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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?

Caterina
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    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? – terdon Sep 04 '22 at 10:06

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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

envs_h_gang_5
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