Fig 1.
The whole workflow of the NGS-PrimerPlex.
Solid arrows denote obligatory steps; dotted arrows denote optional steps that can be applied by the user. The primer design process starts from the list of sequence regions (e.g. regions of a reference genome), however, such region coordinates can be automatically obtained with getGeneRegions.py script. NGS-PrimerPlex tries to design primers for all input regions saving primers constructed to the XLS-file (“draft” file 1). If it is necessary, all primers are verified for the forming secondary structures due to the additional adapter sequences at the 5'-ends of primers. If all target regions are covered, the primers are verified for non-target hybridization and amplification. Primers that do not hybridize to many targets and do not form non-target amplicons are saved to distinct XLS-file (“draft” file 2), others are removed. If all target regions are still covered, the primers are verified for overlapping genome regions with variable positions. Primers that do not overlap variable positions with defined parameters (the frequency of the alternative allele and the distance to the 3'-end) are saved to the new XLS-file (“draft” file 3), others are removed. If all targets are still covered, primers are distributed to the defined number of pools. The diagram was drawn in https://app.diagrams.net.
Fig 2.
The primer design workflow for the foodborne bacterial pathogen detection.
Fig 3.
The NGS-PrimerPlex performance evaluated for the different numbers of BRCA1 and BRCA2 exons on two computers (computer 1: Intel Core i7-8700 3.2GHz, 64 GB RAM, Ubuntu 16.04; computer 2: Intel Core i7-2700K 3.5GHz, 32 GB RAM Ubuntu 18.04).
Horizontal and vertical axes have log2-scale.
Table 1.
Primers for the detection of foodborne bacterial pathogens.
Variable positions in primers from the literature are in bold and underlined. The second variable position for the first S. enterica primer has two alternative alleles (T and A).
Fig 4.
Percent of targets covered by at least 30 reads vs median coverage for samples sequenced for the LRRK2 gene.
Each dot is a distinct DNA sample.