Ciona intestinalis

The bed bug is 1 of 30 i5K pilot genome assemblies that were subjected to automatic gene annotation using a Maker 2.0 (http://www.yandell-lab.org/software/maker.html) annotation pipeline tuned specifically for arthropods. The pipeline is designed to be systematic, providing a single consistent procedure for the species in the pilot study, scalable to handle 100s of genome assemblies, evidence guided using both protein and RNA-seq evidence to guide gene models and targeted to utilize extant information on arthropod gene sets. The core of the pipeline was a Maker 2 instance, modified slightly to enable efficient running on our computational resources. The genome assembly was first subjected to de novo repeat prediction and CEGMA analysis (http://korflab.ucdavis.edu/datasets/cegma/) to generate gene models for initial training of the ab initio gene predictors (Supplementary Data 33). Three rounds of training of the Augustus (http://bioinf.uni-greifswald.de/augustus/) and SNAP (http://korflab.ucdavis.edu/software.html) gene predictors within Maker were used to bootstrap to a high-quality training set. Input protein data included 1 million peptides from a non-redundant (nr) reduction (90% identity) of Uniprot Ecdysozoa (1.25 million peptides) supplemented with proteomes from 18 additional species (Strigamia maritima, Tetranychus urticae, Caenorhabditis elegans, Loa loa, Trichoplax adhaerens, Amphimedon queenslandica, Strongylocentrotus purpuratus, Nematostella vectensis, Branchiostoma floridae, Ciona intestinalis, Ciona savignyi, Homo sapiens, Mus musculus, Capitella teleta, Helobdella robusta, Crassostrea gigas, Lottia gigantea and Schistosoma mansoni) leading to a final nr peptide evidence set of 1.03 million peptides. RNA-seq from C. lectularius adult males and females was used judiciously to identify exon–intron boundaries but with a heuristic script to identify and split erroneously joined gene models. We used CEGMA models for QC purposes: for C. lectularius, of 1,977 CEGMA single-copy orthologue gene models, 1,928 were found in the assembly, and 1,892 in the final predicted gene set. Finally, the pipeline uses a nine-way homology prediction with human, Drosophila and C. elegans, and InterPro Scan5 to allocate gene names. The automated gene set is available from the BCM-HGSC website (https://www.hgsc.bcm.edu/arthropods/bed-bug-genome-project) and at the National Agricultural Library (https://i5k.nal.usda.gov).

The genome sequences of Ciona intestinalis, Branchiostoma floridae, Salmo salar, Danio rerio, Cyprinus carpio, Xenopus tropicalis, Anolis carolinensis, Gallus gallus, Ovis aries, Bos taurus, and Petromyzon marinus were downloaded from Ensembl (https://asia.ensembl.org/index.html). The miRNA sequences of all the other species except C. idella were downloaded from miRbase (https://www.mirbase.org/). The birth, death, and age of miRNA families were estimated based on a phylogenetic tree generated by RAxML(-m PROTGAMMAJTT -f a -# 100) [71 (link)] analysis from single-copy proteins. The birth, death, and age of miRNA families and the ancestral gene contents were assessed using the COUNT software [72 (link)] following the parsimony rule based on the miRNA families of each species. Branch lengths reflect evolutionary divergence times in million years (MY) inferred from timeTree (http://www.timetree.org/).

Specimens of the ascidian species Phallusia philippinensis (misidentified as P. nigra in previous papers [39 (link)]) were collected from the Ginowan Fishery Bay on the west coast of Okinawa Island, Japan. Ciona intestinalis (type A: also called as C. robusta) were supplied by National Bio-Resource Project at Misaki Marine Biological Station, University of Tokyo. P. philippinensis specimens were kept in an aquarium at 20–25 °C in the dark, whereas C. intestinalis specimens were kept at 16 °C under constant light until experimental use. Eggs and semen were collected from the oviduct and spermiduct, respectively, via dissection. Semen was stored on ice or at 4 °C until further use. Eggs were placed in either artificial seawater (ASW) (pH 8.2) or measuring medium after collection and immediately used for experiments. Where dechorionated eggs were required, the vitelline coat and accessory cells were manually removed from the eggs using a sharpened insect pin and fine blade (Shiga Konchu, Tokyo, Japan). Dechorionated eggs were kept in a 1.5% agar-coated dish to avoid disruption.
The ASW consisted of NaCl (462 mM), KCl (9.2 mM), CaCl₂ (9 mM), MgSO₄ (28 mM), MgCl₂ (22 mM), and 10 mM HEPES (Dojindo, Kumamoto, Japan) (pH 8.2). The measuring media were: ASW, whose pH was adjusted with 10 mM good buffers (Dojindo): MES (pH 5.0–6.5); pH PIPES (pH 6.5–7.2); HEPES (pH 7.2–8.2); TAPS (pH 8.2); CHES (pH 9.5) instead of HEPES (pH 8.2).
This study using invertebrates is not regulated by animal welfare, but experiments were performed in accordance with the principle of animal welfare.