Table 1:

best practices for assaying mtDNA methylation

All methodologies
Confirm results with multiple techniques e.g. WGBS, MeDIP, mass spectrometry, PacBio, Nanopore	[13, 14, 19]
Enrich mitochondria or mtDNA prior to assaying to avoid unintentional detection of the nuclear genome	[13, 14, 19]
Present evidence of success of enrichment (e.g. qPCR of mtDNA vs nuclear DNA)	[13, 14]
Bisulphite methodologies
Spike sample with control DNA for evaluation of conversion efficiency	[14, 19]
Linearize mtDNA prior to bisulphite conversion to avoid secondary structure effects	[13, 14, 16, 19, 25]
In WGBS separately analyse and present data from light and heavy strands	[13, 14]
Measure CpG and non-CpG methylation	[10, 13, 14]
Differentiate between 5mC and 5hmC	[14, 19, 26]
Use PCR primers or controls to account for non-CpG methylation	No examples of primers at present. Suitable positive and negative controls in [13, 19, 22]