Annotation Of Mitochondrial Genomes Research Articles

Codon usage bias analysis in the mitochondrial genomes of five Rhingia Scopoli (Diptera, Syrphidae, Eristalinae) species

This study presents the sequencing and annotation of mitochondrial genomes from five Rhingia species of the family Syrphidae, focusing on codon bias. Each species possessed 22 tRNAs genes, 13 protein-coding genes, 2 rRNAs genes, and a control region, without any observed gene rearrangements. Nucleotide composition analysis revealed a higher AT content compared with GC content, indicating AT enrichment. Neutrality plot, Parity rule 2 bias, and effective number of codons plot analyses collectively indicated that natural selection primarily influences the codon usage bias in the five Rhingia species. Relative synonymous codon usage analysis identified the optimal codons for Rhingia binotata, R. fromosana, R. campestris, R. louguanensis, and R. xanthopoda as 10, 14, 10, 11, and 12, respectively, all ending with A/U and exhibiting AT preference. Phylogenetic analysis, based on maximum likelihood and Bayesian inference methods applied to three datasets, confirmed the monophyly of Rhingia. In conclusion, this research establishes a foundation for understanding the phylogenetic evolution and codon usage patterns in Rhingia, offering valuable for future studies.

Fully automated annotation of mitochondrial genomes using a cluster-based approach with de Bruijn graphs.

A wide range of scientific fields, such as forensics, anthropology, medicine, and molecular evolution, benefits from the analysis of mitogenomic data. With the development of new sequencing technologies, the amount of mitochondrial sequence data to be analyzed has increased exponentially over the last few years. The accurate annotation of mitochondrial DNA is a prerequisite for any mitogenomic comparative analysis. To sustain with the growth of the available mitochondrial sequence data, highly efficient automatic computational methods are, hence, needed. Automatic annotation methods are typically based on databases that contain information about already annotated (and often pre-curated) mitogenomes of different species. However, the existing approaches have several shortcomings: 1) they do not scale well with the size of the database; 2) they do not allow for a fast (and easy) update of the database; and 3) they can only be applied to a relatively small taxonomic subset of all species. Here, we present a novel approach that does not have any of these aforementioned shortcomings, (1), (2), and (3). The reference database of mitogenomes is represented as a richly annotated de Bruijn graph. To generate gene predictions for a new user-supplied mitogenome, the method utilizes a clustering routine that uses the mapping information of the provided sequence to this graph. The method is implemented in a software package called DeGeCI (De Bruijn graph Gene Cluster Identification). For a large set of mitogenomes, for which expert-curated annotations are available, DeGeCI generates gene predictions of high conformity. In a comparative evaluation with MITOS2, a state-of-the-art annotation tool for mitochondrial genomes, DeGeCI shows better database scalability while still matching MITOS2 in terms of result quality and providing a fully automated means to update the underlying database. Moreover, unlike MITOS2, DeGeCI can be run in parallel on several processors to make use of modern multi-processor systems.

Mitochondrial genome annotation with MFannot: a critical analysis of gene identification and gene model prediction.

Compared to nuclear genomes, mitochondrial genomes (mitogenomes) are small and usually code for only a few dozen genes. Still, identifying genes and their structure can be challenging and time-consuming. Even automated tools for mitochondrial genome annotation often require manual analysis and curation by skilled experts. The most difficult steps are (i) the structural modelling of intron-containing genes; (ii) the identification and delineation of Group I and II introns; and (iii) the identification of moderately conserved, non-coding RNA (ncRNA) genes specifying 5S rRNAs, tmRNAs and RNase P RNAs. Additional challenges arise through genetic code evolution which can redefine the translational identity of both start and stop codons, thus obscuring protein-coding genes. Further, RNA editing can render gene identification difficult, if not impossible, without additional RNA sequence data. Current automated mito- and plastid-genome annotators are limited as they are typically tailored to specific eukaryotic groups. The MFannot annotator we developed is unique in its applicability to a broad taxonomic scope, its accuracy in gene model inference, and its capabilities in intron identification and classification. The pipeline leverages curated profile Hidden Markov Models (HMMs), covariance (CMs) and ERPIN models to better capture evolutionarily conserved signatures in the primary sequence (HMMs and CMs) as well as secondary structure (CMs and ERPIN). Here we formally describe MFannot, which has been available as a web-accessible service (https://megasun.bch.umontreal.ca/apps/mfannot/) to the research community for nearly 16 years. Further, we report its performance on particularly intron-rich mitogenomes and describe ongoing and future developments.

