Evidence for a duplicated mitochondrial region in Audubon’s shearwater based on MinION sequencing

Abstract

Mitochondrial genetic markers have been extensively used to study the phylogenetics and phylogeography of many birds, including seabirds of the order Procellariiformes. Evidence suggests that part of the mitochondrial genome of Procellariiformes, especially albatrosses, is duplicated, but no DNA fragment covering the entire duplication has been sequenced. We sequenced the complete mitochondrial genome of a non-albatross species of Procellariiformes, Puffinus lherminieri (Audubon's shearwater) using the long-read MinION (ONT) technology. Two mitogenomes were assembled from the same individual, differing by 52 SNPs and in length. The shorter was 19 kb long while the longer was 21 kb, due to the presence of two identical copies of nad6, three tRNA, and two dissimilar copies of the control region (CR). Contrary to albatrosses, cob was not duplicated. We further detected a complex repeated region of undetermined length between the CR and 12S. Long-read sequencing suggests heteroplasmy and a novel arrangement within the duplicated region, indicating a complex evolution of the mitogenome in Procellariiformes.

Sea surface temperature, rather than land mass or geographic distance, may drive genetic differentiation in a species complex of highly dispersive seabirds

“…Contamination in the field was also unlikely since new sampling supplies were used for every sample. Given that only 10% of the cytb sequences presented such ambiguities (which may be due to heteroplasmy; Torres et al, 2018), we removed such sequences for further analyses. However, for the CR, since a third of the total sequences were involved, we kept all CR data in further analyses, considering two haplotype phases for MrBayes and *BEAST analyses (although we recognize that these data are in violation of the assumption that sequences to be phased are under Hardy–Weinberg equilibrium; see discussion below).…”

Sea surface temperature, rather than land mass or geographical distance, may drive genetic differentiation in a species complex of highly-dispersive seabirds

“…The mitochondrial genome of several Procellariiformes presents tandem repeats (Abbott et al 2005;Lounsberry et al 2015), including P. lherminieri (Torres et al 2018). In this taxon, a duplicated region comprising two copies of the CR was found, yielding the presence of double peaks on chromatograms.…”

Rapid mitochondrial genome sequencing based on Oxford Nanopore Sequencing and a proxy for vertebrate species identification

“…Additionally, it is important to note that data analyses to generate mitochondrial genomes (via genome skimming) exclusively using ONT data are not entirely a straightforward process. Other studies have shown complete mtDNA assemblies combining ONT datasets and high‐throughput short‐read datasets from Illumina to improve overall consensus quality (Chandler et al, 2017; Torres et al, 2018). For instance, the complete mitogenome assembly of a clinical‐interest nematode, Nippostrogylus brasiliensis , was improved using dual datasets (Chandler et al, 2017).…”

“…The cost–benefit and accessibility of ONT allow obtaining genome‐wide and organellar DNA sequences in situ without the need (and avoid the associated problems) of PCR‐based amplification. Previous efforts have been made to obtain mitochondrial genomes using ONT sequencing data, but to overcome the read noisiness and improve assembly quality, short‐read data from Illumina platform were also included (Chandler et al, 2017; Torres et al, 2018). Herein, we propose that with enough mitochondrial DNA coverage (genome skimming), the mitogenome could be accurately reconstructed using ONT‐only data combining several computational assembly routines to solve known issues and error‐prone sites in the sequence.…”