The design pipeline

The pipeline is optimized to find the highest coverage with the least number of amplicon pools. In an attempt to design all primer pairs exhaustively, the pipeline creates an exhaustive search by sliding a tiling window one-base at a time across the entire targeted region.

In creating the primers, the pipeline considers:

• A basic filtering

  • Optimal melting temperatures

  • The manufacturability of the resulting primers (the resulting primers should not contain long homopolymers)

  • The nucleotide composition should be between 20%-80% GC

• A biological filtering

  • SNP filtering: no known SNP (dbSNP 138) is allowed in a primer if its Minor Allele Frequency (MAF) is greater than 5%. If MAF < 5%, SNPs are allowed anywhere in the primer.

• An off-target analysis to minimize off-target hybridization

• An overlap analysis to reduce the number of primers (minimal tiling path), because a higher overlap implies a higher cost