MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution.
Nucleic Acids Res
; 49(12): e70, 2021 07 09.
Article
en En
| MEDLINE
| ID: mdl-33849057
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 <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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
VIH
/
Secuenciación de Nanoporos
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos