Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic and 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 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 . 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 and four strains as species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed sp. 1163 and 1168 and sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as and , respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the genome cluster groups, including strains currently named as , , and , would be reclassified either as or .