New PCRpanel software automates design of ultra-multiplex sequencing panels

Designing a polymerase chain reaction assay that reliably amplifies one stretch of DNA is a solved problem in most molecular biology laboratories. Designing hundreds of primer pairs that can all work together in a single reaction, without binding each other, misfiring into the wrong regions of the genome, or falling silent in repetitive DNA, has long remained a laborious, largely manual craft. A team of researchers from the University of Helsinki and Kazakhstan’s National Laboratory Astana and National Center for Biotechnology has now moved that craft decisively toward automation, introducing PCRpanel, a software tool that generates thermodynamically optimised, ultra-multiplex PCR panels for targeted amplicon sequencing in minutes rather than weeks.

The tool, described in the journal BMC Bioinformatics, is a command-line Java application with a companion web interface that jointly optimises the many interacting variables that determine whether a primer panel succeeds or fails. It balances primer melting temperatures and thermodynamic stability, evaluates linguistic sequence complexity to reject primers drawn from low-complexity or repetitive sequence, models primer-dimer interactions across the entire panel, and applies multiplex-aware, reference-guided specificity screening and repeat masking to prevent off-target amplification. The developers report that the software can produce ultra-high-plex panels containing hundreds of amplicons within seconds to minutes on standard hardware, with a worst-case runtime of about fifteen minutes when the full human reference genome must be screened for specificity.

The scope of the software is deliberately broad. PCRpanel supports workflows from a simple two-primer assay up to designs with hundreds of amplicons, and its authors say it accommodates targets ranging from complete viral genomes and eukaryotic gene families to structural-variant breakpoints and environmental metagenomes. Both gene-specific and universal designs are supported across homologous gene families, a capability that matters when the targets are evolutionarily related genes with highly similar exons. The application requires only Java SE 26 or higher, carries no external dependencies, and is distributed under the GNU General Public License, making it freely available to academic and commercial users without registration or licensing fees.

To demonstrate the tool in a demanding real-world setting, the team turned its attention to Alport syndrome, a hereditary kidney disease most commonly caused by pathogenic variants in the collagen IV genes COL4A3, COL4A4 and COL4A5, with the adjacent COL4A6 also included in the design. These genes present a formidable challenge for primer design: they are large, GC-rich in places, riddled with repetitive elements, and members of a homologous family in which a primer intended for one gene can easily amplify a paralogous sequence from another. Using PCRpanel, the researchers designed 237 primer pairs, comprising 474 individual primers, tiled across all exonic regions of the four genes.

Experimental validation on DNA from four clinical Alport syndrome samples confirmed successful amplification for all 237 primer pairs, a striking result for a fully computational design in a gene family notorious for primer cross-reactivity. Because the exercise was structured as a pilot run aimed at initial validation of the design workflow, sequencing was performed on one representative patient sample. Each primer pool was amplified separately and sequenced at two template dilutions, generating fourteen libraries on the Illumina MiSeq platform; one pool was additionally run as a 50-primer sub-pool during protocol optimisation. Reads were aligned to the GRCh38 human reference genome using the DRAGEN Bio-IT Platform, and coverage was quantified over a 45,372-base-pair exon-restricted target interval derived from the GENCODE v49 annotation. Duplicate marking was deliberately disabled, since amplicon molecules generated by a common primer pair are positionally indistinguishable from PCR duplicates.

The analytical performance reported for that representative sample was strong. Read alignment rates ranged from 94.2 to 99.3 percent, between 77.8 and 96.1 percent of aligned reads fell within the exonic target, and mean target depth per library reached 149.1 to 273.7 times. Critically, the fraction of target bases receiving coverage far above the thresholds needed for confident genotype calling was essentially complete: between 97.9 and 100 percent of target bases reached at least 20-fold depth, and all of those also reached at least 50-fold depth. Measured against the genomic footprint each primer pool was designed to cover, the coverage breadth of each library corresponded to roughly 60 to 130 percent, and the authors note that because each library carried only a single pool, coverage normalised to the complete four-gene target reflects the pooling design rather than amplification performance.

