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MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution


Date: 28th January 2020 | Source: BioRxiv

Authors: CM Gallardo, S Wang, DJ Montiel-Garcia, SJ Little, DM Smith, AL Routh, BE Torbett.

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-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range 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 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 <1% frequency.

This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

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