Complete vertebrate mitogenomes reveal widespread repeats and gene duplications

Abstract

Background Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. Results As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100–300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. Conclusions Our results indicate that even in the “simple” case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone.

Mitogenomes of Accipitriformes and Cathartiformes Were Subjected to Ancestral and Recent Duplications Followed by Gradual Degeneration

“…The phylogenetic distribution of mitogenomic gene orders and the reconstruction of ancestral states indicate that the common ancestor of Cathartiformes and Accipitriformes had the fully duplicated gene order, in which the segment of genes for cytochrome b, tRNA-Thr, tRNA-Pro, ND6, and tRNA-Glu as well as the control region were tandemly duplicated. This rearrangement is currently known as the most complete avian duplication, which was previously annotated for Bucerotiformes, Caprimulgiformes, Cariamiformes, Charadriiformes, Ciconiformes, Gaviiformes, Gruidae, Musophagiformes, Passeriformes, Pelecaniformes, Podicipediformes, Procellariiformes, Psittaciformes, Sphenisciformes, Strigiformes, and Suliformes ( Abbott et al 2005 ; Gibb et al 2007 , 2013 ; Morris-Pocock et al 2010 ; Sammler et al 2011 ; Zhou et al 2014 ; Lounsberry et al 2015 ; Akiyama et al 2017 ; Rodrigues et al 2017 ; Zhang et al 2017 ; Urantowka et al 2018 , 2020 ; Formenti et al 2021 ). Since representatives of order Strigiformes, which is sister to Accipitrimorphae, also possess the mitogenomic duplications, we can assume that the common ancestor of these two groups contained the duplicated fragments ( fig.…”

“…So far, different rearrangements including the duplicated control region and genes were identified in representatives of most avian orders ( Eberhard and Wright 2016 ; Urantowka et al 2018 , 2020 ; Mackiewicz et al 2019 ). The most fully duplicated gene order, assigned as GO-FD in figure 1 , was found in the mitogenomes of some Bucerotiformes, Caprimulgiformes, Cariamiformes, Charadriiformes, Psittaciformes, and Strigiformes species ( Sammler et al 2011 ; Urantowka et al 2018 ), some Ciconiformes, Gaviiformes, Musophagiformes, Podicipediformes, Sphenisciformes ( Urantowka et al 2020 ), some Passeriformes ( Formenti et al 2021 ), the majority of Pelecaniformes ( Gibb et al 2007 ; Zhou et al 2014 ) and Procellariiformes ( Abbott et al 2005 ; Gibb et al 2007 ; Lounsberry et al 2015 ) as well as all representatives of Gruidae ( Akiyama et al 2017 ) and Suliformes ( Morris-Pocock et al 2010 ; Gibb et al 2013 ; Rodrigues et al 2017 ; Zhang et al 2017 ).…”

The genome sequence of the common toad, Bufo bufo (Linnaeus, 1758)

“…Two rounds of the Illumina polishing were applied. The mitochondrial genome was assembled at The Rockefeller University using the mitoVGP pipeline ( Formenti et al ., 2021 ). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al ., 2016 ; ( Howe et al ., 2021 )).…”

The genome sequence of the European nightjar, Caprimulgus europaeus (Linnaeus, 1758)

“…The Hi-C scaffolded assembly was polished with arrow using the PacBio data, with merfin ( Formenti et al ., 2021b ) applied to avoid a drop in QV, then polished with the 10X Genomics Illumina data by aligning to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012 ) and applying homozygous non-reference edits using bcftools consensus . A complete mitochondrion was not found using mitoVGP ( Formenti et al ., 2021a ), likely due to the sample being sourced from blood tissue, so mitochondrial sequence NC_025773.1 ( Caprimulgus indicus ) was used during polishing. The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al ., 2016 ) as described previously ( Howe et al ., 2021 ).…”