Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

ABSTRACT: Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also ass

SUBMITTER: Mallory MA 

PROVIDER: S-EPMC10729743 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature