The mitoenrichment method produced fragments of all sizes, including full mitogenomes, because the transposase cleaves the DNA at random places, while the Cas9 in the targeted mitosequencing approach cleaves the DNA at specific sites, hence producing fragments of expected sizes with all the combinations possible with one to five cuts (Fig S3.1). This can be used as an indication of success while monitoring the sequencing run. There are typically peaks of sequences at ~7.5, 9 or 16kb and the number of sequences passing filter on these peaks can be used as an estimation of the coverage of the final assembly.
The number of sequencing pores is low for these two approaches when compared to Nanopore standards, especially for the targeted mitosequencing method, which can be in the order of 0.3% pores sequencing, while the mitoenrichment can have ca 5% of sequencing pores. This is due to the intense selection of mitochondrial DNA that drastically reduces the number of sequences available for each method, with only very few sequences having sequencing adaptors in the case of the targeted mitosequencing approach. Importantly, these low levels are not an indication of failure, but future improvement of methods should look at optimizing a sequencing run with multiple samples analyzed at once by multiplexing / barcoding samples. When using the flongle flow cell, the targeted mitosequencing run carried took only two hours until functional pores were exhausted; however, the mitoenrichment run lasted for 12 h and produced a mitogenome with 640-fold coverage (Table 2, S2).