Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs
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 b...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-05-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/11/5/1372 |
| _version_ | 1797534485830434816 |
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| author | Emily Van Syoc Natália Carrillo Gaeta Erika Ganda |
| author_facet | Emily Van Syoc Natália Carrillo Gaeta Erika Ganda |
| author_sort | Emily Van Syoc |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T11:30:13Z |
| format | Article |
| id | doaj.art-ed079210ed2941e69674ab2a976adc1a |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-10T11:30:13Z |
| publishDate | 2021-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-ed079210ed2941e69674ab2a976adc1a2023-11-21T19:20:18ZengMDPI AGAnimals2076-26152021-05-01115137210.3390/ani11051372Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal SwabsEmily Van Syoc0Natália Carrillo Gaeta1Erika Ganda2Integrative & Biomedical Physiology and Clinical & Translational Sciences Dual-Title Ph.D. Program, Pennsylvania State University, University Park, PA 16802, USADepartment of Internal Medicine, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo 05508-270, BrazilDepartment of Animal Science, College of Agricultural Sciences, Pennsylvania State University, University Park, PA 16801, USAAs 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.https://www.mdpi.com/2076-2615/11/5/1372poultry microbiomecloacal microbiomeanimal microbiomemethod evaluation |
| spellingShingle | Emily Van Syoc Natália Carrillo Gaeta Erika Ganda Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs Animals poultry microbiome cloacal microbiome animal microbiome method evaluation |
| 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 | poultry microbiome cloacal microbiome animal microbiome method evaluation |
| url | https://www.mdpi.com/2076-2615/11/5/1372 |
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