Multiplex PCR Design for Scalable Resequencing.

While conventional PCR applications typically focus on a single PCR assay per reaction, multiplex PCR applications are a convenient and scalable solution becoming more routine. Multiplex methods can be applied to virtually any DNA template source (e.g., plant or human DNA, FFPE DNA isolated from clinical samples, bisulfite-converted DNA for DNA methylation analysis), and offers a cheap, convenient, and scalable solution for experiments that require characterization and analysis of multiple genomic regions.This method will detail the procedures to successfully design, screen, and prepare multiplex amplicon libraries; as well as supporting instructions on how to prepare these libraries for sequencing on Illumina, Ion Torrent, and Oxford Nanopore platforms. The flexibility of assay design allows means that custom multiplex panels can range in size from two assays up to a few hundred amplicons or more. Notably, the method described here is also amenable to whatever PCR buffer system the user prefers to use, making the system globally adaptable to the needs and preferences of the end user.