DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid

ABSTRACT Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing...

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Autores principales:	Erika Ganda, Kristen L. Beck, Niina Haiminen, Justin D. Silverman, Ban Kawas, Brittany D. Cronk, Renee R. Anderson, Laura B. Goodman, Martin Wiedmann
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	host depletion shotgun metagenomics milk DNA RNA biases Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/40a0a49ba60f47f68a9cb5dc661ed0d7
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spelling	oai:doaj.org-article:40a0a49ba60f47f68a9cb5dc661ed0d72021-12-02T18:11:52ZDNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid10.1128/mSystems.00619-212379-5077https://doaj.org/article/40a0a49ba60f47f68a9cb5dc661ed0d72021-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00619-21https://doaj.org/toc/2379-5077ABSTRACT Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.Erika GandaKristen L. BeckNiina HaiminenJustin D. SilvermanBan KawasBrittany D. CronkRenee R. AndersonLaura B. GoodmanMartin WiedmannAmerican Society for Microbiologyarticlehost depletionshotgun metagenomicsmilkDNARNAbiasesMicrobiologyQR1-502ENmSystems, Vol 6, Iss 3 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	host depletion shotgun metagenomics milk DNA RNA biases Microbiology QR1-502
spellingShingle	host depletion shotgun metagenomics milk DNA RNA biases Microbiology QR1-502 Erika Ganda Kristen L. Beck Niina Haiminen Justin D. Silverman Ban Kawas Brittany D. Cronk Renee R. Anderson Laura B. Goodman Martin Wiedmann DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
description	ABSTRACT Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.
format	article
author	Erika Ganda Kristen L. Beck Niina Haiminen Justin D. Silverman Ban Kawas Brittany D. Cronk Renee R. Anderson Laura B. Goodman Martin Wiedmann
author_facet	Erika Ganda Kristen L. Beck Niina Haiminen Justin D. Silverman Ban Kawas Brittany D. Cronk Renee R. Anderson Laura B. Goodman Martin Wiedmann
author_sort	Erika Ganda
title	DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
title_short	DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
title_full	DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
title_fullStr	DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
title_full_unstemmed	DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid
title_sort	dna extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/40a0a49ba60f47f68a9cb5dc661ed0d7
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DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid

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