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High-throughput PCR assay design for targeted resequencing using primerXL

Steve Lefever (UGent) , Filip Pattyn, Bram De Wilde (UGent) , Frauke Coppieters (UGent) , Sarah De Keulenaer (UGent) , JAN HELLEMANS (UGent) and Jo Vandesompele (UGent)
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Abstract
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.
Keywords
Primer design, Targeted resequencing, Variant confirmation next generation sequencing, Sanger sequencing, PCR assay, QUANTITATIVE PCR, ENRICHMENT

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Chicago
Lefever, Steve, Filip Pattyn, Bram De Wilde, Frauke Coppieters, Sarah De Keulenaer, JAN HELLEMANS, and Jo Vandesompele. 2017. “High-throughput PCR Assay Design for Targeted Resequencing Using primerXL.” Bmc Bioinformatics 18.
APA
Lefever, Steve, Pattyn, F., De Wilde, B., Coppieters, F., De Keulenaer, S., HELLEMANS, J., & Vandesompele, J. (2017). High-throughput PCR assay design for targeted resequencing using primerXL. BMC BIOINFORMATICS, 18.
Vancouver
1.
Lefever S, Pattyn F, De Wilde B, Coppieters F, De Keulenaer S, HELLEMANS J, et al. High-throughput PCR assay design for targeted resequencing using primerXL. BMC BIOINFORMATICS. 2017;18.
MLA
Lefever, Steve, Filip Pattyn, Bram De Wilde, et al. “High-throughput PCR Assay Design for Targeted Resequencing Using primerXL.” BMC BIOINFORMATICS 18 (2017): n. pag. Print.
@article{8562983,
  abstract     = {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.},
  articleno    = {400},
  author       = {Lefever, Steve and Pattyn, Filip and De Wilde, Bram and Coppieters, Frauke and De Keulenaer, Sarah and HELLEMANS, JAN and Vandesompele, Jo},
  issn         = {1471-2105},
  journal      = {BMC BIOINFORMATICS},
  keyword      = {Primer design,Targeted resequencing,Variant confirmation next generation sequencing,Sanger sequencing,PCR assay,QUANTITATIVE PCR,ENRICHMENT},
  language     = {eng},
  pages        = {9},
  title        = {High-throughput PCR assay design for targeted resequencing using primerXL},
  url          = {http://dx.doi.org/10.1186/s12859-017-1809-3},
  volume       = {18},
  year         = {2017},
}

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