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Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227252/ https://www.ncbi.nlm.nih.gov/pubmed/34207615 http://dx.doi.org/10.3390/microorganisms9061253 |
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author | Nahar, Akhikun Baker, Anthony L. Nichols, David S. Bowman, John P. Britz, Margaret L. |
author_facet | Nahar, Akhikun Baker, Anthony L. Nichols, David S. Bowman, John P. Britz, Margaret L. |
author_sort | Nahar, Akhikun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8227252 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-82272522021-06-26 Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species Nahar, Akhikun Baker, Anthony L. Nichols, David S. Bowman, John P. Britz, Margaret L. Microorganisms Article 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. MDPI 2021-06-09 /pmc/articles/PMC8227252/ /pubmed/34207615 http://dx.doi.org/10.3390/microorganisms9061253 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Nahar, Akhikun Baker, Anthony L. Nichols, David S. Bowman, John P. Britz, Margaret L. Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title | Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title_full | Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title_fullStr | Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title_full_unstemmed | Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title_short | Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species |
title_sort | benchmarking dna extraction methods for phylogenomic analysis of sub-antarctic rhodococcus and williamsia species |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227252/ https://www.ncbi.nlm.nih.gov/pubmed/34207615 http://dx.doi.org/10.3390/microorganisms9061253 |
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