WES vs ONT DNM allele frequency comparison and parent-of-origin
In total, 77 DNMs of 109 DNMs (71%) were phased and the parent-of-origin determined, with 64 of these (83%) being of paternal origin. Across the 77 phased DNMs there was a DNM allele frequency range of 6% to 78% in the ONT data and 15% to 71 % in the WES DNM base data, clearly showing the presence of postzygotic and prezygotic mutations. From these 77 DNMs, our targeted long-read sequencing approach identified 61 clearly prezygotic DNMs, with a possible third allelic form found in the remaining 16 DNMs. The separation of pre and postzygotic DNMs can be established more clearly through a combined analysis of their base frequencies, allele frequencies, DNM wt allele frequency, and background sequencing error (see Supplementary Table 11). By doing this we confirmed 69 prezygotic DNMs and 8 postzygotic mutations, as depicted in Supplementary Table 11 and 12, and Supplementary Figure 4 and 5. On average a third of allelic data is identified as false in our ONT approach (Supplementary Table 7), and that impacted the allele frequency significantly. After removal of these false allelic data, we noticed that the average ONT allele frequency for prezygotic DNMs were around 50% with an average of 49.6%, with a standard error margin (SEM) of 0.8% (Supplementary Table 13 and Supplementary Figure 4). For germline DNMs, we expect the mutant allele frequency to be approximately 50%. shows that the majority of all ONT DNM allele frequencies were around 50% with the prezygotic average of 49.6% (SEM 0.8%). While raw allele frequencies were expectedly much worse than DNM base frequencies, the polished allele frequencies are slightly more accurate compared to the WES DNM and ONT DNM prezygotic nucleotide base frequencies of 48.6% (SEM 1.0%) and 48.8% (SEM 0.8%), respectively (Supplementary Table 13). As expected, postzygotic frequencies of mutant alleles deviated significantly from this, with an average of 15.9% and a standard error margin of 2.4% in ONT allele data, though the frequencies of postzygotic mutations is less consistent in WES data, where the postzygotic frequency average was 28.8% (SEM 4.9%) (Supplementary Table 13).
The parent-of-origin was also investigated within the pre and postzygotic fractions, where we observe the prezygotic paternal preference of 83% and the postzygotic paternal origin of 62% (5 out of 8), see Supplementary Figure 5. Classification of postzygotic DNMs was determined as per Supplementary Table 11 and a detailed IGV illustration is depicted in Figure 3. All postzygotic calls were corroborated by the DNM base frequencies in the short-read and long-read data, and further supported by all identified allele frequencies and the degree of error in target data (see Supplementary Table 7 and 11).