De novo assembly of transcriptomes of six Hua species (Semisulcospiridae, Cerithioidea, Gastropoda)

Abstract

Species in Semisulcospiridae are important in freshwater ecology and have great research value, yet their genomic resources remain very limited. Here, we present de novo assembled transcriptomes from six species of Hua in Semisulcospiridae, including Hua textrix (Heude, 1888), H. yangi L.-N. Du, J.-X. Yang & Chen, 2023, H. wujiangensis L.-N. Du, J.-X. Yang & Chen, 2023, and three undescribed species. Assembly was performed using Trinity, resulting in average contig lengths ranging from 716.6 to 883.3 bp and transcript numbers ranging from 147,147 to 268,741. Benchmarking Universal Single-Copy Ortholog (BUSCO) analysis was used to assess the transcriptome completeness. The functional annotation of transcripts for each species had over 18,000 BLAST hits, 17,000 GO terms, 15,000 KEGG pathways, 8,000 Pfam accessions, and 140 COG functional categories. This study provides valuable transcriptomic resources for the six Hua species, which can be used for various research of Semisulcospiridae, including biodiversity, phylogeny, and comparative genomics.

Background & Summary

Semisulcospiridae Morrison, 1952 is a group of gastropods that comprises the majority of freshwater benthic fauna^{1, 2, 3–4}, which is important in freshwater ecology⁵. It contains over 90 valid species belonging to four genera: Hua S.-F. Chen, 1943, Juga H. Adams & A. Adams, 1854, Koreoleptoxis J. B. Burch & Y. Jung, 1988, and Semisulcospira O. Boettger, 1886^{3,6, 7–8}. Species in Semisulcospiridae are predominantly found in East Asia and North America, with the majority (over 60 species from three genera) documented in China^2,7,9,10. Species from Semisulcospira, Koreoleptoxis, and Hua can be used as environmental indicators because they can only live in relatively clean water^{10, 11, 12–13}. Moreover, some species of Semisulcospira and Koreoleptoxis have been proven to be intermediate hosts for human and animal parasite Paragonimus westermani Kerbert, 1878^{14, 15–16}.

The evolutionary relationships within Semisulcospiridae are not clear. Most previous studies mainly focused on phylogenetic analyses based on single genes (e.g., COI, 16S, 28S) or their combination, but it is insufficient when explaining the phylogeny in this family^5,17. Semisulcospira was paraphyletic in phylogenetic trees based on COI and 16S, although its reproductive mode (viviparous) differs from the other three genera (oviparous) of Semisulcospiridae^5,9,10,17. Moreover, using only a few genes is not suitable for distinguishing some congeners^7,10. Omics data may help to solve this problem^5,17. However, only a few studies have involved omics research in this family^{18, 19–20}: Lee¹⁸ published the de-novo assembled transcriptome of Semisulcospira coreana (E. von Martens, 1886); Gim¹⁹ published the draft genome of Semisulcospira libertina (A. Gould, 1859); Miura²⁰ published the chromosome-scale genome of Semisulcospira habei G. M. Davis, 1969.

Chen²¹ first described Hua based on specimens collected from Southwest China and designated Melania telonaria Heude, 1889 as its type species. They can be distinguished by their reproductive organs from other genera of Semisulcospiridae. It is characterized by female reproductive organs with an egg-laying groove or an ovipositor under the left tentacle. Comparatively, Koreoleptoxis has both, and Semisulcospira has none². There are 29 species in Hua, and most are distributed endemically in Southwest China^2,7,10. Morphology of shells, radulae, reproductive organs, and DNA barcoding are commonly used to distinguish the species in the genus. The species of Hua show a high variety in shell morphology, such as size, color, shape, and sculpture^2,7,10. Phylogenetic analysis proved that Hua is a monophyletic group in several studies^2,7,10, but not in He⁷. Species Hua are of great research value in the endemism ecology and high biodiversity. However, they are threatened by human activities¹⁰.

With the rapid development and the advancement of high-throughput sequencing technologies, numerous whole-genome sequencing projects are currently undergoing, including the Darwin Tree of Life Project and the Earth BioGenome Project^22,23. However, whole-genome sequencing remains prohibitively expensive and encounters significant technical challenges for small specimens. RNA sequencing (RNA-seq) is a cost-effective alternative that can obtain high-quality coding sequences and has been widely adopted across diverse research fields. For instance, it has been used to study the physiological ecology of aquatic invertebrates^24,25 and contributed to the conservation of endangered species^26,27. Gene expression profiles generated from RNA-seq provided new insights into physiological acclimatization, metabolic trade-offs, resilience, high-mortality events, and niche partitioning between similar species²⁷. RNA-seq has also been used to provide a robust framework for the phylogeny of mollusks^{28, 29, 30–31}. Additionally, high-quality transcriptomes will facilitate the annotation of species’ genomes and promote future studies of evolution, systematics, and functional genomics.

