Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing

Abstract

BackgroundNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps.ResultsOur DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid.ConclusionsOur optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.

Adaptação de um método de extração de DNA a partir de células da mucosa bucal baseado em parâmetros de qualidade / Adaptation of a DNA extraction method from buccal mucosa cells based on quality parameters

“…As pesquisas e inovações tecnológicas em biologia molecular possibilitaram inúmeras técnicas e métodos para a realização da extração do DNA para fins de pesquisa e, mais recentemente, aplicação no diagnóstico de doenças (Nedel et al, 2009). As etapas de extração e purificação do material genético são o ponto de partida para a aplicação dessas técnicas, tendo em vista que métodos insuficientes de obtenção de DNA podem ser responsáveis por comprometer o desempenho de algumas dessas tecnologias (Bouso & Planet, 2019).…”

Whole Genome Sequencing in the Management of Non-Tuberculous Mycobacterial Infections

“…For diagnostic purposes, better treatment results, and expanding our knowledge of mycobacterial diseases, it is necessary to identify and classify new species/subspecies of NTM [28,57,58]. Describing the complete genomes of the novel NTM requires a combination of short-read (e.g., Illumina MiSeq, San Diego, CA, USA) and long-read (e.g., MinION, Oxford, UK; PacBio SMRT, Pacific Biosciences, CA, USA) sequencing technologies (Figure 1 and Table 1) [59]. Short-read sequencing is characterized by high accuracy but does not provide information about the complete genome (including G + C rich regions, recombination, repetitive PE/PPE regions, deletions, and insertions) [60].…”

The respiratory microbiome and nontuberculous mycobacteria: an emerging concern in human health

“…Genotypic methods such as DNA sequence analysis of the 16S rRNA gene are often adequate to identify NTM, although some isolates cannot be differentiated with clear granularity at the species level [4]. DNA extraction may be difficult owing to recalcitrant cell composition and slow colony growth, often requiring extra lytic steps in the laboratory pipeline that may affect efficiency and cost [48]. Finally, NTM usually contain only one or two 16S rRNA genes per genome, and thus may be underrepresented among other taxa with more copies of ribosomal genes using standard 16S rRNA sequencing [49].…”