AMPrimer: An R package for anchored multiplex PCR primer design

Abstract:
Next-generation sequencing (NGS) has revolutionized life science for its ability to massively parallel sequencing. The quality of sequencing highly depends on the quality of the library constructed using PCR. A novel PCR has been developed to tackle some problems in the analysis of NGS termed anchored multiplex PCR (AMP), such as PCR bias. AMP has been shown as a robust and powerful method to enrich targets. Primer design plays an important role in library construction. We implemented an R package, AMPrimer, a panel for designing primers used in AMP. AMPrimer functions as an interface communicating R and Primer3, an opensource primer design software. The input file of AMPrimer contains the location of targets needed to be amplified. Target sequences are classified into two categories according to their length. Longer sequences are split into shorter fragments due to the sequencer’s ability. Boulder-IO file fed into Primer3 is also generated by R. Primer3 would be used twice. The first run of Primer3 is to generate outer primers while the second run of Primer3 is to generate inner primers. Primer pairs are evaluated in terms of directionality, nested primer location, and secondary structure.