High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods

All Rights Reserved. HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypes.