Control of Artifactual Variation in Reported Intersample Relatedness during Clinical Use of a Mycobacterium tuberculosis Sequencing Pipeline.

Wyllie, David H and Sanderson, Nicholas and Myers, Richard and Peto, Tim and Robinson, Esther and Crook, Derrick W and Smith, E Grace and Walker, A Sarah (2018) Control of Artifactual Variation in Reported Intersample Relatedness during Clinical Use of a Mycobacterium tuberculosis Sequencing Pipeline. Journal of clinical microbiology, 56 (8). ISSN 1098-660X. This article is available to all UHB staff and students via ASK Discovery tool http://tinyurl.com/z795c8c by using their UHB Athens login IDs

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Official URL: https://jcm.asm.org/content/56/8

Abstract

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artifactual variation between isolates during routine next-generation sequencing of spp., we investigated its basis in 2,018 consecutive isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted and sequenced, reads were mapped, and consensus sequences were determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 genomic regions, we quantified the relationship between the minor variant depth and the amount of nonmycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of nonmycobacterial bacterial DNA, we found significant increases in minor variant frequencies, of more than 1.5-fold, in 242 regions covering 5.1% of the genome. Included within these were four high-variation regions strongly influenced by the amount of nonmycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we demonstrated an approach identifying critical genomic regions contributing to clinically relevant artifactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multistep laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

Item Type: Article
Additional Information: This article is available to all UHB staff and students via ASK Discovery tool http://tinyurl.com/z795c8c by using their UHB Athens login IDs
Subjects: QW Microbiology. Immunology
Divisions: Clinical Support > Infectious Diseases
Clinical Support > Pathology
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Depositing User: Beth Connors
Date Deposited: 23 Apr 2019 14:56
Last Modified: 23 Apr 2019 14:56
URI: http://www.repository.heartofengland.nhs.uk/id/eprint/2041

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