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 N 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 Received: 14 November 2017; Revised: 11 December 2017; Accepted: 27 December 2017 The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4803946 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Tan et al. studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae. Keywords: clownfish; long reads; genome; transcriptome; hybrid assembly Data Description The clown anemonefish, Amphiprion ocellaris (Fig. 1,NCBITaxon ID: 80 972, Fish Base ID:6509), is a well-known tropical marine fish species among the nonscientific community especially fol- lowing the Pixar film Finding Nemo and its sequel Finding Dory . The visual appeal of A. ocellaris due to its bright coloration and behaviour and ease of husbandry have maintained a strong global demand for this species in the marine aquarium trade, driving a fine balance between positive environmental aware- ness and sustainable ornamental use [1, 2]. Further, given high survival rates and ability to complete their life cycle in captivity, captive-breeding programs to partially sustain their global trade have been successful . For the scientific community, A. ocellaris or anemonefishes in general are actively studied due to their in- triguing reproductive strategy, i.e., sequential hermaphroditism Figure 1: The clown anemonefish ( Amphiprion ocellaris). Photo by Michael P. [4–7] and mutualistic relationships with sea anemones [8–12]. Hammer. Phenotypic body colour variation based on host-anemone use and geography also pose additional questions regarding adap- tive genetic variation . size-selected (8–30 kb) with a BluePippin (Sage Science, Beverly, In recent years, concurrent with the advent of long read se- MA, USA), and processed using the Ligation Sequencing 1D Kit quencing technologies , several studies have explored com- bining short but accurate Illumina reads with long but less ac- (Oxford Nanopore, Oxford, UK) according to the manufacturer’s instructions. Three libraries were prepared and sequenced on curate Nanopore/PacBio reads to obtain genome assemblies that are usually more contiguous with higher completeness than as- 3 different R9.4 flowcells using the MinION portable DNA se- quencer (Oxford Nanopore, Oxford, UK) for 48 hours. semblies based on Illumina-only reads [15–19]. To further con- tribute to the evaluation of long read technology in fish ge- nomics , we sequenced the whole genome of A. ocellaris using Sequence read processing Oxford Nanopore and Illumina technologies and demonstrate that hybrid assembly of long and short reads greatly improved Raw Illumina short reads were adapter-trimmed with Trimmo- the quality of genome assembly. matic v.0.36 (ILLUMINACLIP:2:30:10, MINLEN:100; Trimmomatic, RRID:SCR 011848), followed by a screening for vectors and contaminants, using Kraken v.0.10.5 (Kraken, RRID:SCR 005484) Whole-genome sequencing  based on the MiniKraken DB. Kraken-unclassified reads, i.e., nonmicrobial/viral origin, were aligned to the complete mi- Tissues for genome assembly and as reference material were togenome of NTM A3764 (see the Mitogenome Assembly section) sourced from the collection of the Museum and Art Gallery of to exclude sequences of organellar origin. This results in a total the Northern Territory (NTM). The samples used for DNA ex- of 42.35 Gb of “clean” short reads. Nanopore reads were base- traction and subsequent whole-genome sequencing were from called from their raw FAST5 files using the Oxford Nanopore freshly vouchered captive bred A. ocellaris specimens, repre- proprietary base-caller, Albacore, version 2.0.1. Applying a min- senting a unique black and white colour phenotype found only imum length cutoff of 500 bp, this study produced a total of 8.95 in the Darwin Harbour region, Australia (NTM A3764, A4496, Gbp in 895 672 Nanopore reads (N : 12.7 kb). Sequencing statis- A4497). tics are available in Supplementary Table 1. Genomic DNA was extracted from multiple fin clip and mus- cle samples using the E.Z.N.A. Tissue DNA Kit (Omega