Development and Validation of a High-Throughput Sequencing Test for Mitogenome and rDNA Assembly and Annotation, and Its Use in Support of Nematode Identification of Regulatory Concern

Nematoda is a diverse phylum, and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant-parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 18S, 28S, and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and we defined and validated a standardized test protocol including controls for routine diagnostics (i.e., high-throughput sequencing [HTS] test). The HTS test can be performed on single nematode specimens and outperforms the Sanger-based sequencing by producing less ambiguous consensus sequences and by yielding additional sequence data offering additional diagnostic resolution when needed. Compared with Sanger sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose of the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Genome survey sequencing of the long-legged spiny lobster Panulirus longipes (A. Milne-Edwards, 1868) (Decapoda: Achelata: Palinuridae): improved mitochondrial genome annotation, nuclear repetitive elements classification, and SSR marker discovery

Abstract The long-legged spiny lobster Panulirus longipes (A. Milne-Edwards, 1868) inhabits shallow water rock and coral reefs in the Indo-West Pacific region and is currently targeted by local fisheries along most of its range of distribution. This study conducted, for the first time, a genome survey in P. longipes. Using low coverage short Illumina 150 bp pair-end reads sequencing, we assembled, annotated, and manually curated the mitochondrial genome, described nuclear repetitive elements, and discovered microsatellites (SRR). The pipeline NOVOPlasty assembled the complete mitochondrial genome of P. longipes (coverage = 551×). The mitochondrial genome of P. longipes is 15,739 bp in length and comprised of 13 protein coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. A single 804 bp long intergenic space is assumed to be the control region. The secondary structures of the tRNA genes exhibit a standard “cloverleaf” structure, with the exception of trnS1 that is missing the DHU arm. A large portion of repetitive sequences (~56%) was detected in the nuclear genome of P. longipes and many repetitive elements could not be assigned to known families. Taking into account only annotated repetitive elements, the most common belong to Class I-Long Interspersed Nuclear Element (LINE) and Satellite DNA which are considerably more abundant than Class I-Penelope and Class I-LTR Ty3-gypsy elements. Three clusters were classified as 45S rDNA. A total of 18 SSRs were identified. These newly developed genomic resources will contribute to clarifying the putative existence of cryptic species or subspecies, developing conservation plans, and managing the fishery of the long-legged spiny lobster Panulirus longipes.

Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes.

With the rapid increase of sequenced metazoan mitochondrial genomes, a detailed manual annotation is becoming more and more infeasible. While it is easy to identify the approximate location of protein-coding genes within mitogenomes, the peculiar processing of mitochondrial transcripts, however, makes the determination of precise gene boundaries a surprisingly difficult problem. We have analyzed the properties of annotated start and stop codon positions in detail, and use the inferred patterns to devise a new method for predicting gene boundaries in de novo annotations. Our method benefits from empirically observed prevalances of start/stop codons and gene lengths, and considers the dependence of these features on variations of genetic codes. Albeit not being perfect, our new approach yields a drastic improvement in the accuracy of gene boundaries and upgrades the mitochondrial genome annotation server MITOS to an even more sophisticated tool for fully automatic annotation of metazoan mitochondrial genomes.

Mitochondrial genome annotation and phylogenetic placement of Oreochromis andersonii and O. macrochir among the cichlids of southern Africa.

