BackgroundPCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. High multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently researchers have made some good progress in addressing these shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze.ResultsWe developed a simple protocol, which enables the use of molecular barcodes in high multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we demonstrated the applications in accurate variant calling at very low fraction over a large region and in targeted RNA quantification. We also evaluated the protocol’s utility in profiling FFPE samples.ConclusionsWe demonstrated the successful implementation of molecular barcodes in high multiplex PCR, with multiplex scale many times higher than earlier work. We showed that the new protocol combines the benefits of both high multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1 % mutations with minimal false positives (FP).Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1806-8) contains supplementary material, which is available to authorized users.

For specific detection of somatic variants at very low levels, artifacts from the NGS workflow have to be eliminated. Various approaches using unique molecular identifiers (UMI) to analytically remove NGS artifacts have been described. Among them, Duplex-seq was shown to be highly effective, by leveraging the sequence complementarity of two DNA strands. However, all of the published Duplex-seq implementations so far required pair-end sequencing and in the case of combining duplex sequencing with target enrichment, lengthy hybridization enrichment was required. We developed a simple protocol, which enabled the retrieval of duplex UMI in multiplex PCR based enrichment and sequencing. Using this protocol and reference materials, we demonstrated the accurate detection of known SNVs at 0.1–0.2% allele fractions, aided by duplex UMI. We also observed that low level base substitution artifacts could be introduced when preparing in vitro DNA reference materials, which could limit their utility as a benchmarking tool for variant detection at very low levels. Our new targeted sequencing method offers the benefit of using duplex UMI to remove NGS artifacts in a much more simplified workflow than existing targeted duplex sequencing methods.

Background PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. Highly multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently, people have made good progress at addressing those shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze at one time. Results We described a simple protocol, which enables the use of molecular barcodes in highly multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we studied how molecular barcodes can increase the accuracy of variant calling at very low allelic frequency and reduce PCR amplification bias. We also evaluated its utility in profiling FFPE samples. Conclusions We demonstrated the successful implementation of molecular barcodes in highly multiplex PCR, at a scale many folds beyond earlier work. We showed that the new protocol combined the benefits of both highly multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1% mutation with minimal false positives. Citation Format: Quan Peng, Ravi Vijaya Satya, Marcus Lewis, Pranay Randad, John DiCarlo, Yexun Wang. Reducing amplification artifacts in highly multiplex amplicon sequencing by using molecular barcodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2015-4879