Evaluating coverage bias in next-generation sequencing of Escherichia coli

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.

Strain-level profiling of viable microbial community by selective single-cell genome sequencing

Benchmarking a targeted 16S ribosomal RNA gene enrichment approach to reconstruct ancient microbial communities [PeerJ]

Frontiers 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

Barplot showing the coverage of samples sequenced by HiScanSQ and

Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state, BMC Bioinformatics

Next-Generation Sequencing: From Basic Research to Diagnostics

PDF] Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes

Diagram of phased assembly pipeline. Divided into four primary

PDF] Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes

An Efficient and User-Friendly Software for PCR Primer Design for Detection of Highly Variable Bacteria