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Abstract Background The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods. Objectives To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA. Methods Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients. Results An error‐rate map over all coding positions and all positions reported as mutated in the F8‐specific mutation database was produced. The sequencing produced a mean read depth of >1500X where >97% of positions were covered by >100 reads. Higher error frequencies were observed in positions with A or T as reference allele and in positions surrounded on both sides with C or G. Seventeen of 9319 positions had a mean substitution error frequency >1%. The ability to identify low‐level mosaicism was determined primarily by read depth and error rate of each specific position. Limit of detection (LOD) was 1% require repeated testing and mononucleotide repeats with more than four repeat units need an alternative analysis strategy. Mosaicism was detected in 1 of 16 mothers and confirmed using ddPCR. Conclusions Deep sequencing using an AmpliSeq/Ion Torrent strategy allows for simultaneous identification of disease‐causing mutations in patients and mosaicism in mothers. ddPCR has high sensitivity but is hampered by the need for mutation‐specific design.

Mutations are not identified in ~5% of hemophilia A and 10-35% of type 1 VWD patients. The bleeding tendency also varies among patients carrying the same causative mutation, potentially indicating variants in additional genes modifying the phenotype that cannot be identified by routine single-gene analysis. The F8, F9 and VWF genes were analyzed in parallel using an AmpliSeq strategy and Ion Torrent sequencing. Targeting all exonic positions showed an average read depth of >2000X and coverage close to 100% in 24 male patients with known disease-causing mutations. Discrimination between reference alleles and alternative/indel alleles was adequate at a 25% frequency threshold. In F8, F9 and VWF there was an absolute majority of all reference alleles at allele frequencies >95% and the average alternative allele and indel frequencies never reached above 10% and 15%, respectively. In VWF, 4-5 regions showed lower reference allele frequencies; in two regions covered by the pseudogene close to the 25% cut-off for reference alleles. All known mutations, including indels, gross deletions and substitutions, were identified. Additional VWF variants were identified in three hemophilia patients. The presence of additional mutations in 2 out of 16 (12%) randomly selected hemophilia patients indicates a potential mutational contribution that may affect the disease phenotype and counseling in these patients. Parallel identification of disease-causing mutations in all three genes not only confirms the deficiency, but differentiates phenotypic overlaps and allows for correct genetic counseling.