In either case, each strand in the heteroduplex will be separated. In contrast, in the PASS assay, the individual double-strand DNA molecules will be amplified together in a semi-solid acrylamide gel and both DNA strands in the heteroduplexes can be simultaneously analyzed. This unique feature of the PASS assay will also allow it to determine heterozygous alleles present in the same double-strand DNA molecule in biological materials. Previous studies showed that the recombination frequency could be reduced by the longer extension time during PCR. Analysis of the large number of individual amplicon sequences in this study further demonstrated that extension time had a significant impact on the recombination frequency. The incompletely extended nascent single DNA fragments were considered the main reason for generation recombinants during PCR. At the optimal condition, the Taq polymerase can synthesize 1000 bases in less than 10 seconds. However, the manufactures recommend using 1-minute extension time for 1000 bp of the DNA fragment to ensure the complete synthesis of the target templates. The reduced recombination frequencies at longer extension time strongly suggested that the incompletely extended nascent single DNA fragments were still present at such a level that resulted in higher frequencies of recombination at standard recommended extension time. However, the increased extension time could decrease the recombination frequency by reducing the level of the incompletely extended nascent single DNA fragments in the PCR reaction. Thus, longer extension time should be used whenever possible to prevent generation high levels of recombinants during PCR. When complex microbe quasispecies population or the immunoglobulin repertoire is analyzed, the number of genetically distinct templates in the samples is high. The NGS sequence analysis of two different templates showed that the recombination frequency was 14% under the standard PCR condition, but could be significantly reduced under Crizotinib 877399-52-5 optimized conditions. However, the recombination frequency for less abundant species in a quasispecies population could exceed 70% by NGS analysis. Our NGS analysis results with a mixture of 35 distinct templates showed that as high as 61% of the sequences were recombinants when 3.56106 templates were amplified with 45 thermal cycles. At the PCR conditions that just generated enough amplicons for subsequent NGS analysis, the recombination frequency was still 13%. Our results confirmed that the presence of multiple templates could lead to high recombination frequencies and, as in samples with only two templates, more copies of templates and more thermal cycles could result in higher recombination frequencies. Thus, when the quasispecies samples are analyzed by NGS, PCR conditions should be optimized to minimize the number of PCR-derived recombinants. Recombination among quasispecies templates does not increase the branch length in phylogenetic tree analysis since the mutation rates in the exchanged gene fragments.