Abstract
BackgroundAlthough 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.ResultsTo 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.ConclusionPrimerXL 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.
Highlights
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
Features such as single nucleotide polymorphisms (SNP) and secondary structures, known to have a negative effect on amplification efficiency [12,13,14] are masked in the DNA sequences based on data of the Ensembl variation database [10] and results of a UNAfold analysis [15], respectively
When running the same genes using the latest version of primerXL (v1.0), target coverage increased to 95.7%
Summary
PrimerXL has been extensively used for targeted re-sequencing. One of the first projects entailed the Roche 454 sequencing of 15 genes involved in hereditary deafness (Project 1 using primerXL v0.8 (see Table 1) – 83 Kb) and generated 625 amplicons targeting 91.76% of the coding sequences of all corresponding transcript variants [19]. Whereas 91.76% target coverage was achieved by manually relaxing the design settings, implementation of automatic relaxation increased this percentage to 96.1% Another approach was used in a third project sequencing 23 Leber congenital amaurosis (LCA) and Retinitis Pigmentosa (RP) genes (Project 3 using primerXL v0.9–132 Kb) on the Illumina GAIIx system [22]. Since dynamic optimization was not implemented yet in version 0.9, two primer design rounds with different amplicon sizes (400 bp and 600 bp) were performed from which amplicons were selected to optimally cover all exonic sequences of the corresponding RefSeq transcripts Both design approaches gave a 96% target coverage, the 600 bp designs showed a higher intronic near-target percentage as expected (35% versus 24% for the 400 bp designs). PrimerXL can be accessed at [9]
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