13104-023-06451-7 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Note Sinclair, James West, Nicholas P Cox, Amanda J Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title | Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title_full | Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title_fullStr | Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title_full_unstemmed | Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title_short | Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples |
title_sort | comparison of four dna extraction methods for 16s rrna microbiota profiling of human faecal samples |
topic | Research Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10422837/ https://www.ncbi.nlm.nih.gov/pubmed/37568179 http://dx.doi.org/10.1186/s13104-023-06451-7 |
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