In order to estimate the error rate, train the statistical model and measure the performance of the assay, we prepared calibration samples by pooling four Coriell DNA samples (NA12156, NA12878, NA18507, and NA19240). These Coriell samples have previously been subjected to exome sequencing and thus the positions of coding polymorphisms are known [15]. We pooled these samples four times, permuting the relative concentration of the samples (1%, 5%, 20% and 74%), to obtain four different calibration samples referred to as CAL-A to CAL-D (Figure S2 in Additional file 1). The cancer hotspot amplicon library was supplemented with 158 calibration amplicons, corresponding to 23.2 kb, to detect and measure the prevalence of the alternative allele at 196 to 201 known polymorphic positions in the four CAL samples (Materials and methods). We sequenced CAL-A, CAL-B and CAL-D calibration samples once and CAL-C in duplicate, obtaining more than 30 million pairs of reads per sample, resulting in approximately 24,000-fold coverage depth after mapping (Tables S3 and S4 in Additional file 2). Consistent with our previous report [13], the coverage distribution is uniform (82%
