Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR

The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods. To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA. Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients. 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% for 97% of positions with substitutions and 90% of indel positions. The positions with LOD >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. 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.