to amplify 200-bp fragments around 159 of these SNPs. Because we perform direct sequencing from the amplicon ends (Figure S1b in Additional file 1), we made sure that the calibration SNPs were located at various positions throughout the 200-bp amplicons. This allowed us to estimate the assay performance for calling variants across the 200-bp amplicons. Of the 159 primer pairs tested, 158 successfully amplified the targeted sequences in the four calibration samples. Other SNPs, not initially selected to serve as calibration polymorphisms, are located in these 158 amplicons. We disregarded heterozygous SNPs present only in one of the four Coriell samples, as they would lead to a prevalence of 0.5% in one of the calibration samples. In total this design resulted in 196, 200, 201 and 201 SNPs with known prevalence in the calibration samples CAL-A, CAL-B, CAL-C and CAL-D, respectively. The UDT-Seq assay interrogates a total of 23.2 kb encompassing these calibration SNPs.