Bio-tek, Norcross, GA, USA). For Illumina library prep, approximately 1 Genome size estimation μg of gDNA from isolate A3764 was sheared to 300 bp using a Covaris Focused-Ultrasonicator (Covaris, Woburn, MA, USA) and K-mer counting with the “clean” Illumina reads was performed subsequently processed using the TruSeq DNA Sample Prep Kit with Jellyfish v.2.2.6 (Jellyfish, RRID:SCR 005491), generating (Illumina, San Diego, CA, USA) according to the manufacturer’s k-mer frequency distributions of 17-, 21-, and 25-mers. These instructions. Paired-end sequencing was performed on a sin- histograms were processed by GenomeScope , which esti- gle lane of HiSeq 2000 (Illumina, San Diego, CA, USA) located mated a genome size of 791 to 794 Mbp with approximately 80% at the Malaysian Genomics Resource Centre Berhad. Two ad- of unique content and a heterozygosity level of 0.6% (Supple- ditional libraries were constructed from specimen NTM A3764, mentary Fig. 1). Given that we had previously excluded adapters and both libraries were sequenced on the MiSeq (2 × 300 bp as well as sequences from contaminant or organellar sources, setting), located at the Monash University Malaysia Genomics the max kmer coverage filter was not applied ( max kmer coverage: Facility. -1). A separate estimation performed by BBMap  estimated a To generate Oxford Nanopore long reads, approximately haploid genome size of 967 Mbp. The genome sizes estimated 5 μg of gDNA was extracted from isolates NTM A4496 and A4497, from both approaches are within the range of sizes listed for Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4803946 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Genome sequence of Amphiprion ocellaris 3 Table 1: Genome and transcriptome statistics of the clownfish ( Am- other Amphiprion species (792 Mb–1.2 Gb) as reported on the An- phiprion ocellaris) genome imal Genome Size Database . Illumina Illumina + (≥500 bp) Nanopore Hybrid genome assembly (≥500 bp) Short reads used for assemblies described in this study were only trimmed for adapters, but not for quality. Both short- Genome assembly read-only and hybrid de novo assemblies were performed Contig statistics with the Maryland Super-Read Celera Assembler v.3.2.2 (Ma- Number of contigs 133 997 7810 SuRCA, RRID:SCR 010691). During hybrid assembly, errors Total contig size, bp 851 389 851 880 159 068 were encountered in the fragment correction step of the Cel- Contig N size, bp 15 458 323 678 era Assembler (CA; Celera assembler, RRID:SCR 010750). To Longest contig, bp 204 209 2051 878 overcome this, given that the CA assembler is no longer Scaffold statistics maintained, we disabled the frgcorr step based on one of Number of scaffolds 106 526 6404 the developer’s recommendations, and the hybrid assem- Total scaffold size, bp 852 602 726 880 704 246 bly was subsequently improved with 10 iterations of Pilon Scaffold N size, bp 21 802 401 715 v.1.22 (Pilon, RRID:SCR 014731), using short reads to cor- Longest scaffold, bp 227 111 3111 502 rect bases, fix misassemblies, and fill assembly gaps. To GC/AT/N, % 39.6/60.2/0.14 39.4/60.5/0.06 assess the completeness of the genome, Benchmarking Univer- BUSCO genome completeness sal Single-Copy Orthologs v.3.0.2 (BUSCO, RRID:SCR 015008) Complete 3691 (80.5%) 4417 (96.3%) was used to locate the presence or absence of the Actinopterygii- Complete and single copy 3600 (78.5%) 4269 (93.1%) specific set of 4584 single-copy orthologs (OrthoDB v9). Complete and duplicated 91 (2.0%) 148 (3.2%) The short-read-only and hybrid assemblies yielded total as- Fragmented 534 (11.6%) 63 (1.4%) sembly sizes of 851 Mb and 880 Mb, respectively. Statistics for Missing 359 (7.9%) 104 (2.3%) assemblies for each Pilon iteration are available in Supplemen- Transcriptome assembly tary Table 2. Inclusion of Nanopore long reads for a hybrid as- Number of contigs 25 364 sembly representing approximately ×11 genome coverage led to Total length, bp 68 405 796 a 94% decrease in the number of scaffolds (>500 bp) from 106 Contig N size, bp 3670 526 to 6404 scaffolds and an 18-fold increase in the