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

Nucleic Acids Res. 2021 Jul 9;49(12):e70. doi: 10.1093/nar/gkab231.


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 <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure and enables the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Complementary
  • Drug Resistance, Viral / genetics
  • Evolution, Molecular
  • Fusion Proteins, gag-pol / genetics
  • Genome, Viral
  • HIV / genetics*
  • HIV / isolation & purification
  • HIV Infections / drug therapy
  • HIV Infections / virology
  • Humans
  • Mutation
  • Nanopore Sequencing / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / methods


  • DNA, Complementary
  • Fusion Proteins, gag-pol