Chromosome-level genome assembly of the common chiton, Liolophura japonica (Lischke, 1873)

Chitons (Polyplacophora) are marine molluscs that can be found worldwide from cold waters to the tropics, and play important ecological roles in the environment. However, only two chiton genomes have been sequenced to date. The chiton Liolophura japonica (Lischke, 1873) is one of the most abundant polyplacophorans found throughout East Asia. Our PacBio HiFi reads and Omni-C sequencing data resulted in a high-quality near chromosome-level genome assembly of ∼609 Mb with a scaffold N50 length of 37.34 Mb (96.1% BUSCO). A total of 28,233 genes were predicted, including 28,010 protein-coding ones. The repeat content (27.89%) was similar to that of other Chitonidae species and approximately three times lower than that of the Hanleyidae chiton genome. The genomic resources provided by this work will help to expand our understanding of the evolution of molluscs and the ecological adaptation of chitons.


INTRODUCTION
Mollusca is the second largest animal phylum after Arthropoda and is divided into two subphyla, including the Conchifera (Monoplacophora, Bivalvia, Gastropoda, Scaphopoda, and Cephalopoda) and the Aculifera (Polyplacophora, Caudofoveata, and Solenogastres) [1][2][3].Within the latter, chitons (Polyplacophora) are thought to be a relatively early diverging group of living molluscs [4].They play a crucial role in shaping marine communities in both intertidal and subtidal systems worldwide [5,6].These "living fossils" are characterised by a highly evolutionary-conserved and unique type of shell formed by eight articulating aragonite plates that protect from environmental threats [7][8][9].This biomineralized armour incorporates an unpigmented sensory system known as aesthetes, which is found in all chiton species [10] as a light-sensing adaptation [11].Some members of the families Schizochitonidae and Chitonidae developed shell eyes with aragonite-based lenses, allowing the light to focus onto the pigmented photoreceptive retina [10,12].However, contrary to other molluscs, our understanding of the Polyplacophora is constrained to only two available genomes (Acanthopleura granulata and Hanleya hanleyi) [13,14].
Liolophura japonica (Polyplacophora, Chitonidae) (Lischke, 1873) (Figure 1A) is one of the most abundant polyplacophorans found on the intertidal rocky reefs of the Asian continent, including China, Korea, and Japan.This species diverged from the last common ancestor of Liolophura ∼184 million years ago during the early Pleistocene period [15].On the shore, they are distributed over a wide vertical range from the mid-littoral zone to the low-subtidal zone, where the animals experience periodic fluctuations in environmental conditions with the ebb and flow of the tide [16,17].Unlike many other mobile species inhabiting rocky shores, which migrate towards the low shore areas during summer [18,19], L. japonica does not show any significant seasonal migration behaviour and can survive stressful low tide periods presumably by fitting itself into small refuges via its eight flexible, interlocking plates [17,20,21].In terms of its feeding biology, it is a generalist consumer feeding on a wide range of microalgae and macroalgae [16,22] and, owing to their high density, it is an important grazer in the intertidal zone of East Asia, contributing to the control of on-shore primary productivity [22,23].Given the importance of this species, the high-quality genome presented in this study will help to expand our understanding of the evolution of molluscs and the ecological adaptation of chitons.

CONTEXT
Here, we report the assembled genome of the chiton L. japonica (Polyplacophora, Chitonidae) (Lischke 1873) (Figure 1A).L. japonica was selected to be one of the species sequenced by the Hong Kong Biodiversity Genomics Consortium (also known as EarthBioGenome Project Hong Kong), which is organised by researchers from eight publicly funded universities in Hong Kong.The L. japonica genome presented in this study is of high quality and near chromosomal level, providing a valuable resource for the understanding of the evolutionary biology of polyplacophorans and the adaptation of its resilience under oscillating environmental changes in the intertidal zones.

Collection and storage of samples, isolation of high molecular weight genomic DNA, quantification, and qualification
The chitons L. japonica were collected at the rocky shore in Kau Sai Chau, Hong Kong

DNA shearing, library preparation, and sequencing
A total of 120 μl of DNA sample with 6.2 μg DNA was transferred to a g-tube (Covaris Part No. 520079).The sample was then subjected to six centrifugation steps for 2 min each at 2,000 × g.The resultant DNA was collected and stored in a 2 mL DNA LoBind ® Tube (Eppendorf Cat.No. 022431048) at 4 °C until library preparation.Overnight pulse-field gel electrophoresis was used to examine the molecular weight of the isolated DNA, as described in the previous section.The electrophoresis profile was set as follows: 5 K as the lower end and 100 K as the higher end for the designated molecular weight; Gradient = 6.0 V/cm; Run   HiFi reads were generated and collected for further analysis.One SMRT cell was used for this sequencing.Detailed sequencing data can be found in Table 1.

