Biology subjects

Rathod, P.

Publications and source records attributed to Rathod, P..

5 recordsLinked to original sources

bioRxiv2020

DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

Mycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99{degrees}C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes [≥]1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

microbiology↗

bioRxiv2018

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised mock clinical samples was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

microbiology↗

bioRxiv2018

Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

IntroductionDetecting laboratory cross-contamination and mixed tuberculosis infection are important goals of clinical Mycobacteriology laboratories.\n\nObjectivesTo develop a method detecting mixtures of different M. tuberculosis lineages in laboratories performing Mycobacterial next generation sequencing (NGS).\n\nSettingPublic Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive Mycobacterial Growth Indicator tubes.\n\nMethodsWe analysed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, obtained during development and production use of a diagnostic process using NGS. Counts of the most common (major) variant, and all other variants (non-major variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development.\n\nResultsFor each sample we determined the non-major variant proportions at 55 sets of lineage defining positions. The non-major variant proportion in the two most mixed lineage defining sets (F2 metric) was compared with that in the 47 least mixed lineage defining sets (F47 metric). Three patterns were observed: (i) not mixed by either metric, (ii) high F47 metric suggesting mixtures of multiple lineages, and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevation relative to F47. Pattern (ii) was observed in batches, with similar patterns in the H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in each pattern were 97%, 2.8%, and 0.001%, respectively.\n\nConclusionThe F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis.

microbiology↗

bioRxiv2018

A quantitative evaluation of MIRU-VNTR typing against whole-genome sequencing for identifying Mycobacterium tuberculosis transmission: A prospective observational cohort study

SummaryO_ST_ABSBackgroundC_ST_ABSMycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing is widely used in high-income countries for Mycobacterium tuberculosis typing. Whole-genome sequencing (WGS) is known to deliver greater specificity, but no quantitative prospective comparison has yet been undertaken.\n\nMethodsWe studied isolates from the English Midlands, sampled consecutively between 1 January 2012 and 31 December 2015. In addition to routinely performed MIRU-VNTR typing, DNA was extracted from liquid cultures and sequenced using Illumina technology. Demographic and epidemiological data were extracted from the Enhanced Tuberculosis Surveillance system maintained by Public Health England. Closely related samples, defined using a threshold of five single nucleotide variants (SNVs), were compared to samples with identical MIRU-VNTR profiles, with shared epidemiological risk factors, and to those with both characteristics.\n\nFindings1,999 patients were identified for whom at least one M. tuberculosis isolate had been MIRU-VNTR typed and sequenced. Comparing epidemiological risk factors with close genetic relatedness, only coresidence had a positive predictive value of over 5%. Excluding co-resident individuals, 18.6% of patients with identical MIRU-VNTR profiles were within 5 SNVs. Where patients also shared social risk factors and ethnic group, this rose to 48%. Only 8% of MIRU-VNTR linked pairs in lineage 1 were within 5 SNV, compared to 31% in lineage 4.\n\nInterpretationIn the setting studied, MIRU-VNTR typing and epidemiological risk factors are poorly predictive of close genomic relatedness, assessed by SNV. MIRU-VNTR performance varies markedly by lineage.\n\nFundingPublic Health England, National Institute of Health Research Oxford Biomedical Research Centre.

microbiology↗

bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