Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples.\nDegenerate primers that anneal to conserved regions can facilitate amplification of divergent, lowconcentration variants, evenwhen\nthe strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping\namplicons across multiple whole genomes is described. The new script, run tiled primers, is part of the PriMux software.\nPrimers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses,\nArenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each\ngroup is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross\nreactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to\nspecifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo.\nConclusions.This software should help researchers designmultiplex sets of primers for targeted whole genome enrichment prior to\nsequencing to obtain better coverage of lowtiter, divergent viruses.Applications include viral discovery froma complex background\nand improved sensitivity and coverage of rapidly evolving strains or variants in a gene family
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