Fig. 4

VIS-NGS performance at two different number of PCR amplification cycles and using LM-NEO-PCR alone or combined with NEO-capture. The VIS-NGS performance was evaluated for the two different number of PCR amplification cycles used in the protocol and for the three methods applied. (A)–(D) presents the NGS-findings obtained in single pure clone samples after 25 and 30 PCR amplification cycles. (A) and (B) show the overall reads coverage in reads per million bases (RPMs) (A) and number of VIS loci (B) after LM-NEO-PCR-based VIS-NGS analyses of single clone samples comparing 25 and 30 PCR amplification cycles. The protocol with 30 PCR cycles resulted as expected in significantly more RPMs for all loci compared to 25 PCR cycles (p = 0.030) The number of VIS loci after 30 PCR cycles was also higher than after 25 PCR cycles but not significantly different (p = 0.18). (C) and (D) shows the number of VIS loci called at three different thresholds for read depth coverage after 30 PCR cycles (C) and after 25 PCR cycles (D) No significant differences were observed in the number of VIS loci between 30 and 25 PCR cycles called at > 10 RPM (p = 0.29), > 100 RPM (p = 0.25), and > 1000 RPM (p = 0.23). (E) and (F) show the overall reads coverage in RPMs and number of VIS loci in composed mixed and diluted samples with 2 and 4 clones using LM-NEO-PCR alone or combined with NEO-capture before (capture-PCR) or after LM-NEO-PCR (PCR-capture). No significant differences were seen in read coverage (E) nor in number of VIS loci (F) between methods, however, LM-NEO-PCR alone and PCR-capture resulted in more RPMs than the capture-PCR method (E).