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Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR
- Source :
- Research and Practice in Thrombosis and Haemostasis, Research and Practice in Thrombosis and Haemostasis, Vol 4, Iss 7, Pp 1121-1130 (2020)
- Publication Year :
- 2020
-
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 mutationspecific design.
- Subjects :
- Genetics
polymerase chain reaction
Read depth
factor VIII
hemophilia A
high-throughput nucleotide sequencing
mosaicism
Hematology
Ion semiconductor sequencing
Biology
high‐throughput nucleotide sequencing
Deep sequencing
law.invention
Repeated testing
law
Original Articles ‐ Hemostasis
Biomedicinsk laboratorievetenskap/teknologi
Mutation database
In patient
Digital polymerase chain reaction
Diseases of the blood and blood-forming organs
Biomedical Laboratory Science/Technology
Original Article
RC633-647.5
Polymerase chain reaction
Subjects
Details
- ISSN :
- 24750379
- Volume :
- 4
- Issue :
- 7
- Database :
- OpenAIRE
- Journal :
- Research and practice in thrombosis and haemostasis
- Accession number :
- edsair.doi.dedup.....733848ec5476be74d1b599fb4e25d9dd