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Long-Molecule Sequencing: A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and β-Thalassemia Carriers.
- Source :
-
The Journal of molecular diagnostics : JMD [J Mol Diagn] 2020 Aug; Vol. 22 (8), pp. 1087-1095. Date of Electronic Publication: 2020 May 29. - Publication Year :
- 2020
-
Abstract
- Multiple molecular tests are currently needed for accurate carrier testing for thalassemia. Therefore, long-molecule sequencing (LMS) was evaluated as an alternate on the PacBio Sequel platform for genotyping carriers of α-thalassemia or β-thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and β (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using FreeBayes software version 1.2.0. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common South East Asian, -α3.7, and -α4.2 deletions and four different rare single-nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the most common Chinese insertion and deletion variant c.126&#95;129delCTTT and 14 different SNVs/insertions and deletions and could discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate, and cost-effective genotyping method. With further test coverage to additionally include detection of other clinically significant HBA1/2 copy number variations, such as the Thai, Mediterranean, and Filipino deletions, LMS may eventually serve as a comprehensive method for large-scale thalassemia carrier screening.<br /> (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Asian People genetics
Case-Control Studies
Cost-Benefit Analysis
Data Accuracy
Genetic Carrier Screening economics
Genetic Loci
Genotype
Genotyping Techniques economics
Humans
INDEL Mutation
Multiplex Polymerase Chain Reaction economics
Whole Genome Sequencing economics
alpha-Thalassemia blood
alpha-Thalassemia ethnology
beta-Thalassemia blood
beta-Thalassemia ethnology
DNA Copy Number Variations
Genetic Carrier Screening methods
Genotyping Techniques methods
Multiplex Polymerase Chain Reaction methods
Polymorphism, Single Nucleotide
Whole Genome Sequencing methods
alpha-Thalassemia genetics
beta-Thalassemia genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1943-7811
- Volume :
- 22
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- The Journal of molecular diagnostics : JMD
- Publication Type :
- Academic Journal
- Accession number :
- 32473995
- Full Text :
- https://doi.org/10.1016/j.jmoldx.2020.05.004