Background: 454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.
Results: We constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.
Conclusion: Standard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.