1. 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

2. 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

3. 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

4. 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

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

6. 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

8. 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 kb)
MinION

9. 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

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

11. 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

12. 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

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