Omni-C library preparation and sequencing
An Omni-C library was constructed using the Dovetail ® Omni-C ® Library Preparation Kit ranging between 350 bp and 1,000 bp.At last, the concentration and fragment size of the sequencing library were examined by the Qubit ® Fluorometer, Qubit™ dsDNA HS, and BR Assay Kits, and the TapeStation D5000 HS ScreenTape, respectively.After the quality check, the library was sequenced on an Illumina HiSeq-PE150 platform.Detailed sequencing data information can be found in Table 1.

Transcriptome sequencing
Total RNA and small RNA (<200 nt) from different tissues (i.e., digestive gland, foot, gill, gonad, and heart) of the other individual were isolated using the TRIzol reagent (Invitrogen) and the mirVana™ miRNA Isolation Kit (Ambion) following the manufacturer's protocol respectively.The quality of the extracted RNA was checked using NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (
TEs were annotated as previously described [28] using the automated Earl Grey TE annotation pipeline (version 1.2) with "-r eukarya" to search the initial mask of known elements and other default parameters.Briefly, this pipeline first identified known TEs from Dfam (RRID:SCR_021168) with RBRM (release 3.2) and Repbase (v20181026; RRID:SCR_021169).De novo TEs were then identified, and the consensus boundaries were extended using an automated BLAST (RRID:SCR_004870), Extract, and Extend process with five iterations and 1,000 flanking bases added at each round.Redundant sequences were removed from the consensus library before the genome assembly was annotated with the combined known and de novo TE libraries.Annotations were processed to remove overlap and defragment annotations prior to the final TE quantification.

RESULTS AND DISCUSSION
A total of 8.77 Gb of HiFi bases of the common chiton L. japonica were generated with an average HiFi read length of 8,347 bp with 14X data coverage.After scaffolding with ∼397 Gb Omni-C data, the assembled genome size was 609.5 Mb, with 632 scaffolds, a scaffold N50 of 37.34 Mb, and the complete BUSCO estimation of 96.1% (metazoa_odb10) (Figure 1B; Table 2).A total of 13 pseudomolecules of chromosomal length were anchored from the Omni-C data (Figure 1C; Table 3).This result is close to the karyotype of L. japonica (2n = 24), indicating the assembly is near chromosome-level.The assembled L. japonica genome has a genome size close to the estimation performed by GenomeScope, which was 609.7 Mb with a heterozygosity rate of 1.24% (Figure 1D; Table 4), and similar to the Acanthopleura granulata chiton genome (A.granulate: 606 Mb) [13] (Table 2).Telomeres can also be found in 7 out of 13 pseudomolecules (Table 5).
Total RNA sequencing data from different tissues, including the digestive gland, foot, gill,  were used to predict the gene models, and 28,233 gene models were generated with 28,010 predicted protein-coding genes, a mean coding sequence length of 447 amino acids (AA), and the proteome complete BUSCO estimation of 90.9% (metazoa_odb10) (Table 2).

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380 °N, 114.310 °E) during the summer of 2022.They were kept in 35 ppt artificial seawater at room temperature until DNA isolation.High molecular weight (HMW) genomic DNA was isolated from a single individual.The foot muscle was first frozen in liquid nitrogen and ground to powder.DNA extraction was carried out with the sample powder using a Qiagen MagAttract HMW kit (Qiagen Cat.No. 67563) following the manufacturer's protocol with some modifications.Around 1 g of sample was first put in 200 μl 1× PBS and mixed with RNase A, Proteinase K, and Buffer AL provided in the kit.The mixture was allowed to sit at room temperature (∼22 °C) for 2 h.Next, the mixture was gently flicked every 30 min to allow thorough mixing of samples and digestion solution.The DNA was then isolated from the lysate with the magnetic beads provided in the kit and a magnet rack.Finally, the sample was eluted with 120 μl of elution buffer (PacBio Ref. No. 101-633-500).To prevent unintended DNA shearing during the extraction process, wide-bore tips were consistently employed whenever DNA transfer occurred.Next, the sample was quantified by the Qubit ® Fluorometer, Qubit™ dsDNA HS, and BR Assay Kits (Invitrogen™ Cat.No. Q32851).Overnight pulse-field gel electrophoresis was used to examine the molecular weight of the isolated DNA, together with three DNA markers (-Hind III digest; Takara Cat.No. 3403, DL15,000 DNA Marker; Takara Cat.No. 3582A and CHEF DNA Size Standard-8-48 kb Ladder; Cat.No. 170-3707).The purity of the sample was examined by the NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer, with A260/A280: ∼1.8 and A260/A230: >2.0 as a standard.
time = 15 h:16 min; included angle = 120°; Int.Sw.Tm = 22 s; Fin.Sw.Tm = 0.53 s; Ramping factor: a = Linear.The gel was run in 1.0% PFC agarose in 0.5× TBE buffer at 14 °C.A SMRTbell library was constructed using the SMRTbell ® prep kit 3.0 (PacBio Ref. No. 102-141-700), following the manufacturer's protocol.The genomic DNA was first subjected to DNA repair to remove single-stranded overhangs and repair damages from the shearing step on the DNA backbone.After repair, both ends of 2 were annealed and bound to the SMRTbell library, respectively.The library was loaded at an on-plate concentration of 50-90 pM using the diffusion loading mode.The sequencing was conducted on the Sequel IIe System with an internal control provided in the binding kit.The sequencing was set up and performed in 30-hour movies (with 120 min pre-extension) with the software SMRT Link v11.0 (PacBio).

