A MinION-based Long-Read Sequencing Application With One-Step Polymerase Chain Reaction (PCR) for the Genetic Diagnosis of 21-Hydroxylase Deficiency
Authors' conclusion
We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.
Study context
- Population
- Patients with 21-hydroxylase deficiency (21OHD)
- Participants
- 55 patients
- Comparison
- Long-read sequencing (LRS) vs conventional MLPA and nested PCR
- Outcome
- Genotype concordance with reference method
Methodological detailsone-step PCR, MinION-based long-read sequencing, barcoding
- One-step PCR
- MinION-based long-read sequencing
- Barcoding
Performance
As reported in the abstract
- LRS using Flongle Flow Cell R9.4.1 yielded results consistent with MLPA and nested PCR reference genotypes
- Duplex analysis with Flongle Flow Cell R10.4.1 accurately sequenced a variant in a homopolymer region
- With barcoding, cost comparable to conventional analysis
Further findings
Novel variants
- Novel single-nucleotide variation at intron 7 acceptor site
- c.940-1G > C
- Subtype of classical chimeric junction CH2, "CH2a", from latter part of intron 5 to exon 6
Original publication
A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency. J Clin Endocrinol Metab. Published 6 October 2023. doi:10.
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