The 5′ and 3′ flanking regions for each SNP were obtained from dbSNP. A 32-nucleotide region which contained either the variant or reference nucleotide flanked by nine nucleotides on the 3′ end and 22 nucleotides on the 5′ end was selected as the test sequence. Two-hundred such regions (100 reference and 100 variant) were selected to test 100 SNPs. Universal primer binding regions were added on the 5′ (GTAATTCTAGGAGCTC) and 3′ (CGTTCTAGAGTCGGG) end of each test region. The final test fragment was 63 nucleotides in length (see Supplementary Figure 2). The 200 test fragments were commercially synthesized as pooled single-stranded DNA oligonucleotides (Oligomix®, LC Sciences, Houston, TX, United States). The pool contained 10–50 attomoles of each sequence. The oligonucleotides were synthesized as single-stranded DNA and was diluted 1:5. One μL of the diluted Oligomix® was amplified in a 50 μL PCR reaction using 0.3 μM universal primers and 25 μL 2X CloneAmpTM HiFi PCR premix (Takara, Mountain View, CA, United States). PCR conditions used were: 98°C (10 s), 53°C (5 s), and 72°C (5 s) for 35 cycles.
