Biology subjects

Mann, B. C.

Publications and source records attributed to Mann, B. C..

3 recordsLinked to original sources

bioRxiv2024

Systematic review and meta-analysis of protocols and yield of direct from sputum sequencing of Mycobacterium tuberculosis

Direct sputum whole genome sequencing (dsWGS) can revolutionize Mycobacterium tuberculosis (Mtb) diagnosis by enabling rapid detection of drug resistance and strain diversity without the biohazard of culture. We searched PubMed, Web of Science and Google scholar, and identified 8 studies that met inclusion criteria for testing protocols for dsWGS. Utilising meta-regression we identify several key factors positively associated with dsWGS success, including higher Mtb bacillary load, mechanical disruption, and enzymatic/chemical lysis. Specifically, smear grades of 3+ (OR = 14.7, 95% CI: 3.5, 62.1; p = 0.0005) were strongly associated with improved outcomes, whereas decontamination with sodium hydroxide (NaOH) was negatively associated (OR = 0.005, 95% CI: 0.001, 0.03; p = 7e-06), likely due to its harsh effects on Mtb cells. Furthermore, mechanical lysis (OR = 193.3, 95% CI: 11.7, 3197.8; p = 0.008) and enzymatic/chemical lysis (OR = 18.5, 95% CI: 1.9, 183.1; p = 0.02) were also strongly associated with improved dsWGS. Across the studies, we observed a high degree of variability in approaches to sputum pre-processing prior to dsWGS highlighting the need for standardized best practices. In particular we conclude that optimizing pre-processing steps including decontamination with the exploration of alternatives to NaOH to better preserve Mtb cells and DNA, and best practices for cell lysis during DNA extraction as priorities. Further and considering the strong association between Mtb load and successful dsWGS, protocol improvements for optimal sputum sample collection, handling, and storage could also further enhance the success rate of dsWGS.

microbiology↗

bioRxiv2024

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis (Mtb) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

bioinformatics↗

bioRxiv2023

Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples

BackgroundWithin-host M. tuberculosis (Mtb) diversity may capture antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here we compared three sample preprocessing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Design/MethodsMtb positive NALC - NaOH treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I or Benzonase digestion. Levels of contaminating host DNA and target Mtb DNA was assessed by Quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. ResultsThe pre-treatment sample has a high host-to-target ratio of DNA (6 168 {+/-} 1 638 host copies/ng to 212,3 {+/-} 59,4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3 422 {+/-} 2 162 host copies/ng to 11 721 {+/-} 7 096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum after benzonase treatment the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb to 90,95% from 1,18 % compared with benzonase treatment without enrichment. ConclusionWe demonstrate a low limit of detection (LoD) for a new custom dsDNA Mtb target enrichment panel that has a favourable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

molecular biology↗