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Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs

SIMPLE SUMMARY: The cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, an...

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Autores principales: Van Syoc, Emily, Carrillo Gaeta, Natália, Ganda, Erika
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151189/
https://www.ncbi.nlm.nih.gov/pubmed/34065976
http://dx.doi.org/10.3390/ani11051372
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author Van Syoc, Emily
Carrillo Gaeta, Natália
Ganda, Erika
author_facet Van Syoc, Emily
Carrillo Gaeta, Natália
Ganda, Erika
author_sort Van Syoc, Emily
collection PubMed
description SIMPLE SUMMARY: The cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, and cloacal swabs are often used. There is a need to evaluate laboratory methods for 16S rRNA sequencing in cloacal swab samples to ensure reproducible and trustworthy downstream results. We compared four DNA extraction methods from two commercially available magnetic-based DNA extraction kits. Mock communities and negative controls were included for each method and subjected to 16S rRNA sequencing. While extraction quality and yield differed between each extraction method, overall sequencing results were not affected, including alpha and beta diversity. Positive and negative controls are an important aspect of microbiome science and our findings lend guidance to future microbiome research in poultry. ABSTRACT: As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.
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spelling pubmed-81511892021-05-27 Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs Van Syoc, Emily Carrillo Gaeta, Natália Ganda, Erika Animals (Basel) Article SIMPLE SUMMARY: The cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, and cloacal swabs are often used. There is a need to evaluate laboratory methods for 16S rRNA sequencing in cloacal swab samples to ensure reproducible and trustworthy downstream results. We compared four DNA extraction methods from two commercially available magnetic-based DNA extraction kits. Mock communities and negative controls were included for each method and subjected to 16S rRNA sequencing. While extraction quality and yield differed between each extraction method, overall sequencing results were not affected, including alpha and beta diversity. Positive and negative controls are an important aspect of microbiome science and our findings lend guidance to future microbiome research in poultry. ABSTRACT: As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry. MDPI 2021-05-12 /pmc/articles/PMC8151189/ /pubmed/34065976 http://dx.doi.org/10.3390/ani11051372 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Van Syoc, Emily
Carrillo Gaeta, Natália
Ganda, Erika
Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title_full Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title_fullStr Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title_full_unstemmed Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title_short Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
title_sort choice of commercial dna extraction method does not affect 16s sequencing outcomes in cloacal swabs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151189/
https://www.ncbi.nlm.nih.gov/pubmed/34065976
http://dx.doi.org/10.3390/ani11051372
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