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Detection of mosaic...
Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR
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- Manderstedt, Eric (författare)
- Kristianstad University,Högskolan Kristianstad,Avdelningen för miljö- och biovetenskap,Forskningsmiljön Biomedicin,Plattformen för molekylär analys
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- Nilsson, Rosanna (författare)
- Kristianstad University,Högskolan Kristianstad,Fakulteten för naturvetenskap
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- Ljung, Rolf (författare)
- Lund University,Lunds universitet,Pediatrik, Lund,Sektion V,Institutionen för kliniska vetenskaper, Lund,Medicinska fakulteten,Pediatrisk hematologi,Forskargrupper vid Lunds universitet,Paediatrics (Lund),Section V,Department of Clinical Sciences, Lund,Faculty of Medicine,Paediatric Haematology Research Unit,Lund University Research Groups,Skåne University Hospital
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- Lind-Halldén, Christina, 1959- (författare)
- Kristianstad University,Högskolan Kristianstad,Forskningsmiljön Biomedicin,Avdelningen för miljö- och biovetenskap
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- Astermark, Jan (författare)
- Skånes universitetssjukhus, Malmö,Skåne University Hospital
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- Halldén, Christer, 1957- (författare)
- Kristianstad University,Högskolan Kristianstad,Forskningsmiljön Biomedicin,Avdelningen för miljö- och biovetenskap
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(creator_code:org_t)
- Wiley, 2020
- 2020
- Engelska.
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Ingår i: Research and practice in thrombosis and haemostasis. - : Wiley. - 2475-0379. ; 4:7, s. 1121-1130
- Relaterad länk:
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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% 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. 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.
Ämnesord
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Biomedical Laboratory Science/Technology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Biomedicinsk laboratorievetenskap/teknologi (hsv//swe)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin -- Hematologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine -- Hematology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine (hsv//eng)
Nyckelord
- factor VIII; hemophilia A; high-throughput nucleotide sequencing; mosaicism; polymerase chain reaction
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)