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Benchmarking DNA extraction methods for phylogenomic analysis of sub-Antarctic Rhodococcus and Williamsia species


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Nahar, A, Baker, AL ORCID: 0000-0001-5455-2271, Nichols, DS ORCID: 0000-0002-8066-3132, Bowman, JP ORCID: 0000-0002-4528-9333 and Britz, ML 2021 , 'Benchmarking DNA extraction methods for phylogenomic analysis of sub-Antarctic Rhodococcus and Williamsia species' , Microorganisms, vol. 9, no. 6 , pp. 1-19 , doi: 10.3390/microorganisms9061253.

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Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

Item Type: Article
Authors/Creators:Nahar, A and Baker, AL and Nichols, DS and Bowman, JP and Britz, ML
Keywords: Actinobacteria, Rhodococcus erythropolis, Rhodococcus qingshengii, Williamsia, DNA extraction, next-generation sequencing, mycolic acids, ANI; psychrotrophic, lipids
Journal or Publication Title: Microorganisms
Publisher: MDPI
ISSN: 2076-2607
DOI / ID Number: 10.3390/microorganisms9061253
Copyright Information:

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) License, ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

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