Exact mapping of Illumina blind spots in theMycobacterium tuberculosisgenome reveals platform-wide and workflow-specific biases

Abstract

Whole genome sequencing (WGS) is fundamental to M. 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 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 GC-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 GC-rich and homopolymeric sequences, expanding the "Illumina-sequencable" 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.

Novel and Reported Compensatory Mutations in rpoABC Associate Specifically with Predominant Mycobacterium tuberculosis Rifampicin Resistance Marker rpoB:S450L

“…The study combined sets of isolates sequenced by different platforms, each with different sequencing biases. For example, the RS1 SMRT sequenced isolates had lower than average coverage, and the Illumina sequenced isolates have known difficulties with homopolymers and high GC regions 31 . Additionally, the novel putative compensatory mutations identified in this study were implicated only by association and require future mutagenesis experiments to confirm whether they truly compensate for fitness costs.…”

A pragmatic pipeline for drug resistance identification inMycobacterium tuberculosisusing whole genome sequencing

“…The mycobacterial genome has a high GC content (>60%) and multiple repeat regions. These structural features create challenges when considering sequencing library preparation and data analysis (18,19). On the other hand, an advantage in the analysis of the DR genotype for M. tuberculosis is that mycobacterial resistance is mainly dependent on the evolution of mutations such as single nucleotide polymorphisms (SNPs) and insertions or deletions (INDELs) within chromosomal genes rather than horizontal gene transmission (20).…”