MitoHooker: A PCR-free enrichment strategy using RNA baits for targeted detection of mitochondrial base modifications on nanopore sequencing devices

Date

2021

Authors

Hall, Courtney
Reid, Danielle
Phillips, Nicole
Planz, John

ORCID

0000-0002-6115-1027 (Hall, Courtney)

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Abstract

Purpose: Aberrant methylation and increased oxidative damage throughout the mitochondrial genome (mtDNA) have been implicated in numerous diseases ranging from cancer to neurodegeneration. Current understanding, however, is obscured by the inherent limitations of traditional detection techniques. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of mtDNA are present within a sample, these sequences represent a small fraction of total genetic material competing for pore access. Therefore, this project aimed to evaluate RNA baits hybridization capture for enrichment of mtDNA prior to nanopore sequencing. Methods: Heavy and light mtDNA strands in cell-free plasma extracts were individually captured using the Arbor Biosciences myBaits Expert Mito kit. Elutant from the first capture served as input for rebaiting with the opposite probe set. Following complement synthesis by Klenow fragment, double-stranded products were multiplexed and sequenced on the MinION device. Resultant basecalled reads were mapped to the human reference genome to assess on- and off-target coverage. Base modifications in the raw data were detected using a combination of available bioinformatics tools and in-house algorithms. Results: Although overall input and throughput were significantly lower than a typical whole genome sequencing run, read count and coverage data indicate that this technique allowed mtDNA to outcompete background DNA while maintaining modified bases within the native strands. Conclusion: The workflow developed herein could provide novel insights into the complete collection of mtDNA base modifications and enable identification of disease-relevant alterations in this landscape.

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