Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly.
ABSTRACT: Background:Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. Results:We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. Conclusions:We present the first genome of any anemonefish and demonstrate the value of low coverage (?11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.
Project description:Many animal societies have dominance hierarchies in which social rank is correlated with size. In such societies, the growth and size of individuals can be a strategic response to their social environment: in fishes, individuals may decrease their growth rate to remain small and retain a subordinate position; in mammals, individuals may increase their growth rate to become large and attain a dominant position-a strategy called competitive growth. Here, we investigate whether the clown anemonefish, Amphiprion percula, exhibits competitive growth also. We show that juvenile clownfish paired with a size-matched reproductive rival increase their growth rate and size relative to solitary controls. Remarkably, paired individuals achieved this, despite being provided with the same amount of food as solitary controls. Our results demonstrate that clownfish are able to increase their growth rate in response to social competition. This study adds to the growing body of evidence that the growth of social vertebrates can be a fine-tuned plastic response to their social environment.
Project description:Picasso clownfish belong to the subfamily Amphiprioninae and are considered a variant of the genus <i>Amphiprion</i>. In this study, we first sequenced the complete mitochondrial genome of the Picasso clownfish by Illumina next-generation sequencing technology. The length of the whole mitogenome is 16,727?bp long, with a gene arrangement and composition similar to those of two other <i>Amphiprion</i> species (<i>Amphiprion ocellaris</i> and <i>Amphiprion percula</i>). The topological structure of the phylogenetic tree shows that the Picasso clownfish is more closelyrelated to <i>A. percula</i> than it is to <i>A. ocellaris</i>, suggesting that the Picasso clownfish may be a variant of <i>A. percula</i>.
Project description:Anemonefishes (genera: Amphiprion and Premnas; family Pomacentridae) are a group of 28 species of coral reef fishes that are found in obligate symbiosis with large tropical sea anemones. A phylogenetic hypothesis based on morphological analyses of this group suggests that the ancestral anemonefish was a generalist with similar morphology to other pomacentrids, and that it gave rise to other anemonefish species that were more specialized for living with particular species of host anemones. To test this hypothesis we constructed a molecular phylogeny for the anemonefishes by sequencing 1140 base pairs of the cytochrome b gene and 522 base pairs of the 16S rRNA gene for six species of anemonefishes (representatives of all subgenera and species complexes) and two other pomacentrid species. Three methods of phylogenetic analysis all strongly supported the conclusion that anemonefishes are a monophyletic group. The molecular phylogeny differs from the tree based on morphological data in that the two species of specialized anemonefishes (Premnas biaculeatus and Amphiprion ocellaris) were assigned to a basal position within the clade, and the extreme host generalist (Amphiprion clarkii) to a more derived position. Thus, the initial anemonefish ancestors were probably host specialists and subsequent speciation events led to a combination of generalist and specialist groups. Further phylogenetic studies of additional anemonefish species are required to substantiate this hypothesis.
Project description:Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.
Project description:The Amphiprion (anemonefish or clownfish) family of teleost fish, which is not a common model species, exhibits multiple unique characteristics, including social control of body size and protandrous sex change. The social changes in sex and body size are modulated by neuropeptide signaling pathways. These neuropeptides are formed from complex processing from larger prohormone proteins; understanding the neuropeptide complement requires information on complete prohormones sequences. Genome and transcriptome information within and across 22 teleost fish species, including 11 Amphiprion species, were assembled and integrated to achieve the first comprehensive survey of their prohormone genes. This information enabled the identification of 175 prohormone isoforms from 159 prohormone proteins across all species. This included identification of 9 CART prepropeptide genes and the loss of insulin-like 5B and tachykinin precursor 1B genes in Pomacentridae species. Transcriptome assemblies generally detected most prohormone genes but provided fewer prohormone genes than genome assemblies due to the lack of expression of prohormone genes or specific isoforms and tissue sampled. Comparisons between duplicate genes indicated that subfunctionalization, degradation, and neofunctionalization may be occurring between all copies. Characterization of the prohormone complement lays the foundation for future peptidomic investigation of the molecular basis of social physiology and behavior in the teleost fish.
Project description:Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300?bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.
Project description:BACKGROUND:Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1-2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained. RESULTS:Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness). CONCLUSIONS:These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.
