Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes |
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Authors: | Quan Peng Ravi Vijaya Satya Marcus Lewis Pranay Randad Yexun Wang |
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Affiliation: | Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland USA |
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Abstract: | 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. |
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