Andrea Legati from The Foundation of the Carlo BEsta Neurological Institute, IRCCS, Italy, spoke at the Nanopore Community Meeting 2021 on “Mitochondrial DNA analysis by long-read NGS in patients affected by mitochondrial diseases.” Legati spoke about the role of mitochondria in ATP production and metabolites and the significance of mitochondrial DNA (mtDNA) deletions. There are limitations with short-read sequencing in the identification of mtDNA rearrangements. Legati and team extracted DNA from muscle biopsies of mitochondrial disease patients previously diagnosed as carriers of mtDNA large deletions. DNA was extracted by phenol chloroform use, and library preparation used the rapid sequencing kit and the ligation sequencing kit. Libraries were sequenced on R9.4.1 flow cells. For data analysis, mtDNA was aligned using minimap2 and ngmlr. The Bam files generated were analyzed using the Sniffles tool. Legati and team presented results from the use of the rapid sequencing kit for mtDNA analysis. The average length for mtDNA was 4.4 kb! Reads were analyzed including those suggesting mtDNA rearrangement. For this, they validated with PCR primers. Results were visualized with Circos to identify rearrangements. They also sequenced four mtDNA samples with the ligation kit. Similar values were obtained as those from short-read sequencing and PCR. The last slide of results Legati presented was a comparison of the rapid and ligation kit outputs. The results were similar. Legati concluded that their experimental plan suggests both kits produced high mtDNA coverage. They then compared the pros and cons of each kit. Eack kit/workflow has advantages and challenges because of the way the DNA is prepared for sequencing. The comparison of the rapid and ligation sequencing kits was thought-provoking, since we use both kits often, though we don’t sequence mtDNA.