The results also offered an instructive lesson in how parameter choices shape outcomes in repetitive genomic terrain. Under the amplicon-size window and genome-wide specificity screen used for the deposited panel, in silico exonic coverage reached 65.6 percent for COL4A3, 41.4 percent for COL4A4 and 64.4 percent for COL4A6, but only 30.8 percent for COL4A5. A systematic sweep of 1,688 design runs, however, raised coverage to 92.8 percent of the exonic target in a single configuration with repeat masking enforced in full and the genome-wide specificity screen omitted, and placed an amplicon over all 53 exons of the gene across the sweep. The authors argue that this shortfall therefore reflects the specific parameters used for the deposited panel together with the stringency of the specificity screen, not an inherent limit of the method. They provide a step-by-step gap-filling procedure that restricts redesign to uncovered intervals and merges rescue primers into the panel under dimer control, allowing regions that initially fail to be recovered at a stated and explicitly accepted off-target risk.

PCRpanel was benchmarked in silico against two existing tools, NGS-PrimerPlex and Olivar, positioning it within a growing ecosystem of amplicon design software but distinguishing itself by the joint optimisation of sequence complexity, thermodynamic stability, dimer interactions and off-target risk in a single computational pass. That integrated approach is what allows the tool to remain robust even in repetitive or homologous regions, the environments where conventional pipelines most often surrender. The practical consequence is a substantial compression of the design cycle: what previously required iterative manual refinement by an experienced practitioner can now be generated on a desktop computer while the user waits.

The clinical implications of the work extend well beyond Alport syndrome. Targeted amplicon sequencing is a mainstay of molecular diagnostics because it concentrates sequencing capacity on the regions that matter, keeping costs low and interpretation straightforward, but building custom panels has been a bottleneck for laboratories facing rare diseases, local pathogen surveillance, or rapidly evolving viral genomes. The study’s authors emphasise that PCRpanel offers a scalable, platform-agnostic solution for ultra-multiplex PCR panel construction, and the software’s design range, from complete viral genomes to metagenomes, suggests applications in infectious disease genomics, biodiversity monitoring through environmental DNA, and routine diagnostic resequencing alike. Because the tool supports both short-read and long-read sequencing platforms, including Oxford Nanopore Technologies instruments, laboratories are not locked into a single sequencing ecosystem when adopting a panel design.

For the Alport syndrome community specifically, the demonstration that a single computational design can amplify every exon across three established disease genes and a fourth homolog, verified experimentally in patient DNA with sequencing depth far exceeding the requirements for variant calling, offers a template for genetic testing programs in settings where commercial capture kits are financially out of reach. The study was approved by the Ethics Committee of the National Center for Biotechnology in Astana and conducted under the Declaration of Helsinki with written informed consent from all participants. Funded by the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan and supported by open access funding from the University of Helsinki, the work reflects a growing effort to move advanced genomic tooling out of well-resourced centers and into broader clinical and research practice. With PCRpanel freely available on GitHub, the barrier to building a custom, several-hundred-amplicon sequencing panel has shifted from weeks of specialist labor to a single afternoon of computation, and the resulting panels, the authors show, hold up under the unforgiving scrutiny of real patient genomes.

Subject of Research: Automated design software for ultra-multiplex PCR primer panels used in targeted amplicon sequencing

Article Title: PCRpanel: automated design of ultra-multiplex PCR panels for targeted amplicon sequencing

Article References: Kalendar, R., Daniyarov, A., Serikzhan, A., Kairov, U., Basharova, D., Toleuzhanova, A., Bekbayeva, A., Romanova, A., & Zholdybayeva, E. (2026). PCRpanel: automated design of ultra-multiplex PCR panels for targeted amplicon sequencing. BMC Bioinformatics. https://doi.org/10.1186/s12859-026-06648-0

Image Credits: AI Generated

DOI: 10.1186/s12859-026-06648-0

Keywords: PCRpanel, primer design software, ultra-multiplex PCR, targeted amplicon sequencing, next-generation sequencing, Alport syndrome, COL4A3, COL4A4, COL4A5, sequence complexity, off-target prediction, bioinformatics tool