Error rates, PCR recombination, and sampling depth in HIV-1 whole genome deep sequencing

•Quantification of error rates of Illumina deep sequencing protocols.•Quantification of in-vitro recombination during RT-PCR.•Intrinsic estimation of sequencing depth from redundantly sequenced variants.

Deep sequencing is a powerful and cost-effective tool to characterize the genetic diversity and evolution of virus populations. While modern sequencing instruments readily cover viral genomes many thousand fold and very rare variants can in principle be detected, sequencing errors, amplification biases, and other artifacts can limit sensitivity and complicate data interpretation. For this reason, the number of studies using whole genome deep sequencing to characterize viral quasi-species in clinical samples is still limited. We have previously undertaken a large scale whole genome deep sequencing study of HIV-1 populations. Here we discuss the challenges, error profiles, control experiments, and computational test we developed to quantify the accuracy of variant frequency estimation.