Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

Abstract

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.

Novel Nanobiocomposites Based on Natural Polysaccharides as Universal Trophic Low-Dose Micronutrients

“…Thus, they are similar microorganisms and belong the same order Actinomycetales of Gram-positive bacteria. However, R. erythropolis contains mycolic acids in its cell wall [ 70 , 71 ]. Mycolic acids are long-chain branched fatty acids [ 72 ].…”

Introduction to the principles and methods underlying the recovery of metagenome‐assembled genomes from metagenomic data

“…Although out of the scope, it is important to highlight the importance of these steps (Pollock et al, 2018 ). In particular, care should be taken to avoid contamination and biases due to DNA extraction and degradation during processing and storage (Han et al, 2019 ; Nahar et al, 2021 ; Sar et al, 2018 ). Comparative studies indicated that the choice of DNA extraction method affects the outcome, with mechanical and/or enzymatic pretreatments being often superior (Gryp et al, 2020 ; Henderson et al, 2013 ).…”

Physical Insight into the Conditions Required in the Solid-Phase Molecular Self-Assembly of SDS Revealed by Coarse-Grained Molecular Dynamics Simulation

“…Sodium dodecyl sulfate (SDS) is widely applied in many commercial cleaning products, such as soaps, toothpaste, etc . It is also used for DNA and protein extraction in polyacrylamide gel electrophoresis. Due to its common use, SDS has been extensively studied experimentally over the past century via different experimental methods. , From the perspective of colloidal chemistry, the preassemble behavior of SDS is clearly known: the formation of tail-to-tail aligned structures through hydrophobic interaction.…”

“…Sodium dodecyl sulfate (SDS) is widely applied in many commercial cleaning products, such as soaps, toothpaste, etc. 17 It is also used for DNA and protein extraction 21 in polyacrylamide gel electrophoresis. Due to its common use, SDS has been extensively studied experimentally over the past century via different experimental methods.…”