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Sequencing of mRNA from Whole Blood using Nanopore Sequencing
Published on: June 3, 2019
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Rh Blood Group D Antigen Genotyping Using a Portable Nanopore-based Sequencing Device: Proof of Principle
Wajnat A Tounsi1,2, Vasileios P Lenis2,3, Silja M Tammi4
1Faculty of Applied Medical Sciences, Department of Medical Laboratory Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.
Clinical Chemistry
|June 2, 2022
Summary
Oxford Nanopore
Area of Science:
- Genetics
- Molecular Biology
Background:
- Nanopore sequencing offers direct, real-time DNA analysis using biological pores.
- The portable MinION device from Oxford Nanopore Technologies utilizes voltage to drive DNA through nanopores, measuring current changes to determine nucleobases.
Purpose of the Study:
- To evaluate the efficacy of nanopore sequencing for Rh blood group D antigen (RHD) gene zygosity testing.
- To determine RHD allele variations using MinION sequencing.
Main Methods:
- Genomic DNA from 13 samples was analyzed for RHD gene zygosity using droplet digital PCR.
- Long-range PCR amplified the RHD gene, followed by library preparation and 1D Nanopore sequencing (R9 flow cells).
- Data underwent basecalling, alignment, and variant calling, with results compared to Ion PGM sequencing.
Main Results:
- High sequence coverage (up to 500×) enabled accurate variant calling in the RHD gene.
- RHD allele determination was successful for most samples, identifying 3 known and 6 novel variant alleles.
- One sample was suspected to be homozygous RHD with two heterozygous RHD variant alleles.
Conclusions:
- MinION sequencing proved effective for blood group genotyping.
- Sufficient sequencing data facilitated high RHD gene coverage, enabling confident variant calling and allele determination.
