Evaluating Coverage Bias in Next-generation Sequencing of Escherichia Coli

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2021 Jun 24

PMID 34166413

Citations 5

Authors

Samantha Gunasekera

Sam Abraham

Marc Stegger

Stanley Pang

Penghao Wang

Shafi Sahibzada

Mark ODea

Affiliations

Soon will be listed here.

Abstract

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content.

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The efficiency of Nextera XT tagmentation depends on G and C bases in the binding motif leading to uneven coverage in bacterial species with low and neutral GC-content.

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