MtGenome for the cost of a gene: Long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

Abstract Background: Mitochondrial genome sequences have become critical to the study of biodiversity, though the cost of doing so with genome skimming or other methods has been difficult to reduce beyond ~$15–20 per sample making it costly to scale up. Here, we report on an approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION as a proof-of-concept. We implemented several cost-saving measures to reach a per-sample cost as low as $7 per mtGenome on the PacBio platform. Results: With the PacBio data we recovered at least one of the two fragments for 96% of samples (~80% of input amplicons) with mean coverage ~1,500x. The ONT data recovered less than 50% of input fragments due to the use of PacBio Barcoded Universal Primers (BUPs) and low throughput, though we are confident results will improve with ONT-optimized BUPs. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, significant improvement in tree support with longer alignments. Conclusions: This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.