Genetic characterization of southern African cichlids has not received much attention. Here, we describe the mitogenome sequences and phylogenetic positioning of Oreochromis andersonii and O. macrochir among the African cichlids. The complete mitochondrial DNA sequences were determined for O. andersonii and O. macrochir, two important aquaculture and fisheries species endemic to southern Africa. The complete mitogenome sequence lengths were 16642 bp and 16644 bp for O. andersonii and O. macrochir respectively. The general structural organization follows that of other teleost species with 13 protein–coding genes, 2 rRNAs, 22 tRNAs and a non-coding control region. Phylogenetic placement of the two species among other African cichlids was performed using Maximum Likelihood (ML) and Bayesian Markov-Chain-Monte-Carlo (MCMC). The consensus trees confirmed the relative positions of the two cichlid species with O. andersonii being very closely related to O. mossambicus and O. macrochir showing a close relation to both species. Among the 13 mitochondrial DNA protein coding genes ND6 may have evolved more rapidly and COIII was the most conserved. There are signs that ND6 may have been subjected to positive selection in order for these cichlid lineages to diversify and adapt to new environments. More work is needed to characterize the southern Africa cichlids as they are important species for capture fisheries, aquaculture development and understanding biogeographic history of African cichlids. Bio-conservation of some endangered cichlids is also essential due to the threat by invasive species.

Fungal Genome Annotation.

The term "genome annotation" includes identification of protein-coding and noncoding sequences (e.g., repeats, rDNA, and ncRNA) in genome assemblies and attaching functional information (metadata) to these annotated features. Here, we describe the basic outline of fungal nuclear and mitochondrial genome annotation as performed at the US Department of Energy Joint Genome Institute (JGI).

The diversity of mtDNA rns introns among strains of Ophiostoma piliferum, Ophiostoma pluriannulatum and related species.

BackgroundBased on previous studies, it was suspected that the mitochondrial rns gene within the Ophiostomatales is rich in introns. This study focused on a collection of strains representing Ophiostoma piliferum, Ophiostoma pluriannulatum and related species that cause blue-stain; these fungi colonize the sapwood of trees and impart a dark stain. This reduces the value of the lumber. The goal was to examine the mtDNA rns intron landscape for these important blue stain fungi in order to facilitate future annotation of mitochondrial genomes (mtDNA) and to potentially identify mtDNA introns that can encode homing endonucleases which may have applications in biotechnology.ResultsComparative sequence analysis identified five intron insertion sites among the ophiostomatoid fungi examined. Positions mS379 and mS952 harbor group II introns, the mS379 intron encodes a reverse transcriptase, and the mS952 intron encodes a potential homing endonuclease. Positions mS569, mS1224, and mS1247 have group I introns inserted and these encode intact or eroded homing endonuclease open reading frames (ORF). Phylogenetic analysis of the intron ORFs showed that they can be found in the same insertion site in closely and distantly related species.ConclusionsBased on the molecular markers examined (rDNA internal transcribed spacers and rns introns), strains representing O. pilifera, O. pluriannulatum and Ophiostoma novae-zelandiae could not be resolved. Phylogenetic studies suggest that introns are gained and lost and that horizontal transfer could explain the presence of related intron in distantly related fungi. With regard to the mS379 group II intron, this study shows that mitochondrial group II introns and their reverse transcriptases may also follow the life cycle previously proposed for group I introns and their homing endonucleases. This consists of intron invasion, decay of intron ORF, loss of intron, and possible reinvasion.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3076-6) contains supplementary material, which is available to authorized users.

Complete mitochondrial genome annotation of the giant intestinal fluke, Fasciolopsis buski (Indian isolate) as revealed by ion torrent and illuminanext-generation sequencing

The complete mitogenome sequences of the intestinal fluke Fasciolopsis buski are presented for the first time in this study. It is 14,119 bp long and is thus the shortest trematode mitochondrial genome sequenced to date. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The overall base composition of F. buski mitogenome is 17.89% for A, 9.16% for C, 27.59% for G and 45.36% for T, and has a GC content of 36.75%. The assembled mitogenome (GenBank accession number KX449331) consists of 12 protein-coding genes (PCGs), 22 transfer RNAs and two ribosomal RNA genes. The mtDNA for the intestinal fluke reported herein would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data.

