The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing.

Ultra-deep pyrosequencing (UDPS) of targeted amplicons allows to determine a large number of individual sequence reads from a single PCR product, and this approach is thus extremely valuable for analysis of mixed viral populations. A mixture of genetically distinct DNA templates, however, may lead to the generation of recombinant sequences during the initial PCR amplification step. In the present study, the frequency at which these misleading PCR artefacts are formed has been estimated by using artificial mixtures of genetically distinct human cytomegalovirus (HCMV) DNA templates for a given cycling profile. The presence of similar copy numbers of non-identical HCMV target sequences, combined with high levels of input HCMV DNA, as is found in some clinical samples, favored the formation of recombinant PCR products. Thus, to estimate the full natural diversity within mixed viral populations using UDPS, artificial chimeras generated during the PCR step should be taken into account as a potential artefact. Copyright (c) 2010 Elsevier B.V. All rights reserved.