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• W3123673090 abstract "Whole-genome sequencing (WGS) is fundamental to Mycobacterium tuberculosis basic research and many clinical applications. Coverage across Illumina-sequenced M. tuberculosis genomes is known to vary with sequence context, but this bias is poorly characterized. Here, through a novel application of phylogenomics that distinguishes genuine coverage bias from deletions, we discern Illumina ‘blind spots’ in the M. tuberculosis reference genome for seven sequencing workflows. We find blind spots to be widespread, affecting 529 genes, and provide their exact coordinates, enabling salvage of unaffected regions. Fifty-seven pe/ppe genes (the primary families assumed to exhibit Illumina bias) lack blind spots entirely, while the remaining pe/ppe genes account for 55.1 % of blind spots. Surprisingly, we find coverage bias persists in homopolymers as short as 6 bp, shorter tracts than previously reported. While G+C-rich regions challenge all Illumina sequencing workflows, a modified Nextera library preparation that amplifies DNA with a high-fidelity polymerase markedly attenuates coverage bias in G+C-rich and homopolymeric sequences, expanding the ‘Illumina-sequenceable’ genome. Through these findings, and by defining workflow-specific exclusion criteria, we spotlight effective strategies for handling bias in M. tuberculosis Illumina WGS. This empirical analysis framework may be used to systematically evaluate coverage bias in other species using existing sequencing data." @default.
• W3123673090 created "2021-02-01" @default.
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• W3123673090 date "2021-03-01" @default.
• W3123673090 modified "2023-09-27" @default.
• W3123673090 title "Exact mapping of Illumina blind spots in the Mycobacterium tuberculosis genome reveals platform-wide and workflow-specific biases" @default.
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• W3123673090 doi "https://doi.org/10.1099/mgen.0.000465" @default.
• W3123673090 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8190613" @default.
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