Project description:Anemonefishes (Pomacentridae Amphiprioninae) are a group of 30 valid coral reef fish species with their phylogenetic relationships still under debate. The eight available mitogenomes of anemonefishes were used to reconstruct the molecular phylogenetic tree; six were obtained from this study (Amphiprion clarkii, A. frenatus, A. percula, A. perideraion, A. polymnus and Premnas biaculeatus) and two from GenBank (A. bicinctus and A. ocellaris). The seven Amphiprion species represent all four subgenera and P. biaculeatus is the only species from Premnas. The eight mitogenomes of anemonefishes encoded 13 protein-coding genes, two rRNA genes, 22 tRNA genes and two main non-coding regions, with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. Among the 13 protein-coding genes, A. ocellaris (AP006017) and A. percula (KJ174497) had the same length in ND5 with 1,866 bp, which were three nucleotides less than the other six anemonefishes. Both structures of ND5, however, could translate to amino acid successfully. Only four mitogenomes had the tandem repeats in D-loop; the tandem repeats were located in downstream after Conserved Sequence Block rather than the upstream and repeated in a simply way. The phylogenetic utility was tested with Bayesian and Maximum Likelihood methods using all 13 protein-coding genes. The results strongly supported that the subfamily Amphiprioninae was monophyletic and P. biaculeatus should be assigned to the genus Amphiprion. Premnas biaculeatus with the percula complex were revealed to be the ancient anemonefish species. The tree forms of ND1, COIII, ND4, Cytb, Cytb+12S rRNA, Cytb+COI and Cytb+COI+12S rRNA were similar to that 13 protein-coding genes, therefore, we suggested that the suitable single mitochondrial gene for phylogenetic analysis of anemonefishes maybe Cytb. Additional mitogenomes of anemonefishes with a combination of nuclear markers will be useful to substantiate these conclusions in future studies.
Project description:In the present work, a highly pathogenic isolate of Fusarium oxysporum f. sp. lini, which is the most harmful pathogen affecting flax (Linum usitatissimum L.), was sequenced for the first time. To achieve a high-quality genome assembly, we used the combination of two sequencing platforms - Oxford Nanopore Technologies (MinION system) with long noisy reads and Illumina (HiSeq 2500 instrument) with short accurate reads. Given the quality of DNA is crucial for Nanopore sequencing, we developed the protocol for extraction of pure high-molecular-weight DNA from fungi. Sequencing of DNA extracted using this protocol allowed us to obtain about 85x genome coverage with long (N50 = 29 kb) MinION reads and 30x coverage with 2 × 250 bp HiSeq reads. Several tools were developed for genome assembly; however, they provide different results depending on genome complexity, sequencing data volume, read length and quality. We benchmarked the most requested assemblers (Canu, Flye, Shasta, wtdbg2, and MaSuRCA), Nanopore polishers (Medaka and Racon), and Illumina polishers (Pilon and POLCA) on our sequencing data. The assembly performed with Canu and polished with Medaka and POLCA was considered the most full and accurate. After further elimination of redundant contigs using Purge Haplotigs, we achieved a high-quality genome of F. oxysporum f. sp. lini with a total length of 59 Mb, N50 of 3.3 Mb, and 99.5% completeness according to BUSCO. We also obtained a complete circular mitochondrial genome with a length of 38.7 kb. The achieved assembly expands studies on F. oxysporum and plant-pathogen interaction in flax.
Project description:Long-read sequencing techniques, such as the Oxford Nanopore Technology, can generate reads that are tens of kilobases in length and are therefore particularly relevant for microbiome studies. However, owing to the higher per-base error rates than typical short-read sequencing, the application of long-read sequencing on microbiomes remains largely unexplored. Here we deeply sequenced two human microbiota mock community samples (HM-276D and HM-277D) from the Human Microbiome Project. We showed that assembly programs consistently achieved high accuracy (?99%) and completeness (?99%) for bacterial strains with adequate coverage. We also found that long-read sequencing provides accurate estimates of species-level abundance (R = 0.94 for 20 bacteria with abundance ranging from 0.005% to 64%). Our results not only demonstrate the feasibility of characterizing complete microbial genomes and populations from error-prone Nanopore sequencing data but also highlight necessary bioinformatics improvements for future metagenomics tool development.