title: Reducing amplification artifacts in high 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. 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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 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).
authors.affiliation: Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland, USA. quan.peng@qiagen.com.
authors.forename: Quan
authors.initials: Q
authors.lastname: Peng
authors.affiliation: Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland, USA. ravi.vijayasatya@gmail.com.
authors.forename: Ravi
authors.initials: R
authors.lastname: Vijaya Satya
authors.affiliation: Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland, USA. marcus.lewis@qiagen.com.
authors.forename: Marcus
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland, USA. pranay.randad@qiagen.com.
authors.forename: Pranay
authors.initials: P
authors.lastname: Randad
authors.affiliation: Life Science Research and Foundation, QIAGEN Sciences, Inc., Frederick, Maryland, USA. yexun.wang@qiagen.com.
authors.forename: Yexun
authors.initials: Y
authors.lastname: Wang
mesh_terms: D016477:Artifacts; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics; D015203:Reproducibility of Results; D017421:Sequence Analysis / Q000379:methods
chemical_list: D017931:DNA Primers; D012313:RNA