Abstract
PCR-based target enrichment is widely used to prepare libraries for next generation sequencing (NGS). Amplicon tiling is often used for assay design but can result in amplicon overlap that reduces assay sensitivity. Pillar Biosciences™ SLIMamp® enrichment technology enables efficient amplicon tiling even with overlapping amplicons. Here, we present an automated primer design tool (ampPD) for amplicon tiling and evaluate the performance of an assay designed against the TP53 tumor suppressor gene.
The ampPD workflow includes target preparation, candidate primer generation, primer selection and pooling. All 11 TP53 coding exons were targeted for amplification and performance of the assay design was evaluated by synthesizing and pooling the ampPD output primers to prepare SLIMamp libraries from 10ng, 5ng and 1ng input DNA from FFPE tumor samples. Resulting libraries were normalized, pooled and sequenced on an Illumina MiSeq.
Automated TP53 primer design generated an initial pool of 627 compatible primers that was reduced to an optimized pool of 19 amplicons, of which 16 amplicons were overlapping. The total design took roughly 10 seconds to complete. The assay displayed high coverage uniformity, with 100% of targeted based covered >0.2x mean coverage for all samples. For the 5ng and 10ng samples, variant detection was highly reproducible and 100% concordant with known outcome and a median 86% on-target rate. For the 1ng samples, all positive variants were detected but with increased background noise and a lower median on-target rate.
The ampPD tool is a rapid and robust primer design pipeline for amplicon tiling assays. Using TP53 as a model, the resulting primers provided uniform coverage and sensitive variant detection at FFPE-derived DNA inputs >1ng. Future work will extend the design pipeline to larger and more complex panels, enabling rapid creation of single-tube targeted sequencing assays for a variety of applications.