Mitochondrial genomes help us to understand and study biodiversity
Mitogenomes have ample phylogenetic information that can be used to build robust phylogenetic trees. They are also used for human medicine, and for research on bioenergetics, adaptation, gene rearrangements, and mito-nuclear incompatabilities.
Workflow
Hundreds of mitogenomes are amplified in two slightly overlapping fragments that completely encircle the structure.
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First-round PCR with tailed primers targeting primer binding sites and resulting in universal adapters attached to PCR fragments
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Second-round PCR targeting the universal adapters and resulting in indexed fragments
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Equimolar pooling of asymmetrically indexed fragments (note that Fragment 1 and 2 of the same sample received an identical index pair)
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Separate library preparation protocols for PacBio and Oxford Nanopore sequencing.
Results
Both Oxford Nanopore (ONT) and PacBio were successful. We recovered exceptionally high coverage with PacBio, indicating that we could easily scale this up to thousands of samples. Reads were mostly in the size of the desired amplicons.
Phylogenetic Information
As expected, increasingly larger mitochondrial alignments produce better-supported gene trees! Most of the gains were from one gene to a single 9kb fragment, with only limited gains from there to the full mtGenome (15 kb).
