Chloroplot: An Online Program for the Versatile Plotting of Organelle Genomes

About 2000 completely sequenced mitochondrial genomes are available from the NCBI RefSeq data base together with manually curated annotations of their protein-coding genes, rRNAs, and tRNAs. This annotation information, which has accumulated over two decades, has been obtained with a diverse set of computational tools and annotation strategies. Despite all efforts of manual curation it is still plagued by misassignments of reading directions, erroneous gene names, and missing as well as false positive annotations in particular for the RNA genes. Taken together, this causes substantial problems for fully automatic pipelines that aim to use these data comprehensively for studies of animal phylogenetics and the molecular evolution of mitogenomes. The MITOS pipeline is designed to compute a consistent de novo annotation of the mitogenomic sequences. We show that the results of MITOS match RefSeq and MitoZoa in terms of annotation coverage and quality. At the same time we avoid biases, inconsistencies of nomenclature, and typos originating from manual curation strategies. The MITOS pipeline is accessible online at http://mitos.bioinf.uni-leipzig.de. Copyright © 2012 Elsevier Inc. All rights reserved.

Abstract We have developed the web application GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) for the rapid and accurate annotation of organellar genome sequences, in particular chloroplast genomes. In contrast to existing tools, GeSeq combines batch processing with a fully customizable reference sequence selection of organellar genome records from NCBI and/or references uploaded by the user. For the annotation of chloroplast genomes, the application additionally provides an integrated database of manually curated reference sequences. GeSeq identifies genes or other feature-encoding regions by BLAT-based homology searches and additionally, by profile HMM searches for protein and rRNA coding genes and two de novo predictors for tRNA genes. These unique features enable the user to conveniently compare the annotations of different state-of-the-art methods, thus supporting high-quality annotations. The main output of GeSeq is a GenBank file that usually requires only little curation and is instantly visualized by OGDRAW. GeSeq also offers a variety of optional additional outputs that facilitate downstream analyzes, for example comparative genomic or phylogenetic studies.

We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data—mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.