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 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 hard-to-lyse bacteria. We benchmarked three spin-column-based against a classical extraction method employing lysozyme, proteinase K SDS six lysozyme-resistant, sub-Antarctic strains Corynebaceriales. Prior cultivation broths glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved lysis both kit methods. The produced average fragment sizes 27–59 Kbp tight size ranges, meeting standards sequencing, phylogenomic analyses. By 16S rRNA gene we classified two as Williamsia four Rhodococcus species. Pairwise comparison nucleotide identity (ANI) alignment fraction (AF), plus clustering analysis, confirmed sp. 1163 1168 1135 1138 novel Phylogenetic, lipidomic biochemical analyses psychrotrophic 1139 1159 R. qingshengii erythropolis, respectively, ANI similarity >98% AF >60% species delineation. On this basis, some members the erythropolis cluster groups, including currently named enclensis, baikonurensis, opacus rhodochrous, would be reclassified either or qingshengii.