scaffold N BUSCO completeness length from 21 802 bp to 401 715 bp (Table 1). In addition, the Complete 4253 (92.8%) genome completeness was also substantially improved in the Complete and single-copy 4128 (90.1%) hybrid assembly, with BUSCO detecting complete sequences of Complete and duplicated 125 (2.7%) 96.3% (4417/4584) of single-copy orthologs in the Actinopterygii- Fragmented 127 (2.8%) specific dataset. Missing 204 (4.4%) Genome annotation Transcriptome sequencing and assembly Number of protein-coding genes 27 420 Number of functionally 26 211 Total RNA extraction from RNAshield-preserved whole-body annotated proteins and muscle tissues of isolate A4496 used Quick-RNA MicroPrep Mean protein length 514 aa (Zymo Research Corpt, Irvine, CA, USA) according to the man- Longest protein 29 084 aa ufacturer’s protocols. After assessing total RNA intactness on (titin protein) the Tapestation2100 (Agilent), mRNA was enriched using NEB- Average number (length) of exon 9 (355 bp) Next Poly(A) mRNA Magnetic Isolation Kit (NEB, Ipwich, MA, per gene USA) and processed with NEBNext Ultra RNA Library Prep Kit Average number (length) of 8 (1532 bp) for Illumina (NEB, Ipwich, MA, USA). Libraries from both whole- intron per gene body and muscle tissues were sequenced on a fraction of MiSeq V3 flowcell (1 × 150 bp). Single-end reads from both libraries in addition to 2 publicly available A. ocellaris transcriptome se- total of 3 passes were run with MAKER2; the first pass was quencing data (SRR5253145 and SRR5253146, Bioproject ID: PR- based on hints from the assembled transcripts as RNA-seq ev- JNA374650) were individually assembled using Scallop v0.10.2 idence (est2genome) and protein sequences from 11 fish species  based on HiSat2  alignment of RNA-sequencing reads to downloaded from Ensembl (Ensembl, RRID:SCR 002344)(pro- the newly generated A. ocellaris genome. The transcriptome as- tein2genome), whereas the second and third passes included gene semblies were subsequently merged using the tr2aacds pipeline models trained from the first (and then second) passes with from the EvidentialGene  package and similarly assessed for ab initio gene predictors SNAP (SNAP, RRID:SCR 002127)and completeness using BUSCO, version 3 . The final nonredun- Augustus (Augustus: Gene Prediction, RRID:SCR 008417). In dant transcriptome assembly, which was subsequently used to the final set of genes predicted, sequences with annotation edit annotate the A. ocellaris genome, contains 25 264 contigs/isotigs distance (AED) values of less than 0.5 were retained. A small (putative transcripts) with an accumulated length of 68.4 Mb and AED value suggests a lesser degree of difference between the BUSCO-calculated completeness of 92.8% (Table 1). predicted protein and the evidence used in the prediction (i.e., fish proteins, transcripts). This resulted in a final set of 27 240 protein-coding genes with an average AED of 0.14 (Table 1). A Genome annotation BUSCO analysis on the completeness of the predicted protein Protein-coding genes were predicted with the MAKER v.2.31.9 dataset detected the presence of 4259 (92.9%) single-copy or- genome annotation pipeline (MAKER, RRID:SCR 005309). A thologs from the Actinopterygii-specific dataset. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4803946 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Tan et al. Figure 2: Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 . Further, to infer the putative function of these predicted pro- AMPOCE 00 012675-RA (71.5% protein identity to O42145), was teins, NCBI’s blastp v.2.6.0 (-evalue 1e-10, -seg yes, -soft masking searched (tblastn) against the NCBI TSA database (Taxon: true, -lcase masking;BLASTP, RRID:SCR 001010) was used to Amphirion) and showed strikingly high protein identity (99%) find homology to existing vertebrate sequences in the nonre- to a translated RNA transcript from Amphiprion bicinctus dundant (NR) database. Applying a hit fraction filter to include (c183337 g1 i2: GDCV01327693) . The cyp19a1a gene codes for only hits with ≥70% target length fraction, the