Although some genomic resources on Semisulcospiridae have been published, there is still not much literature on transcriptome research, and there is no transcriptomics data for Hua^{18, 19–20}. In this study, we report de novo assembled transcriptomes for six Hua species collected from China, facilitating multifaceted research on Semisulcospiridae.

Materials and Methods

Sample collection

Specimens of Hua were collected from the streams in Yunnan and Guizhou of China (Table 1, Fig. 1). They were transported alive to the laboratory, and the foot was cut and stored in RNAlater Stabilization Reagent (Coolaber, Beijing, China) for RNA extraction. The morphological and molecular information (COI and 16S rRNA; Table 2) was integrated for species identification. Live individuals were identified using morphological criteria (Fig. 1) in Du¹⁰ and Du² by Yuanzheng Meng, a taxonomic specialist in Semisulcospiridae.

Table 1. Collection information of six Hua species and SRA Acc. No. of the raw data generated from RNA-Seq.

Species	Specimen catalog	Collection date	Collection station	SRA Acc. No.
Hua textrix Heude, 1888	SEM-A1	2023-09	Dianchi Lake, Kunming, Yunnan Province, China	SRR32814904
Hua yangi Du, Yang & Chen, 2023	SEM-B1	2023-09	Luoping County, Qujing, Yunnan Province, China	SRR32814972
Hua sp. 1	SEM-C1	2023-09	Shizong County, Qujing, Yunnan Province, China	SRR32835171
Hua sp. 2	SEM-D1	2023-09	Wenshan, Yunnan Province, China	SRR32835202
Hua sp. 3	GZA-001A	2024-01	Kaiyang County, Guiyang, Guizhou Province, China	SRR32834839
Hua wujiangensis Du, Yang & Chen, 2023	GZB-001A	2024-01	Kaiyang County, Guiyang, Guizhou Province, China	SRR32833658

[See PDF for image]

Fig. 1

Specimens used in this study. (A) Hua textrix (Heude, 1888), SEM-A1; (B) Hua yangi L.-N. Du, J.-X. Yang & Chen, 2023, SEM-B1; (C) Hua sp. 1, SEM-C1; (D) Hua sp. 2, SEM-D1; (E) Hua sp. 3, GZA-001A; (F) Hua wujiangensis L.-N. Du, J.-X. Yang & Chen, 2023, GZB-001A.

Table 2. The Blast results of COI and 16S (Top 5 blast results are shown).

