Abstract 4879: Reducing amplification artifacts in highly multiplex amplicon sequencing by using molecular barcodes

Abstract

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

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