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Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research
BACKGROUND: Aerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbi...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067373/ https://www.ncbi.nlm.nih.gov/pubmed/33902731 http://dx.doi.org/10.1186/s40793-019-0349-z |
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author | Bøifot, Kari Oline Gohli, Jostein Moen, Line Victoria Dybwad, Marius |
author_facet | Bøifot, Kari Oline Gohli, Jostein Moen, Line Victoria Dybwad, Marius |
author_sort | Bøifot, Kari Oline |
collection | PubMed |
description | BACKGROUND: Aerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations. RESULTS: Here, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures a more comprehensive microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract. CONCLUSIONS: By demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments. |
format | Online Article Text |
id | pubmed-8067373 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-80673732021-04-26 Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research Bøifot, Kari Oline Gohli, Jostein Moen, Line Victoria Dybwad, Marius Environ Microbiome Methodology BACKGROUND: Aerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations. RESULTS: Here, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures a more comprehensive microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract. CONCLUSIONS: By demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments. BioMed Central 2020-01-13 /pmc/articles/PMC8067373/ /pubmed/33902731 http://dx.doi.org/10.1186/s40793-019-0349-z Text en © The Author(s). 2020 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. |
spellingShingle | Methodology Bøifot, Kari Oline Gohli, Jostein Moen, Line Victoria Dybwad, Marius Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title | Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title_full | Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title_fullStr | Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title_full_unstemmed | Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title_short | Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
title_sort | performance evaluation of a new custom, multi-component dna isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067373/ https://www.ncbi.nlm.nih.gov/pubmed/33902731 http://dx.doi.org/10.1186/s40793-019-0349-z |
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