Fig. 5

Detection of reported and new VIS loci in single pure clones and by VIS-NGS LM-NEO-PCR alone. Detection of reported and new VIS loci in single pure clones after LM-NEO-PCR. The number and read depth coverage of the previously reported VIS loci in the single pure clones are presented in (A) and (B) for both 25 and 30 PCR amplification cycles. (A) The number of loci detected per clone at four thresholds for read depth coverage. It shows that > 1 and > 10 RPM calling (grey bars) result in more loci in each clone than reported (red bar), irrespective 25 or 30 PCR cycles, but also in off-target calling in negative controls (parental). The thresholds > 100 and > 1000 RPM (blue bars) result in numbers of VIS loci called (most) like the number reported. (B) Read depth coverage per detected locus after 25 and 30 PCR cycles. It shows two separate groups of loci, one group with low read depth coverage (< 100 RPM) and the other group of loci with high read depth coverage (> 1000 RPM). (C) Presents the read depth coverage for each reported and new VIS locus, showing that reported VIS loci were correctly called at > 1000 RPM, while a-specific calling occurs around 100 RPM in other clones than reported. Moreover, it shows that as expected 30 PCR cycles (squares) resulted generally in higher read depth coverage than 25 PCR cycles (circles). All novel VIS loci were found based on the > 1000 RPM calling threshold. Only MSL1 was detected in clone VIII-20, but also a-specifically at lower read depth coverage in clone VIII-24. (D) Illustrates the PCR design with the universal neomycin primer (neoP) which can act as forward and reverse primer depending on the VIS-locus orientation. Forward and reverse locus-specific primers (LSPs) were developed but only one LSP together with neoP resulted in PCR amplification products. The locus-specific PCR and gel electrophoresis analyses were performed for one reported locus (BNIP3L) and six novel VIS loci identified by VIS-NGS in clones VIII-18 (DPM3, MIR1908, KB1930G5.4, SNORA63, GLB1L) and VIII-20 (MSL1) (E). PCR amplicons were only obtained when the neomycin primer and locus-specific primer had an opposite orientation. The expected > 1000 bp PCR amplicons were seen for all except GLB1L in only the correct clones (indicated by red arrows). No amplicons were observed in the negative template control (NTC) nor in parental without VIS (PAR), nor in clones lacking the VIS locus.