primerXL

BACKGROUND: Although the sequencing landscape is rapidly evolving and sequencing
costs are continuously decreasing, whole genome sequencing is still too expensive
for use on a routine basis. Targeted resequencing of only the regions of interest
decreases both costs and the complexity of the downstream data-analysis. Various
target enrichment strategies are available, but none of them obtain the degree of
coverage uniformity, flexibility and specificity of PCR-based enrichment. On the
other hand, the biggest limitation of target enrichment by PCR is the need to
design large numbers of partially overlapping assays to cover the target.
RESULTS: To overcome the aforementioned hurdles, we have developed primerXL, a
state-of-the-art PCR primer design pipeline for targeted resequencing. It uses an
optimized design criteria relaxation cascade and a thorough downstream in silico
evaluation process to generate high quality singleplex PCR assays, reducing the
need for amplicon normalization, and outperforming other target enrichment
strategies and similar primer design tools when considering assay quality,
coverage uniformity and target coverage. Results of four different sequencing
projects with 2348 amplicons in total covering 470 kb are presented. PrimerXL can
be accessed at www.primerxl.org . CONCLUSION: PrimerXL is an state-of-the-art,
easy to use primer design webtool capable of generating high-quality targeted
resequencing assays. The workflow is fully customizable to suit every
researchers' needs, while an innovative relaxation cascade ensures maximal target
coverage.

High-throughput PCR assay design for targeted resequencing using primerXL.

primerXL is a web-based tool for designing sequence primers for targeted re-sequencing.

primerXL

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