Discussion
This study evaluates simple and cost-effective methods for the discovery
of fish mitogenomes for the purpose of building up fish mitochondrial
databases. We combined long fragment sequencing and de novo assembly to
generate unbiased consensus sequences that do not miss gene
duplications, order variations, homopolymeric or tandem repeat regions.
We used two mitochondrial DNA enrichment approaches in combination with
two different Oxford Nanopore sequencing kits: the mitoenrichment
enhances the amount of mtDNA at the extraction and post-extraction steps
and can be applied to all fish while the targeted mitosequencing favors
the sequencing of mtDNA from previously dephosphorylated genomic DNA
with the aid of a CRISPR Cas9 enzyme in combination with five guide RNA
sequences that direct the cuts and can be applied to a majority of fish
species. Indeed, we obtained novel complete mitogenomes, some following
the typical vertebrate mitogenome, some with extremely repetitive
segments in the control region and others with gene rearrangements and
indels. We generated a new reference mitogenome for chinook
(Oncorhynchus tshawytscha ) and complete mitogenome of another two
key species of economical and conservation importance, the Pacific hake
(Merluccius productus ) and eulachon (Thaleichthys
pacificus ). We see the methods tested here as an immediate, speedy,
practical sequencing approach that provides certainty and quality to the
mitogenome contigs.
Myctophids (lanterfishes and blackchins) are a common finding in our
laboratory metabarcoding studies from oceanic samples, being the most
abundant family of fishes in the oceans in terms of biomass. Adding
resolution further than Myctophidae ASV beyond the phylogenetic effort
by Poulsen et al. (2013) is important to resolve species within eDNA
metabarcoding work. Myctophiformes is a very diverse family with 250
species described with very divergent larval forms that is far from
complete in the molecular databases (Poulsen et al., 2013, also see Fig
S3.2). Moreover, very few specimens remain identifiable in survey net
hauls because their scales and photophores pattern are often damaged.
Getting more voucher specimens sequenced is thus important and de novo
assembly of this group of fishes is key because the gene rearrangement
made mapping to a close relative mitogenome insufficient, resulting in
shorter genomes with missing genes (data not shown). The complete
mitogenomes of the blue lanternfish (Tarletonbeania crenularis ),
Northern lampfish (Stenobrachius leucopsarus ), and California
headlighfish (Diaphus theta ) were generated for the first time in
this study. Prior myctophid mitogenomes (Poulsen et al., 2013) were
almost complete but were missing the last ca. 2000 bp from the tRNA-Thr,
t-RNA-Pro and the control region, due to the difficulty of sequencing
such highly polymorphic and repetitive region (e.g. 44 G and
>30 repetitions of 18bp on S. leucopsarus ).
Figure 4. Sequential order of the genes found in the six species
studied. The tRNA are noted with their single-letter aminoacid code and
there are either they are placed on above or below their line to denote
in which strand they are transcribed.