Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2

PLoS One. 2017 Jul 12;12(7):e0181062. doi: 10.1371/journal.pone.0181062. eCollection 2017.

Abstract

Current PCR-based target enrichment methods for next generation sequencing (NGS) of overlapping amplicons often requires separate PCR reactions and subsequent pooling of amplicons from the different reactions. The study presents a novel method, deemed stem-loop inhibition mediated amplification (SLIMamp), for amplifying overlapping or tiled amplicons in a single multiplex PCR reaction. During a SLIMamp PCR reaction, a stem loop structure formed by the overlapping amplicon suppresses additional amplification of itself by preventing the annealing of the primers. Using the SLIMamp strategy, we designed a next-generation sequencing (NGS) assay to enrich the exon regions of BRCA1 and BRCA2 for sequencing on an Illumina MiSeq system. We used 35 cell line DNAs and 6 patient blood DNAs in the study to evaluate the assay performance. For each sample, all targeted regions were successfully amplified and sequenced with excellent coverage uniformity and specificity. >99% of the total sequencing reads were mapped to the human reference genome (hg19) and >99% of the mapped reads were on the targeted exons. >98% of bases were covered at >0.20x of the mean coverage and >99% are covered at >0.15x of the mean depth. Among 34 independently sequenced samples, all variants were reliably detected with no false positives or false negatives. SLIMamp provides a robust method for single-tube multiplex PCR amplification of numerous, overlapping amplicons that tile for targeted next-generation sequencing.

MeSH terms

  • BRCA1 Protein / chemistry
  • BRCA1 Protein / genetics*
  • BRCA2 Protein / chemistry
  • BRCA2 Protein / genetics*
  • Cell Line, Tumor
  • Female
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Nucleotide Sequencing / standards
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Molecular Diagnostic Techniques / standards
  • Multiplex Polymerase Chain Reaction / methods*
  • Multiplex Polymerase Chain Reaction / standards
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, DNA / standards

Substances

  • BRCA1 Protein
  • BRCA1 protein, human
  • BRCA2 Protein
  • BRCA2 protein, human

Grant support

This research was supported by Pillar Biosciences. No individual authors received specific funding for this work. Pillar Biosciences provided support in the form of salaries to DS, GS, YK, and ZW.