Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR
Manderstedt, Eric LU ; Nilsson, Rosanna ; Ljung, Rolf LU
; Lind-Halldén, Christina
LU
; Astermark, Jan
LU
and Halldén, Christer
LU
(2020)
In Research and practice in thrombosis and haemostasis
4(7).
p.1121-1130
- 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... (More)
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.
(Less)
- author
- Manderstedt, Eric
LU
; Nilsson, Rosanna
; Ljung, Rolf
LU
; Lind-Halldén, Christina
LU
; Astermark, Jan
LU
and Halldén, Christer
LU
- organization
- publishing date
- 2020-10
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Research and practice in thrombosis and haemostasis
- volume
- 4
- issue
- 7
- pages
- 10 pages
- publisher
- Wiley
- external identifiers
-
- scopus:85106541243
- pmid:33134778
- ISSN
- 2475-0379
- DOI
- 10.1002/rth2.12425
- language
- English
- LU publication?
- yes
- additional info
- © 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis.
- id
- 801ab3cc-cf5f-488c-ac65-ef05a85a881d
- date added to LUP
- 2020-11-08 12:19:03
- date last changed
- 2024-04-03 17:16:31
@article{801ab3cc-cf5f-488c-ac65-ef05a85a881d,
abstract = {{<p>Background: The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods.</p><p>Objectives: To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA.</p><p>Methods: Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients.</p><p>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.</p><p>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.</p>}},
author = {{Manderstedt, Eric and Nilsson, Rosanna and Ljung, Rolf and Lind-Halldén, Christina and Astermark, Jan and Halldén, Christer}},
issn = {{2475-0379}},
language = {{eng}},
number = {{7}},
pages = {{1121--1130}},
publisher = {{Wiley}},
series = {{Research and practice in thrombosis and haemostasis}},
title = {{Detection of mosaics in hemophilia A by deep Ion Torrent sequencing and droplet digital PCR}},
url = {{http://dx.doi.org/10.1002/rth2.12425}},
doi = {{10.1002/rth2.12425}},
volume = {{4}},
year = {{2020}},
}