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

Date Published: July 12, 2017

Publisher: Public Library of Science

Author(s): Desiree Schenk, Gang Song, Yue Ke, Zhaohui Wang, Arnar Palsson.


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.

Partial Text

Target enrichment methods in next generation sequencing can be categorized into two main classes: hybrid capture enrichment and amplicon-based enrichment[1–3]. Hybrid capture based methods such as SureSelect (Agilent Technologies) and SeqCap (Roche Nimblegen) are highly scalable and have advantages for large gene panels and whole exome sequencing[4,5]. However, hybrid capture methods typically require high DNA input amounts, a complicated and lengthy library preparation process, and high cost.

We have developed SLIMamp PCR, a novel method for multiplex PCR, that enables amplification of overlapping amplicons in a single-tube format. SLIMamp inhibits amplification of overlapping amplicons by forming a stem that competes with primer annealing for extension. A portion of the forward primer sequence, (F2^) is added to the reverse primer, or vice versa (reverse sequence on the forward primer) that results in the overlapping amplicon forming a stem-loop structure. The stem reduces the availability of the sequence needed for primer annealing and with two-step cycling conditions during PCR, the amplification of the overlapping amplicon is inhibited.




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