1887

Abstract

Whole-genome sequencing (WGS) is fundamental to basic research and many clinical applications. Coverage across Illumina-sequenced 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 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 genes (the primary families assumed to exhibit Illumina bias) lack blind spots entirely, while the remaining 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 Illumina WGS. This empirical analysis framework may be used to systematically evaluate coverage bias in other species using existing sequencing data.

Funding
This study was supported by the:
  • National Institute of Allergy and Infectious Diseases (Award R01AI105185)
    • Principle Award Recipient: Faramarz Valafar
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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/content/journal/mgen/10.1099/mgen.0.000465
2021-01-27
2021-10-18
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