Ravi Sachidanandam: Exploring mitochondrial genetics with nanopore long-read sequencing

Ravi Sachidanandam, from Girihlet inc. spoke about mitochondrial genetics and how overcoming heteroplasmy, that being having multiple types of a cellular organelle within an individual cell, is important for accurately identifying haplotypes. Furthermore, Ravi spoke of how targeted sequencing of mitochondria has significant clinical applications in terms of identifying and interpreting low abundant pathogenic variants.

Starting his talk, Ravi gave some background on how mtDNA is passed down on the maternal side and how heteroplasmy patterns can change during this transmission. Next, he described how heteroplasmy occurs in disease systems, whereby a continuous gradient of disease severity is related to the uneven distribution of mitochondria in daughter cells.

Moving on, Ravi said that some of the main confounding effects of trying to measure heteroplasmy in mitochondria is that mtDNA is typically of low abundance and copy number will vary between cells and individuals. All the common methods used to analyse mtDNA suffer from significant contamination from genomic material and that to avoid this, complex organelle purifications must be used. Ravi spoke about a method he and his team were developing called Mseek for target enrichment of small circular genomes from cellular organelles. Similar to enzymatic plasmid purification methods, nucleases were used to degrade linear and nicked mtDNA leaving only fully circularised mtDNA.

As the mitochondrial genome is only 16kb long, Ravi said that you don’t need much sequence data to get great coverage. Moving on, Ravi spoke about how Heteroplasmy seems to be consistently passed for a parent cell to a daughter cell when in pure culture but and he suggested that because of this, heteroplasmy could potentially be used for identification methods.

Moving on to discuss some experimental results from the oxford nanopore platform, the purified mtDNA was cut using a single restriction endonuclease and used as a template for long range PCR to amplify prior to library prep and sequencing. Ravi noted that the PCR did not cause chimeric molecules, but more variants were detected in the nanopore data compared with short read data and some of the frequencies were different. In terms of on target percentages, across the replicate samples 19.6 % to 78 % of reads aligned to the mitochondrial genome with most samples giving over 50 % of reads aligned. Examining the variants called in both short and long read sequencing data generated from the same samples, there was good concordance between the two platforms but the long read nanopore data picked up a more. Examining the technical variability between a number of different sample Ravi said it was remarkably consistent and to convince himself of this he needed to use an extra barcoded, non-mitochondrial sample as a control to convince himself there was no significant barcode misclassification.