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 could 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 primary material, laboratory methods, data analysis, and interpretation. The focus of this study is in gathering the primary material, in this case, DNA. We used bovine milk as a model to 1) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk; 2) evaluate host DNA depletion methods for use with milk, and 3) develop and evaluate a selective lysis-PMA based protocol for host DNA depletion in milk. Our results suggest that magnetic-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 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 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.

Evaluation of Host Depletion and Extraction Methods for Shotgun Metagenomic Analysis of Bovine Vaginal Samples

“…Propidium monoazide unbiasedly digests the exposed DNA. Hence, the selective impacts of PMA-based host depletion treatments depend greatly on the lysis methods and the permeability of microbial cell membrane ( 38 , 48 , 49 ). Potentially, the integrity of the microbial cells in vaginal swab samples was not preserved in vaginal mucus during the sample collection and transfer.…”