Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes

BMC Genomics
Quan PengYexun Wang

Abstract

PCR 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. We 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. We 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 bot...Continue Reading

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Related Concepts

RNA
Reproducibility of Results
Morphologic Artifacts
Sequence Determinations
Oligonucleotide Primers
DNA Barcode, Taxonomic
High-Throughput Nucleotide Sequencing
Multiplex Ligation-Dependent Probe Amplification
DNA
Polymerase Chain Reaction

Related Feeds

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

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