Primer Design Pipeline for Large Scale Sanger Validation

When secondary confirmatory testing is required for the verification of next-generation sequencing (NGS) results, Sanger sequencing methodology is utilized at HGSC/HGSC-CL. A crucial step for accurate Sanger validation is the... [ view full abstract ]

When secondary confirmatory testing is required for the verification of next-generation sequencing (NGS) results, Sanger sequencing methodology is utilized at HGSC/HGSC-CL. A crucial step for accurate Sanger validation is the primer design process. Although there are a number of web-based tools available, none of them are suitable for large-scale high throughput clinical settings. Thus, primers are designed for a selected group of variants with the assistance of an in-house automated tool, which stores the prepared primer designs in a database schema system.

We have built a custom pipeline that generates PCR primer sequences for a region of interest. The underlying architecture relies upon major tools such as Primer3 for generating primer sequences, and insilico-Pcr (isPcr) for determining primer specificity. Variant inputs can be in either VCF format or tab-delimited format. We use a lower-case repeat masked and SNP masked reference genome in our pipeline to avoid primer mispriming and allele-specific primer binding. If a single amplicon is obtained while using isPcr, we confirm the primers are on the same chromosome as our target and cover the target accurately. Our pipeline cycles through 6 primer design tiers, which consist of criteria for product size, GC %, Tm etc. and have been tailored based on our lab protocols. The characteristics set by these tiers gradually become less restrictive to allow the design of difficult genomic regions. If due to genome characteristics, primers cannot be designed, the regions are sent to an in-house expert for manual primer design. The pipeline reports the final primer sequences with their relevant attributes and amplicon information.

We have used our pipeline to successfully design primers across various research and clinical projects to validate sites with 90.92% accuracy. The future plan is to expand our pipeline to implement nested PCR for difficult regions of the genome. For variant inputs, a plan is to add support for HGVS notation with reference to coding DNA (cDNA) transcripts.