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Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation.
Amy R Vandiver1, Brittany Pielstick2, Timothy Gilpatrick3
1Division of Dermatology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, United States.
Mitochondrion
|July 5, 2022
Summary
This study presents a new Cas9-based sequencing method for mitochondrial DNA (mtDNA). The technique enables full-length reads, improving mutation phasing and deletion detection for better understanding of mtDNA diseases.
Area of Science:
- Genomics
- Molecular Biology
- Mitochondrial Biology
Background:
- Mitochondrial DNA (mtDNA) harbors genetic variations linked to numerous diseases.
- Current sequencing technologies face limitations in accurately phasing mutations and detecting large deletions within the mtDNA.
Purpose of the Study:
- To develop and validate an amplification-free sequencing method for obtaining long nanopore reads of the entire mitochondrial genome.
- To enhance the understanding of mtDNA variation, particularly in disease contexts.
Main Methods:
- Adaptation of Cas9-mediated cleavage for sequence enrichment of mtDNA.
- Utilizing long-read nanopore sequencing technology.
- Application of the method to phase mutations in a patient with an mtDNA-linked syndrome.
Main Results:
- Successful generation of full-length nanopore reads for mtDNA.
- Accurate phasing of mutations in an individual with an mtDNA-related disorder.
- Demonstrated capability to map age-induced large sporadic deletions in mtDNA.
Conclusions:
- The developed Cas9-based, amplification-free method significantly improves the analysis of mtDNA variation.
- This technique offers deeper insights into mtDNA-linked diseases and age-related mtDNA alterations.