remaining unan- a steroidogenic enzyme that converts androgens into estrogens notated sequences were subsequently aligned to all sequences  were recently shown to be instrumental during sex change in the NR database. With this method, 20 107 proteins (74%) were in Amphiprion bicinctus, as evidenced by significant correlation annotated with a putative function based on homology. Addi- and differential expression of this gene between males and ma- tionally, InterProScan v.5.26.65 (InterProScan, RRID:SCR 005829) ture females . We also observed a similar profile based on  was used to examine protein domains, signatures, and mo- mapping of RNA reads from the publicly available male and fe- tifs present in the predicted protein sequences. This analysis de- male transcriptomes of A. ocellaris to the cyp19a1a gene region tected domains, signatures, or motifs for 26 211 proteins (96%). as visualized using the Integrative Genomics Viewer (Fig. 2). Overall, 96% of the predicted clownfish protein-coding genes The A. ocellaris cyp19a1a gene is located on a 419-kb scaffold and were functionally annotated with information from at least 1 of is spanned by multiple Minimap2-aligned Nanopore reads . the 2 approaches. It is noteworthy that in the Illumina-only assembly, this gene is fragmented and located on 3 relatively short scaffolds (Fig. 2). Mitogenome recovery via genome skimming Genome skimming [38, 39] was performed on 3 additional Conclusion A. ocellaris individuals from known localities (Supplementary We present the first clownfish genome co-assembled with Table 3). Mitogenome assembly was performed with MITObim, high-coverage Illumina short reads and low-coverage (∼11×) version 1.9 (MITObim, RRID:SCR 015056), using the complete Nanopore long reads. Hybrid assembly of Illumina and mitogenome of A. ocellaris (GenBank: NC009065.1) as the bait for Nanopore reads is one of the new features of the MaSuRCA read mapping. The assembled mitogenomes were subsequently assembler, version 3.2.2, which works by constructing long annotated with MitoAnnotator . Consistent with the original and accurate mega-reads from the combination of long and broodstock collection from northern Australia, the captive-bred short read data. Although this is a relatively computationally black and white A. ocellaris NTM A3764 exhibits strikingly high intensive strategy with long run times, we observed substantial whole-mitogenome nucleotide identity (99.98%) to sample NTM improvement in the genome statistics when compared with A3708 as a wild collection from Darwin Harbour, Australia. In Illumina-only assembly. As Nanopore technology becomes more addition, the overall high pair-wise nucleotide identity (>98%) of mature, it is likely that future de novo genome assembly will NTM A3764 to newly generated and publicly available A. ocellaris shift toward high-coverage long read–only assembly, followed whole mitogenomes further supports its morphological identi- by multiple iterations of genome polishing using Illumina reads. fication as A. ocellaris (Supplementary Table 3). Identification of the cyp19a1a gene associated Availability of supporting data with sexual differentiation Data supporting the results of this article are available in the The validated cyp19a1a enzyme of Danio rerio (Uniprot: O42145) GigaDB repository . Raw Illumina and Nanopore reads gen- was used as the query (E-value = 1e-10) for blastp search erated in this study are available in the Sequence Read Archive against the predicted A. ocellaris proteins. The top blast hit, (SRP123679), whereas the Whole Genome Shotgun project has Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4803946 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Genome sequence of Amphiprion ocellaris 5 been deposited at DDBJ/EMBL/GenBank under the accession 9. Arvedlund M, Nielsen LE. Do the anemonefish Amphiprion NXFZ00000000, both under BioProject PRJNA407816. ocellaris (Pisces: Pomacentridae) imprint themselves to their host sea anemone Heteractis magnifica (Anthozoa: Actinidae)? 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