The complete mitochondrial genome of the Sara Longwing Heliconius sara (Insecta: Lepidoptera: Nymphalidae)

The complete mitochondrial genome of the Sara Longwing Heliconius sara has been reconstructed from the whole-genome Illumina sequencing data. The mitochondrial genome is 15,372 bp in size with the highly asymmetric overall A + T content of 80.6%. Annotation of mitochondrial genome revealed a total of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and 1 D-loop region. Most PCGs are initiated with the ATN codons, while COX1 and ND1 start with CGA and TTG, respectively. COX1, COX2, and ND4 harbor the incomplete stop codon T, while all the others are terminated with the TAR codons. The complete mitochondrial genome of H. Sara would contribute to our further understanding of the phylogeny and evolution of the genus Heliconius and related taxa.

Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.

Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.

Community annotation and bioinformatics workforce development in concert--Little Skate Genome Annotation Workshops and Jamborees

Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome.

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.

Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae).

We report the assembly of the 14,054 bp near complete sequencing of the mitochondrial genome of the legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae), which we subsequently used to estimate divergence and relationships within the lepidopteran lineage. The arrangement and orientation of the 13 protein-coding, 2 rRNA, and 19 tRNA genes sequenced was typical of insect mitochondrial DNA sequences described to date. The sequence contained a high A+T content of 80.1% and a bias for the use of codons with A or T nucleotides in the 3rd position. Transcript mapping with midgut and salivary gland ESTs for mitochondrial genome annotation showed that translation from protein-coding genes initiates and terminates at standard mitochondrial codons, except for the coxI gene, which may start from an arginine CGA codon. The genomic copy of coxII terminates at a T nucleotide, and a proposed polyadenylation mechanism for completion of the TAA stop codon was confirmed by comparisons to EST data. EST contig data further showed that mature M. vitrata mitochondrial transcripts are monocistronic, except for bicistronic transcripts for overlapping genes nd4/nd4L and nd6/cytb, and a tricistronic transcript for atp8/atp6/coxIII. This processing of polycistronic mitochondrial transcripts adheres to the tRNA punctuated cleavage mechanism, whereby mature transcripts are cleaved only at intervening tRNA gene sequences. In contrast, the tricistronic atp8/atp6/coxIII in Drosophila is present as separate atp8/atp6 and coxIII transcripts despite the lack of an intervening tRNA. Our results indicate that mitochondrial processing mechanisms vary between arthropod species, and that it is crucial to use transcriptional information to obtain full annotation of mitochondrial genomes.

An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes

Mitochondrial (mt) genomics represents an understudied but important field of molecular biology. Increasingly, mt dysfunction is being linked to a range of human diseases, including neurodegenerative disorders, diabetes and impairment of childhood development. In addition, mt genomes provide important markers for systematic, evolutionary and population genetic studies. Some technological limitations have prevented the expanded generation and utilization of mt genomic data for some groups of organisms. These obstacles most acutely impede, but are not limited to, studies requiring the determination of complete mt genomic data from minute amounts of material (e.g. biopsy samples or microscopic organisms). Furthermore, post-sequencing bioinformatic annotation and analyses of mt genomes are time consuming and inefficient. Herein, we describe a high-throughput sequencing and bioinformatic pipeline for mt genomics, which will have implications for the annotation and analysis of other organellar (e.g. plastid or apicoplast genomes) and virus genomes as well as long, contiguous regions in nuclear genomes. We utilize this pipeline to sequence and annotate the complete mt genomes of 12 species of parasitic nematode (order Strongylida) simultaneously, each from an individual organism. These mt genomic data provide a rich source of markers for studies of the systematics and population genetics of a group of socioeconomically important pathogens of humans and other animals.