PloS one
Sub-consensus haploid variant calling in long-read sequencing technology
By Zair X, Wilm A, Benton MC and 6 others.
Abstract
Background
Next-generation sequencing (NGS) is vital for tracking viral evolution during outbreaks. Oxford Nanopore Technologies (ONT) offers long-read, cost-effective sequencing, but accurate sub-consensus variant detection remains challenging. LoFreq, a widely used caller for sub-consensus variant calling in haploid genomes, was designed for short reads. This study assesses and benchmarks LoFreq variant identification accuracy on long-read data and proposes a calibration method to improve accuracy.
Methods
We created truth sets using three SARS-CoV-2 spike gene plasmids (7179 bases, 100 SNVs) and whole Escherichia coli genomes. Libraries were sequenced using R9.4.1 and R10.4.1 flow cells. LoFreq's recall was benchmarked across chemistries and library sizes. We also introduced a method to adjust Phred quality scores for enhanced accuracy.