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

Eric Manderstedt, Rosanna Nilsson, Rolf Ljung, Christina Lind-Halldén, Jan Astermark, Christer Halldén

Research output: Contribution to journalArticlepeer-review


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 mutation-specific design.

Original languageEnglish
Pages (from-to)1121-1130
Number of pages10
JournalResearch and practice in thrombosis and haemostasis
Issue number7
Publication statusPublished - 2020 Oct

Bibliographical note

© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis.

Subject classification (UKÄ)

  • Hematology


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