Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR.

ABSTRACT: The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy^® PowerFood^® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library

SUBMITTER: Siebert A 

PROVIDER: S-EPMC8195793 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature