Mitogenome assembly
Sequences that passed the filter of the Guppy5sup base caller were then
assembled de novo using Flye v2.7 (Kolmogorov et al., 2019) using
Geneious [flye –nano-raw input_0_Unpaired.fastq –threads 8
–genome-size 0.016m –min-overlap 1000 –iterations 1 –out-dir
out]. In the instances in which this de novo assembly failed with the
raw data, sequences from 10-18 kbp were extracted and used for de novo
assembly in Flye. The resulting circular mitogenome was then used as the
reference to map the sequences from its own run to polish the contig and
revise the sequencing depth parameters using minimap2 (Li, 2018) within
Geneious. Two iterations of the consensus of the contig were built with
the highest quality of bases matching at least 60 % and removing the
reference genome. This consensus sequence was then annotated using
MitoFish (Iwasaki et al., 2013). All consensuses from the different
tissues, treatments and runs for chinook salmon were aligned using MAFFT
in Geneious and compared. A genome skimming approach was conducted to
the two whole genome sequencing runs (C in Table 3) by mapping the
sequencing data to the mitogenome of a closely related species using
minimap2 (Osmerus mordax dentex (MH370836) in the case ofT. pacificus and Merluccius merluccius in the case ofM. productus ) and the resulting consensus sequence without the
reference genome was then used as the reference for the second mapping
iteration.
Figure 1. Outline of the two enrichment methods from DNA
extraction to de novo assembly of the mitogenomes. A.
Mitoenrichment: Mitochondrial DNA (mtDNA) enrichment by isolating
intact mitochondria followed by DNA extraction. Any residual nuclear DNA
(nDNA) co-purification is depleted based on the ability of the
exonuclease V of cleaving the 5’ and 3’ termini of any linear DNA
(single or double stranded) while leaving circular DNA intact. The
circular DNA is then linearized with a transposase that facilitates the
addition of the sequencing adaptors. B. Targeted mitosequencing :
Targeted Sequencing that combines the CRISPR Cas9 nuclease cutting
ability directed by a so-called guide RNA or crRNA for targeted
scissions of the DNA. The DNA has been previously treated with a
phosphatase enzyme that dephosphorylates the free ends of DNA strands
preventing any sequencing adapters binding the non-target DNA but
allowing the adapters binding to the newly free phosphorylated ends in
the selected regions. Both approaches are followed by sequencing on an
Oxford Nanopore platform. Illustration by Su Kim, NWFSC/NOAA Fisheries.