The recombinants generated during PCR can lead to the false interpretation of genetic diversity in the sample, incorrect identification of novel gene species, and new biological phenotypes that do not exist in vivo. To avoid such PF-4217903 artificial recombinants, SGS techniques were developed to obtain sequences free of recombination from a quasispecies population by amplification of individual genomic templates. However, the quasispecies genomes of human pathogens and immunoglobulin repertoires were recently characterized by NGS, which requires a bulk PCR amplification of highly complex quasispecies populations. Therefore, understanding how the recombination frequency is affected by PCR conditions will help to minimize the PCR-mediated recombinants in bulk PCR amplification. Previous studies have showed that the thermal cycles, templates inputs, extension time and enzymes could impact generation of recombinants during PCR. However, how exactly those factors affect recombination have not fully understood since a large number of relatively long sequences from individual amplicons were not available from various PCR conditions for analysis. In this study, we demonstrated that the higher numbers of thermal cycles and templates could significantly increase the proportions of artificial recombinants in the PCR products. In a standard PCR condition, 41.7% of the PCR products were recombinants within an 870 bp gene fragment. Such a higher level of artificial recombinants can significantly affect accurate analysis of a quasispecies genome population obtained by the bulk PCR amplification. However, the longer extension time can significantly reduced recombination frequencies. Thus, when it is not possible to characterize a quasispecies genomic population by SGS, it is important to use minimum numbers of templates and thermal cycles as well as longer extension time to minimize the PCRmediated recombination. By directly characterizing PCR amplicons using the PASS assay that can simultaneously analyze thousands of genomes and determine the linkage of bases at multiple sites in each individual genome, we found that heteroduplexes in the PCR amplicon population continuously increased, and the recombination frequency were significantly higher in heteroduplexes than homoduplexes. Thus, our results demonstrated that disassociation of the incompletely extended primer from one template and annealing to a different template was the main mechanism for frequent recombination during PCR. The heteroduplex as the cause for generation PCR-mediated recombinants was previously hypothesized but not proven since all previous methods could not directly analyze heteroduplexes. When the numbers of amplicons were low in the PCR reaction, the chance for heteroduplexes to form was small and recombination occurred rarely. Recombinants present in individual heteroduplexes would not be detected by other sequencing methods in which the individual double-strand.