Fig. 1

Formation of chimeric products during PCR using two-plasmid template and test PCR for their detection. a A scheme of formation of chimeric DNA molecules. b Amplification of BC–ROI fragments from plasmid templates by two consecutive rounds of PCR. Products of round #2 PCR contain all necessary elements for Illumina NGS: sequences necessary for attachment of amplicons to a flow cell surface (“P5” and “P7”), sequencing primer sites (“seq1” and “seq2”) and a custom 8-bp sample index sequence (“i”). “Target specific forward” and “target specific reverse” denote sequences immediately flanking BC and ROI. Black horizontal arrow indicates a fragment of PCR products analyzed by NGS in this study. c Structures of all possible PCR products for the two-plasmid template system. Red and blue horizontal arrows indicate positions of specific primers used for the test PCR. d Agarose gel electrophoresis analysis of PCR products. Conventional PCR products were generated using either 1 ng of an equimolar mixture of plasmid#1 and plasmid#2 (lane 1) or water (no template control, “NTC”; lane 2) as template and primers Libr-A₁-for/Libr-rev. Test PCR were set up using either ¹/₁₀₀th of the purified products of conventional one-round PCR (lanes 3–6) or water (no template controls, “NTCs”; lanes 7–10) as template and the following primers: BC1/ROI1 (lanes 3 and 7), BC2/ROI2 (lanes 4 and 8), BC1/ROI2 (lanes 5 and 9) and BC2/ROI1 (lanes 6 and 10). M stands for GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific)