Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing

Abstract

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.

Molecular Biology and Diversification of Human Retroviruses

“…However, even in the presence of Vif expression, sublethal levels of G-to-A mutations are observed within the HIV-1 genome, indicating that suppression of APOBEC3-mediated restriction is incomplete (142,143,158,206,207). These mutations have important implications within the context of viral infection, and have been linked to changes in coreceptor usage, immune evasion, and the failure of ART (208)(209)(210)(211)(212)(213)(214).…”

“…This also results in the introduction of A-to-G mutations in proviral DNA when these editing events occur during reverse transcription (154,231). A-to-G, and U-to-C, hypermutation have been observed in many negative-sense RNA viruses but are observed relatively infrequently in retroviruses such as HIV-1 and HIV-2 (142,154,207,232,233).…”