Primers for each candidate SNP were designed as nested pairs using Primer3 (refs. 54 (link),55 (link)). External amplicons were fixed at 400–800 bp, and internal amplicons were fixed at 250–300 bp. Internal primer pairs had partial Illumina adaptor sequences added to allow the construction of barcoded Illumina libraries. Tail sequences are: left adaptor 5′-ACACTCTTTCCCTACAC GACGCTCTTCCGATCT-3′, right adaptor 5′-TCGGCATTCCTGCTGAACC GCTCTTCCGATCT-3′. PCR was performed using hot start Taq DNA polymer-ase according to the manufacturer’s instructions (Thermo Scientific). PCR1 (external primers) used a touchdown PCR approach for increased specificity. The products of PCR1 were diluted 100× and used as a template for PCR2, which added the partial Illumina adaptor tails. Candidate SNP PCRs for the same tumor were then pooled, and each tumor was barcoded with a different full-length Illumina adaptor barcode in a short PCR3 (15 cycles), so that each tumor could be decoded after sequencing. Resulting tumor-specific PCRs were pooled, cleaned up with 0.8× AmpureXP beads (Beckman Coulter), quantified and run on an Illumina MiSeq 150-bp PE run.