Figure 6. Recombination frequency during simultaneous amplification of multiple distinct HIV-1 genomes by next generation sequencing.
A mixture of 35 genetically distinct HIV-1 genomes was subjected to PCR amplification. The PCR was performed with different copies of templates (3.5×104, 3.5×105 or 3.5×106 copies) using different thermal cycle numbers (30, 35, 40 and 45). The PCR products were sequenced using a two-direction 600 cycle reagent kit on MiSeq. The merged sequences from two overlapping reads of the same cluster were then aligned to the HIV-1 reference sequence. The frequencies of all 35 parental sequence and their recombinants were determined by linkage analysis of 139 informative sites in each amplicon sequence using Nautilus [36].