The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles
Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM pr...
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| Format: | Article |
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MDPI AG
2020-05-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/3/2/39 |
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| author | Anna Ojo-Okunola Shantelle Claassen-Weitz Kilaza S. Mwaikono Sugnet Gardner-Lubbe Heather J. Zar Mark P. Nicol Elloise du Toit |
| author_facet | Anna Ojo-Okunola Shantelle Claassen-Weitz Kilaza S. Mwaikono Sugnet Gardner-Lubbe Heather J. Zar Mark P. Nicol Elloise du Toit |
| author_sort | Anna Ojo-Okunola |
| collection | DOAJ |
| description | Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp<sup>®</sup> DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of <i>Enhydrobacter</i> and <i>Acinetobacter</i>. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa. |
| first_indexed | 2024-03-10T19:48:27Z |
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| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-10T19:48:27Z |
| publishDate | 2020-05-01 |
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| series | Methods and Protocols |
| spelling | doaj.art-3510ad4bf29a488f85aad11e96799ede2023-11-20T00:35:13ZengMDPI AGMethods and Protocols2409-92792020-05-01323910.3390/mps3020039The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial ProfilesAnna Ojo-Okunola0Shantelle Claassen-Weitz1Kilaza S. Mwaikono2Sugnet Gardner-Lubbe3Heather J. Zar4Mark P. Nicol5Elloise du Toit6Division of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaDivision of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaComputational Biology Group and H3ABioNet, Department of Integrative Biomedical Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaDepartment of Statistics and Actuarial Science, Faculty of Economic and Management Sciences, Stellenbosch University, Matieland 7602, Stellenbosch, South AfricaInstitute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaDivision of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaDivision of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South AfricaCulture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp<sup>®</sup> DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of <i>Enhydrobacter</i> and <i>Acinetobacter</i>. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.https://www.mdpi.com/2409-9279/3/2/3916S rRNA gene sequencingbacterial profilesDNA extractionhuman milkskim milkwhole milk |
| spellingShingle | Anna Ojo-Okunola Shantelle Claassen-Weitz Kilaza S. Mwaikono Sugnet Gardner-Lubbe Heather J. Zar Mark P. Nicol Elloise du Toit The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles Methods and Protocols 16S rRNA gene sequencing bacterial profiles DNA extraction human milk skim milk whole milk |
| title | The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles |
| title_full | The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles |
| title_fullStr | The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles |
| title_full_unstemmed | The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles |
| title_short | The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles |
| title_sort | influence of dna extraction and lipid removal on human milk bacterial profiles |
| topic | 16S rRNA gene sequencing bacterial profiles DNA extraction human milk skim milk whole milk |
| url | https://www.mdpi.com/2409-9279/3/2/39 |
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