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Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples
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  • Julia Schwenker,
  • Meike Friedrichsen,
  • Silvio Waschina,
  • Corinna Bang,
  • A. Franke,
  • Ricarda Mayer,
  • Christina Hölzel
Julia Schwenker
Christian-Albrechts-Universitat zu Kiel

Corresponding Author:[email protected]

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Meike Friedrichsen
Christian-Albrechts-Universitat zu Kiel
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Silvio Waschina
Christian-Albrechts-Universitat zu Kiel
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Corinna Bang
Christian-Albrechts-Universitat zu Kiel
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A. Franke
Christian-Albrechts-Universitat zu Kiel
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Ricarda Mayer
GNA Biosolutions GmbH
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Christina Hölzel
Christian-Albrechts-Universitat zu Kiel
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Abstract

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S rRNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony-forming units (cfu) and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. The different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were re-sequenced in triplicates (n=120). The most reproducible results for alpha- and beta-diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pre-treatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pre-treatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.
12 Nov 2021Submitted to MicrobiologyOpen
12 Nov 2021Submission Checks Completed
12 Nov 2021Assigned to Editor
12 Nov 2021Reviewer(s) Assigned
26 Nov 2021Review(s) Completed, Editorial Evaluation Pending
26 Nov 2021Editorial Decision: Revise Minor
25 Jan 20221st Revision Received
27 Jan 2022Submission Checks Completed
27 Jan 2022Assigned to Editor
27 Jan 2022Review(s) Completed, Editorial Evaluation Pending
27 Jan 2022Reviewer(s) Assigned
07 Feb 2022Editorial Decision: Revise Minor
21 Feb 20222nd Revision Received
23 Feb 2022Submission Checks Completed
23 Feb 2022Assigned to Editor
05 Mar 2022Review(s) Completed, Editorial Evaluation Pending
10 Mar 2022Editorial Decision: Accept
Apr 2022Published in MicrobiologyOpen volume 11 issue 2. 10.1002/mbo3.1275