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Dovetail Cat.No. 21005) according to the manufacturer's instructions.Around 20 mg of flash-freezing powered tissue sample was added into 1 mL 1× PBS, where the genomic DNA was crosslinked with formaldehyde, and the fixed DNA was digested with endonuclease DNase I. Next, the concentration and fragment size of the digested sample were checked by the Qubit ® Fluorometer, Qubit™ dsDNA HS, and BR Assay Kits (Invitrogen™ Cat.No. Q32851), and the TapeStation D5000 HS ScreenTape, respectively.Following the quality examination, both ends of the DNA were polished.Ligation of a biotinylated bridge adaptor was conducted at 22 °C for 30 min, and the subsequent proximity ligation between crosslinked DNA fragments was performed at 22 °C for 1 h.After the ligation events, the DNA was reverse crosslinked and then purified with SPRIselect™ Beads (Beckman Coulter Product No. B23317) to remove the biotin that was not internal to the ligated fragments.The Dovetail™ Library Module for Illumina (Dovetail Cat.No. 21004) was used for the end repair and adapter ligation.During this process, the DNA was tailed with an A-overhang, which allowed Illumina-compatible adapters to ligate to the DNA fragments at 20 °C for 15 min.The Omni-C library was then sheared into small fragments with USER Enzyme Mix and purified with SPRIselect™ Beads.Next, Streptavidin Beads were added to isolate the DNA fragments with internal biotin.Universal and Index PCR Primers from the Dovetail™ Primer Set for Illumina (Dovetail Cat.No. 25005) were used to amplify the library.The final size selection step was carried out with SPRIselect™ Beads to pick only the DNA fragments gonad, and heart, was used to assemble the transcriptome of L. japonica.The final transcriptome assembly contained 294,118,260 transcripts, with 192,010 Trinity-annotated genes (average length of 1,100 bp and N50 length of 2,373 bp).The resultant transcriptomes

Figure 2 .
Figure 2. Genome assembly quality control and contaminant/cobiont detection for the Liolophura japonica.

Table 1 .
Details of genome and transcriptome sequencing data.DNA were polished and tailed with an A-overhang.The ligation of T-overhang SMRTbell adapters was performed at 20 °C for 30 min.Next, the SMRTbell library was purified with SMRTbell ® cleanup beads (PacBio Ref. No. 102158-300).The concentration and size of the library were examined using the Qubit [24]® Fluorometer, Qubit™ dsDNA HS, and BR Assay Kits (Invitrogen™ Cat.No.Q32851), and the pulse-field gel electrophoresis, respectively.A subsequent nuclease treatment step was performed to remove any non-SMRTbell structure in the library mixture.A final size-selection step was carried out to remove the small DNA fragments in the library with 35% AMPure PB beads[24].The Sequel ® II binding kit 3.2 (PacBio Ref. No. 102-194-100) was used for the final preparation for sequencing.Sequel II primer 3.2 and Sequel II DNA polymerase 2.
Thermo Scientific™ Cat.No.ND-ONE-W) and gel electrophoresis.The qualified transcriptome samples were sent to Novogene Co. Ltd (Hong Kong, China) for the library construction for polyA-selected RNA sequencing using the TruSeq RNA Sample Prep Kit v2 (Illumina Cat.No.RS-122-2001), and 150 bp paired-end sequencing.Agilent 2100 Bioanalyser (Agilent DNA 1000 Reagents) was used to measure the insert size and concentration of the final libraries.Details of the sequencing data are listed in Table1.

Table 5 .
List of telomeric repeats found in nine scaffolds.

Table 6 .
Catalogue of repeat elements in the Liolophura japonica genome.