1. MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution
- Author
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Gallardo, Christian M, Wang, Shiyi, Montiel-Garcia, Daniel J, Little, Susan J, Smith, Davey M, Routh, Andrew L, and Torbett, Bruce E
- Subjects
Biological Sciences ,Bioinformatics and Computational Biology ,Genetics ,Human Genome ,Infectious Diseases ,Immunization ,Biotechnology ,Vaccine Related ,Prevention ,HIV/AIDS ,Infection ,Good Health and Well Being ,DNA ,Complementary ,Drug Resistance ,Viral ,Evolution ,Molecular ,Fusion Proteins ,gag-pol ,Genome ,Viral ,HIV ,HIV Infections ,Humans ,Mutation ,Nanopore Sequencing ,Reverse Transcriptase Polymerase Chain Reaction ,Environmental Sciences ,Information and Computing Sciences ,Developmental Biology ,Biological sciences ,Chemical sciences ,Environmental sciences - Abstract
Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolved genetic interactions given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of these genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s of viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at
- Published
- 2021