Long Amplicon Mitochondrial Genomes

I developed a new method to sequence hundreds of mitochondrial genomes in parallel using long amplicon sequencing on both PacBio and Oxford Nanoproe platforms.

 
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MtGenomes help us to understand and study biodiversity.

Mtgenomes have ample phylogenetic information and can be used to build phylogenetic trees and also to study bioenergetics, adaptation, gene rearrangements, and mito-nuclear incompatabilities.

 

A. The mtGenome is amplified in two slightly overlapping fragments that completely encircle the mtGenome.


B. Overview of lab workflow:

  1. first-round PCR with tailed primers targeting primer binding sites and resulting in universal adapters attached to PCR fragments

  2. second-round PCR targeting the universal adapters and resulting in indexed fragments

  3. equimolar pooling of asymmetrically indexed fragments (note that Fragment 1 and 2 of the same sample received an identical index pair)

  4. separate library preparation protocols for PacBio and Oxford Nanopore sequencing.

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Lab Results

Both Oxford Nanopore (ONT) and PacBio producded good results. We got really 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.

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Phyogenetic Information content

As expected, increasingly larger mitochondrial alignments produce better-supported gene trees! Most of the gains was from one gene to a single 9kb fragment.

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Interested in Trying?

This work is currently in review. Please contact me for a draft of the manuscript and lab protocol.