QoALa: a comprehensive workflow for viral quasispecies diversity comparison using long-read sequencing data

The concept of viral quasispecies refers to a constantly mutating viral population occurring within hosts, which is essential for grasping the micro-evolutionary patterns of viruses. Despite its high error rate, long-read sequencing holds potential for advancing viral quasispecies research by resolving coverage limitations in next-generation sequencing. We introduce a refined workflow, QoALa, implemented in the longreadvqs R package. This workflow begins with nucleotide position-wise noise minimization of read alignments and sample size standardization, and extends to viral quasispecies comparison across related samples. Raw read samples from five studies of different viruses (HCV, HBV, HIV, SARS-CoV-2, and IAV), sequenced by major long-read platforms, were used to evaluate these approaches. The comparative results provide novel insights into intra- and inter-host diversity dynamics in various scenarios and unveil rare haplotypes not reported in the original study, underscoring the versatility and practicality of our methodology.