I'm interested in doing de-novo sequencing but also phylogenetic analysis. In particular, after de-novo sequencing and annotating the genome, I need to align the CO1 gene and the nuclear 28S rRNA gene of several species. When extracting DNA and sequencing with PacBio for example, how does the assembler know what corresponds to nuclear and what to mitochondrial DNA?
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4$\begingroup$ Does it need to? I mean, and I stress that I have never done de-novo sequencing so I may be completely wrong here, but won't the assembler simply try to assemble the longest contiguous contigs it can? And, if so, won't that naturally result in the mitochondrial sequences getting assembled together in one set of contigs and the non-mito sequences in others? I would expect this to be even easier with long read technology like PacBio. Finally, if you're only interested in a couple of genes, can't you directly target the highly conserved parts of them to amplify and sequence? $\endgroup$– terdon ♦Sep 4, 2022 at 10:06
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$\begingroup$ Cannot comment on CO1, but rrRNA should be different enough between the mitochondria and the nucleus as to not be confused. For one, mitochondria (like bacteria) should have 23S subunits instead of 28S. Many programs (e.g. barrnap, whatever is used by Prokka) automatically distinguish them. $\endgroup$– LauraJun 2 at 12:06
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1$\begingroup$ Could you kindly review the answer provided? Please do consider upvoting or accepting an answer. $\endgroup$– M__ ♦Sep 8 at 8:59
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According to this pdf, from pacb.com
isolation with mitoISO MITOISO
The kit will include three steps: 1) mechanical rupturing of tissues or cells 2) removing cellular debris and nuclei by low speed centrifugation and 3) harvesting mitochondria by high speed centrifugation.
Then, you can follow the DNA extraction with mitochondrial DNA, and then sequenced on the PacBio Sequel.
If you are doing whole genome sequencing, the mitogenome will have a higher read depth according to this article stating that lower read depth of the nuclear genome separating the two different origins:
those of mitochondrial origin & those of nuclear origin
