Abstract 2283: PCR-based validation of novel cancer-predisposing mutations identified upon exome sequencing: An emphasis on primer design

Abstract

Abstract Whole exome sequencing (WES) is a robust instrument for the identification of novel genes for hereditary diseases. WES experiments usually produce hundreds or thousands of candidates, which require validation in primary DNA samples and subsequent case-control studies. The analysis of each candidate includes individual primer design for allele-specific polymerase chain reaction (PCR) assay or high-resolution melting (HRM) coupled with Sanger sequencing. For the time being, the selection of primers for PCR remains a time-consuming procedure. None of the available software instruments allows to generate a pool of primers for the entire coding region of the gene, therefore each coding fragment has to be selected and analyzed manually. Furthermore, the existing tools have limited capability to ensure that primers will not amplify a non-target fragment of the genome. The consideration of single nucleotide polymorphisms located within primer sequence is usually done without adjustment for their frequency. We developed a pipeline, which facilitates the process of large-scale primer design in an automatic mode. We named the tool PrimerWay and made it available at http://github.com/zoldrax/primer-way under the GNU General Public License. The developed pipeline is provided as a standalone python-based script which requires installed Primer3, tntBLAST and HTSlib. We created automatic upload of the sequence of interest with flanking regions using protein_id or genome coordinates from the reference genome. We utilized the ThermonucleotideBLAST (tntBLAST) instrument in order to ensure PCR specificity. While other computer tools focus mainly on the heuristic definition of sequence similarity, this software runs in-silico PCR based on the thermodynamic similarity and minimizes the risk of the amplification form a non-target template. Our pipeline detects polymorphic sites within the potential primer (5'-end or 3'-end) using a common subset of the dbSNP public database. If the SNP is rare and located at the 5'-end of the primer, it is highly unlikely to compromise further experiments. PrimerWay is capable to suggest the design for the analysis of long DNA sequences. It breaks the sequence for short consecutive fragments and generates a pool of candidate primers for each fragment. It suggests, which of the chosen fragments and primer pairs are most reliable for analysis, by considering the risks of forming dimers, recognizing the non-target sequences, or being compromised by SNPs; these risks are measured by the comprehensive penalty scores. Subsequently, it considers several variants of breaking the entire sequence for overlapping DNA fragments. The utility of PrimerWay tool was validated in a WES study for hereditary breast cancer, which utilized, inter alia, 23 candidate mutations and entire coding regions of two candidate genes. The research is supported by RSF grant 16-45-02011. Citation Format: Ekaterina Sh Kuligina, Anna P. Sokolenko, Ilya V. Bizin. PCR-based validation of novel cancer-predisposing mutations identified upon exome sequencing: An emphasis on primer design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2283.