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

Annemarie Siebert,Lena Staib,Siegfried Scherer,Etienne V Doll,Katharina Hofmann,Mareike Wenning

doi:10.1007/s00253-021-11353-4

Annemarie Siebert, Lena Staib + Show 4 more

Open Access

https://doi.org/10.1007/s00253-021-11353-4

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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-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers.Key points• Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota.• Reduction of eukaryotic DNA by enzymatic digestion.• Shift of detected microbiome caused by high cycle numbers in library-PCR.

Highlights

Amplicon-based high-throughput sequencing undoubtedly facilitates the in-depth characterization of individual complex raw milk (RM) microbiota
The study focused on three steps: (i) reduction of eukaryotic DNA from the cow’s somatic cells by removal of casein and subsequent selective lysis; (ii) bacterial lysis and DNA extraction by either enzymatic lysis, mechanical treatment, or a combination of both to enhance lysis in particular of Gram-positive cells; and (iii) the number of cycles in the library-PCR to check for PCR bias
Reduction of eukaryotic DNA by removal of casein and selective lysis of somatic cells As the cow’s genome is approximately 1000-fold larger than a bacterial genome, there is a large excess of eukaryotic over bacterial DNA

Summary

Introduction

Amplicon-based high-throughput sequencing undoubtedly facilitates the in-depth characterization of individual complex raw milk (RM) microbiota. It has been applied in recent years to unravel the impact of weather conditions (Li et al 2018), the health status of the dairy cow (Lima et al 2018), farm. In European fresh raw cow’s milk, the aerobic, mesophilic cell count is restricted to a maximum of 5 log colony forming unit (cfu) mL-1 (Regulation (EC) No 853/2004), but densities usually range from 3 to 5 log cfu mL-1 (Fricker et al 2011; Mallet et al 2012; von Neubeck et al 2015; Fretin et al 2018; Skeie et al 2019). In addition to the sample matrix properties, the disadvantageous ratio between bacterial and eukaryotic DNA further poses an enormous challenge to bacterial DNA isolation

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