The genome sequence of a solitary sea squirt, Ascidia mentula (Müller, 1776)

We present a genome assembly from an individual Ascidia mentula (the (a solitary sea squirt); Chordata; Ascidiacea; Phlebobranchia; Ascidiidae). The genome sequence is 197.0 megabases in span. Most of the assembly is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 19.46 kilobases in length.


Background
Ascidia mentula is a unitary (= 'solitary', non-budding) ascidian (sea squirt) of the order Phlebobranchia.The body is oblong or elongate-ovoid and grows to a size of about 180 mm.The external covering, the tunic, is thick and translucent, with irregular swellings.The openings of the two siphons are very well separated, the inhalant siphon being at the extreme end of the body and the exhalant one-half to two-thirds of the way back; the siphon openings are barely raised above the general outline of the tunic.A network of fine vascular sinuses, with multiple branches each ending in a minute bulb near the surface, imparts pink or red colouration to the tunic.This colouration appears to be influenced by the amount of light experienced by the individual, ranging from red when well-lit through pink to grey (but still with tiny pink/red spots) in dark sites.As is typical of unitary ascidians, eggs and sperm are spawned for external fertilisation.

Genome sequence report
The genome was sequenced from one Ascidia mentula (Figure 1) collected from Torquay (50.46,.A total of 74-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 27 missing joins or mis-joins, reducing the scaffold number by 12.73%, and decreasing the scaffold N50 by 7.49%. The final assembly has a total length of 197.0 Mb in 95 sequence scaffolds with a scaffold N50 of 21.2 Mb (Table 1).A summary of the assembly statistics is shown in Figure 2, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.9%) of the assembly sequence was assigned to 9 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/38561.

Sample acquisition and nucleic acid extraction
An Ascidia mentula (specimen ID MBA-201019-015A, ToLID kaAscMent1) was collected by hand from Torquay Marina,     RNA was extracted from tissue of kaAscMent in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (https://dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/v1).The RNA concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination  and corrected as described previously (Howe et al., 2021).
Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al., 2017)

Angelica Crottini
Universidade do Porto, Porto, Porto District, Portugal This note reports on the genome assembly of Ascidia mentula, a solitary ascidian belonging to the order Phlebobranchia.The genome of Ascidia mentula is assembled in 9 chromosomes and all steps, from collecting to genome sequencing and assembly, are reported in a clear and straightforward way.
Here below are a few minor suggestions: "(50.46,-3.53)" change to "(latitude 50.46, longitude -3.53)"This note presents the genome assembly of the solitary tunicate Ascidia mentula.The genome is of high quality, assembled into 9 chromosomes representing 99.9% of the total assembly, and will be useful for the community interested in comparative genomics and tunicate.
In what follows, I am listing a few minor elements: Background: "the amount of light": is it artificial or sun light?I am not sure is very relevant, but as tunicates are very common in artificial habitat, this information could be added.I guess a reference could be included too.

Figure 1 .
Figure 1.Photographs of the Ascidia mentula (kaAscMent1) specimen used for genome sequencing.A: Three specimens of Ascidia mentula collected at the same time from Torquay Marina.The specimen sequenced, ' A', is on the left.B: Dissection showing anterior portion of branchial basket with dorsal lamina, circumpharyngeal band, dorsal tubercle and parasitic copepod (green).C: Dissection showing portion of branchial basket posterior to that shown in B, with mucus string (food string) adjacent to dorsal lamina.

Figure 2 .
Figure 2. Genome assembly of Ascidia mentula, kaAscMent1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 196,975,169 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (27,920,005 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,171,019 and 15,452,589 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the metazoa_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ascidia%20mentula/dataset/CANNZX01/snail.

Figure 5 .
Figure 5. Genome assembly of Ascidia mentula, kaAscMent1.1:Hi-C contact map of the kaAscMent1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=Io-Iy42sQcqV0NQQVaocWQ.

Table 1 . Genome data for Ascidia mentula, kaAscMent1.1. Project accession data
(Rhie et al., 2021)enchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).** BUSCO scores based on the metazoa_odb10 BUSCO set using v5.3.2.C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison.A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/Ascidia%20mentula/dataset/CANNZX01/busco.Devon, UK (latitude 50.46, longitude -3.53) on 2020-10-19.The specimen was taken from the marina pontoon (on submerged plastic settlement panels) by Christine Wood and John Bishop (Marine Biological Association).The specimen was identified by John Bishop and preserved in liquid nitrogen.The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation; DNA extraction; HMW DNA fragmentation; and fragmented DNA clean-up.The kaAscMent1 sample was

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests
: No competing interests were disclosed.Reviewer Expertise: Taxonomy, Systematics, Evolutionary Genetics/Genomics I

confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

"
Subsequent studies have addressed … natural products" I am not sure that I see what this meant here Method: In the sentence: "In brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA." to sample or to eliminate -> choose one not both?Probably to eliminate "Protocols employed by the Tree of Life laboratory are publicly available on protocols.io:https://dx.doi.org/10.17504/protocols.io.8epv5xxy6g1b/v1."Iamnotverysurethis is needed here as they are cited throughout the text?"The mitochondrial genome was assembled using MitoHiFi(Uliano-Silva et al., 2023), which runs MitoFinder(Allio et al., 2020)or MITOS(Bernt et al., 2013)and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence."I am not sure this is clear enough here.In the end, did you use both Mitofinder and MITOS?And I am not sure what is the purpose of having both.

the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.