The PCR technique largely depends on several factors, such as polymerase, number of cycles, probe degradation, template volume, and size of the reaction mixture (Chen, 2014; Kuang et al., 2018). In contrast, virtual sequencing is a fully independent system, mimicking the sequencing strategy and identifying novel features of genomes, namely the satellite repeats, variations, and single-nucleotide polymorphism. Furthermore, our primers were designed to amplify the specific 100, 400 and 800 bp DNA parts, in. In turn, the virtual sequencing allowed targeting the random areas, which enables it to be used for de novo sequencing of random DNA fragments. For the experimental generation of truly novel plasmids in their native host, where the genetic material requires a correct assembly, it might be necessary to enrich and purify the plasmid DNA (Smalla et al., 2015). This can be achieved by closing the sequence gaps between contigs by PCR-amplification and subsequent Sanger sequencing of the PCR-product (Smalla et al., 2015). To provide the consistent experimental results, we used the same conditions for each experiment, including the design of primers with the same melting temperature, reaction time, and amount of reaction mixture. In addition, we used the fixed number of DNA copies (10 ng), contributing to the sequencing accuracy in our experiments.
As an outcome, the numerical and experimental data (Supplementary material 2) corroborated reasonably for the number of DNA copies, which was minimal at 800 nt of sequence length, representing a relationship with average r2 and p-values of 0.92 and 0.012 for all the analyzed genetic elements (Figure 5 [A-C]). This demonstrates that the number of amplicons in the PCR experiments and virtual sequencing correlate with the numbers of cycles of coverage rate consistently. Finally, this novel methodology can be applied as an inexpensive quality control technique for standard sequencing analysis, and as a support for the user with a reduced sequencing budget.