A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Short-read sequencing methods for mitochondrial DNA (mtDNA) reconstruction face limitations including amplification bias, mapping challenges, and difficulties in phasing variants, capturing multiple deletions, and achieving even genome coverage.
• Existing techniques struggle to accurately represent the full spectrum of mtDNA variation due to inherent biases and technical constraints.

Purpose of the Study:

• To develop and validate a novel method for high-coverage, full-length human mitochondrial genome sequencing using native single molecules.
• To overcome the limitations of short-read sequencing in analyzing mtDNA, particularly in determining variant phasing and disentangling complex structural variations.

Main Methods:

• Utilized RNA-guided DNA endonuclease Cas9 to target and induce breaks at specific sites, defining the start and end of mitochondrial DNA molecules for long-read sequencing.
• Developed a custom bioinformatics pipeline featuring the software 'baldur' for analyzing long-read sequencing data, enabling high-resolution detection of heteroplasmy and structural variants.

Main Results:

• Achieved high demultiplexing specificity and accurate delineation of full-length mitochondrial genomes, irrespective of structural variant complexity.
• The 'baldur' software efficiently detects single nucleotide heteroplasmy down to 1%, physically determines variant phasing, and accurately resolves complex deletions in mitochondrial DNA.

Conclusions:

• The Cas9-based single-molecule sequencing workflow provides a robust tool for comprehensive mitochondrial DNA analysis.
• This method significantly advances the study of mtDNA variation and is poised to accelerate research in mitochondrial genomics and related fields.