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).