Recombination frequency between sites at different length.

<p>Recombination frequency between sites at different length.</p

Recombination frequencies at different conditions during PCR.

<p>(A) Recombination frequencies were determined at different thermal cycles. Equal amount of NL4-3 and 89.6 plasmids (10⁷ copies per template) were mixed together and co-amplified. The PCR was carried with 5, 10, 15, 20, 25 or 30 thermal cycles. (B) Recombination frequencies were determined with different numbers of templates. Equal amount of NL4-3 and 89.6 plasmids (10¹, 10³, 10⁵ or 10⁷ copies each) was mixed together and co-amplified by 30 cycles of PCR. (C) Recombination frequencies were determined with different extension time. Equal amount of NL4-3 and 89.6 plasmids (10⁷ copies per template) were mixed together and co-amplified. The PCR was carried with different extension time (1, 2, 4 or 8 minutes). The PCR products were analyzed by the PASS assay and the recombination frequency at each condition was determined by linkage analysis of six bases.</p

Recombination analysis of two low genetic diversity templates during PCR.

<p>A partial <i>pol</i> gene (870 bp) was amplified from two genetic variants (1B7 and 1D1) of WEAU. Nucleotides that are distinct at six positions in 1B7 and 1D1 are shown. The regions between two neighbor nucleotides are named as A through E and the genetic distances between them are indicated.</p

PCR error rate with individual template.

<p>PCR error rate with individual template.</p

Frequency of recombinants with different recombination breakpoints.

<p>Frequencies of recombinants with different breakpoints were determined for PCR with different thermal cycles (A), different template concentrations (B), and different extension time (C). Recombinants with one (diamond), two (square) or three (triangle) breakpoints were determined. No amplicons contained more than three recombination breakpoints.</p

Comparison of recombination frequencies between templates with high and low genetic diversities.

<p>Note: 30 cycles of PCR with 1000 copies of each template.</p><p>Comparison of recombination frequencies between templates with high and low genetic diversities.</p

Determination of recombinant frequencies in PCR with 35 distinct templates by next generation sequencing.

<p>Determination of recombinant frequencies in PCR with 35 distinct templates by next generation sequencing.</p

Detection of PCR-mediated recombinants by PASS.

<p>(A) Nucleotides used for linkage analysis to identify recombinants are indicated. A partial <i>pol</i> gene (870 bp) was amplified. Nucleotides that are distinct at six positions between 89.6 and NL4-3 are shown. The regions between two neighbor nucleotides are named as A through E and the genetic distances between them are shown. (B) Linkage analysis of nucleotides at six positions by PASS. The polonies in the same PASS gel were probed by six sequential SBEs to identify recombinants. Each image represents the results from one SBE. The sequencing primers were named according to the base positions and are indicated at the bottom of the image. Each spot represents an amplicon from a single DNA molecule. The bases in 89.6 were detected by SBE with Cy5-labeled nucleotides (red) and the bases in NL4-3 were detected by SBE with Cy3-labeled nucleotides (green). The numbered arrows indicate linkage analysis results from different double-stranded DNA molecules: (1), homoduplex without recombination; (2), heteroduplex without recombination; (3), homoduplex with a recombination breakpoint between nt384 and nt585; (4), heteroduplex with a recombination breakpoint between nt384 and nt585.</p

Recombination frequency during simultaneous amplification of multiple distinct HIV-1 genomes by next generation sequencing.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106658#pone.0106658-Kijak1" target="_blank">[36]</a>.</p

Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

<div><p>Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist <i>in vivo</i>. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 10¹³–10¹⁴ using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.</p></div