Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Area of Science:

• Genomics
• Molecular Biology
• Neurology

Background:

• Mitochondrial genome (mtDNA) variants are linked to neurological disorders like MELAS.
• The 16 kb mtDNA is suitable for continuous sequencing via long-read sequencing (LRS).
• Current mtDNA sequencing methods can be complex and costly.

Purpose of the Study:

• To demonstrate a simple, accessible method for comprehensive mtDNA sequencing using Flongle.
• To evaluate Flongle-based sequencing for diagnosing mitochondrial diseases.
• To identify potential mtDNA variants associated with Parkinson's disease (PD).

Main Methods:

• Whole mtDNA amplification (WMA) followed by LRS on Flongle.
• Comparison of Flongle results with Cas9 enrichment methods.
• Assessment of heteroplasmy rate accuracy using equimolar variants.
• Sequencing of mtDNA from PD patients.

Main Results:

• mtDNA variants were detected in all tested mitochondrial disease patients.
• Flongle sequencing results were comparable to Cas9 enrichment.
• Accurate heteroplasmy level detection (r² > 0.99) was achieved with WMA on Flongle.
• A known variant was found in three PD patients.

Conclusions:

• Flongle provides a streamlined and cost-effective approach for mtDNA sequencing.
• This method simplifies the diagnosis of mitochondrial diseases.
• Flongle-based sequencing shows potential for identifying disease-associated mtDNA variants.