The characterisation of mtDNA deletions using long-read sequencing STP Research Project The characterisation of mtDNA deletions using long-read sequencing Lizzie Wood Oxford Medical Genetics Laboratories

Introduction Mitochondria mtDNA Organelles found in large copy numbers within cells Inner membrane is the site of the mitochondrial respiratory chain and ATP production mtDNA 16.6kb circular molecule 37 genes 100-10,000 copies per cell

Mitochondrial disorders Biochemical abnormalities of the respiratory chain Caused by pathogenic mutations in mtDNA or nuclear DNA mtDNA mutations can be point mutations or rearrangements mtDNA deletions cause diseases such as Pearson Syndrome, Kearns-Sayre Syndrome, and Progressive External Ophthalmoplegia Schon, E. et al. Nature Rev. Genet. 13, 2012

Project aim Current testing strategy for mtDNA deletions: Project aim: Long-range PCR (13.8kb product) and gel electrophoresis Southern blot for further characterisation Project aim: Sequencing strategy to define breakpoints Compare long-read sequencing to current NGS technology 2-8kb deletion

Oxford Nanopore Technologies Long range PCR 12 patients: 9 single deletions 3 normal controls Oxford Nanopore Technologies MinION Illumina MiSeq

One protein unzips the DNA helix into two strands. 1 A second protein creates a pore in the membrane and holds an ‘adapter’ molecule. 2 A flow of ions through the pore creates a current. Each base blocks the flow to a different degree, altering the current. 3 The adapter molecule keeps bases in place long enough for them to be identified electronically. 4 Eisenstein, M. Nature 550, 2017

Normal patient Good coverage across mitochondrial genome MiSeq Normal patient Good coverage across mitochondrial genome MinION data shows increased error rate (reported as 5-10% in literature) Point mutations identifiable by both techniques MinION data – see single reads covering full amplicon (mean read length 2.5-6.5kb) MinION

Single deletion patient MiSeq Single deletion patient Clear drop in read depth corresponding to deletion MinION data shows long reads spanning entire deletion – indicating exact breakpoint positions MiSeq data shows more gradual decline, possibly due to exclusion of short reads spanning the breakpoints during alignment MinION

Breakpoint analysis Manual read-depth analysis method Breakpoints successfully mapped for all single deletion patients Confirmed by Sanger sequencing

Conclusions Long-range PCR successfully used to amplify the majority of the mitochondrial genome Long-read sequencing using the ONT MinION generated single reads of the full 16.1 kb amplicon Both Illumina NGS and Nanopore long-read sequencing strategies enabled breakpoint mapping for all single deletion patients Sanger sequencing used for breakpoint confirmation Currently, no advantage to using long-read sequencing over traditional NGS technology

Further work Breakpoint mapping Explore automated methods for breakpoint analysis To integrate into existing pipeline Would involve exploring different bioinformatics tools Nanopore sequencing Maximising read length during library preparation Extending the investigation to point mutation analysis

Acknowledgements Oxford Medical Genetics Laboratories Carl Fratter Sirisha Hesketh Philip Hodsdon Oxford Brookes University Ryan Pink