Sample name	Gene	SubjectAcc	Species	Alignment length (bp)	Identity (%)	Reference
Hua textrix (SEM-A1)	COI	MK251701	Hua textoria	768	99.48	Du10
		MK969005	Hua textrix	759	99.47	Du9
		MK969107	Hua textrix	762	98.43	Du9
		MK251700	Hua sp. D292	768	94.79	Du10
		MK251699	Hua scrupea	768	94.53	Du10
	16S	MK944141	Hua textrix	550	100.00	Du9
		MK251613	Hua textoria	797	99.88	Du10
		MK251612	Hua textoria	801	99.38	Du10
		MK944245	Hua textrix	550	99.09	Du9
		MK251610	Hua sp. D292	797	97.62	Du10
Hua yangi (SEM-B1)	COI	MK969082	Hua sp. 1 LD-2019	762	99.87	Du10
		MK969071	Hua sp. 1 LD-2019	762	99.74	Du10
		MK969084	Hua sp. 1 LD-2019	729	99.73	Du10
		MK969070	Hua sp. 1 LD-2019	729	99.73	Du10
		MK969086	Hua sp. 1 LD-2019	720	97.36	Du10
	16S	MK944219	Hua sp. 1 LD-2019	548	99.82	Du10
		MK944207	Hua sp. 1 LD-2019	548	99.82	Du10
		MK944218	Hua sp. 1 LD-2019	545	99.82	Du10
		MK944220	Hua sp. 1 LD-2019	548	99.64	Du10
		MK944206	Hua sp. 1 LD-2019	545	99.27	Du10
Hua sp. 1 (SEM-C1)	COI	MK251763	Hua aubryana	768	99.61	Du10
		MK251717	Hua sp. tc LD-2019	768	99.61	Du10
		MK251762	Hua aubryana	765	99.61	Du10
		MK251715	Hua sp. tc LD-2019	765	99.61	Du10
		MK251714	Hua sp. tc LD-2019	762	99.61	Du10
	16S	MK251625	Hua sp. tc LD-2019	796	99.87	Du10
		MK251628	Hua sp. tc LD-2019	655	99.85	Du10
		MK251683	Hua aubryana	591	99.83	Du10
		MK251687	Hua aubryana	590	99.83	Du10
		MK251630	Hua sp. tc LD-2019	797	99.75	Du10
Hua sp. 2 (SEM-D1)	COI	NC_085155	Hua aristarchorum	1,533	87.35	Xu52
		NC_037771	Semisulcospira coreana	1,521	86.65	Kim53
		NC_046494	Koreoleptoxis nodifila	1,533	86.63	Choi54
		MK559478	Semisulcospira gottschei	1,533	86.30	Lee55
		KJ696780	Koreoleptoxis nodifila	1,533	85.98	Unpublished
	16S	MK251653	Hua bailleti	802	92.39	Du10
		MK251655	Hua bailleti	782	92.33	Du10
		MK251654	Hua bailleti	802	92.27	Du10
		MK251625	Hua sp. tc LD-2019	801	92.26	Du10
		FJ471494	Semisulcospira jacqueti	808	92.20	Strong56
Hua sp. 3 (GZA-001A)	COI	NC_085155	Hua aristarchorum	1,533	89.04	Xu52
		HM016250	Juga (Calibasis) sp. 7 DC-2011	1,394	87.88	Campbell57
		EF587013	Juga orickensis	1,160	87.33	Campbell57
		HM016256	Juga silicula silicula	1,386	87.30	Campbell57
		MK559478	Semisulcospira gottschei	1,534	87.09	Lee55
	16S	OQ025227	Hua sp. q HC-2022	565	99.47	Unpublished
		OQ025228	Hua sp. q HC-2022	565	99.47	Unpublished
		OQ147345	Hua sp. q HC-2022	565	99.47	Unpublished
		OQ025206	Hua sp. q HC-2022	564	99.29	Unpublished
		OQ025223	Hua sp. q HC-2022	564	99.29	Unpublished
Hua wujiangensis (GZB-001A)	COI	MK251712	Hua sp. D256	768	99.48	Du10
		NC_085155	Hua aristarchorum	1,533	87.54	Xu52
		NC_046494	Koreoleptoxis nodifila	1,533	87.02	Choi54
		KJ696780	Koreoleptoxis nodifila	1,533	86.56	Unpublished
		PQ334808	Koreoleptoxis amurensis	1,533	86.56	Unpublished
	16S	MK251623	Hua sp. D256	796	98.49	Du10
		MK251685	Hua aubryana	623	96.95	Du10
		FJ471494	Semisulcospira jacqueti	804	96.14	Strong56
		FJ471495	Semisulcospira jacqueti	805	95.90	Strong56
		MK251625	Hua sp. tc LD-2019	798	95.87	Du10

Species identification

Hua textrix(Heude, 1888), SEM-A1 (Fig. 1A): Shell medium-sized, solid, conical, with seven whorls. Surface brown, with a dark brown band on the bottom of each whorl, with spiral lines and axial ribs crossing each other, forming checkerboard patterns. Apex pointed. Aperture ovate. The blast results show high identity values (over 99%) with H. textrix^2,9.

Hua yangiL.-N. Du, J.-X. Yang & Chen, 2023, SEM-B1 (Fig. 1B): Shell small, solid, ovate, with four whorls. Surface brown, smooth. Body whorl inflated. Apex blunt. Aperture round. The blast results show high identity values (over 99%) with H. yangi^2,9.

Huasp. 1, SEM-C1 (Fig. 1C): Shell medium-sized, solid, conical, with seven whorls. Surface brown, with spiral lines and axial ribs crossing each other, forming checkerboard patterns. Apex pointed. Aperture ovate. According to blast results, sequences from Hua aubryana (Heude, 1888) and Hua tchangsii Du et al., 2019 both show high identity values (over 99%), but morphological characters differ from both species: H. aubryana has two lines of nodules on the body whorl, and H. tchangsii has smooth shells^2,9. Therefore, it is marked as an undetermined species (Hua sp. 1) here.

Huasp. 2, SEM-D1, (Fig. 1D): Shell small, solid, ovate, with four whorls. Surface yellow-brownish, smooth. Body whorl inflated. Aperture round. The blast results show low identity values (less than 87.35% of COI and 92.39% of 16S), and morphological features cannot match any species either. Based on the above information, it may be a potential new species. Therefore, it is marked as an undetermined species (Hua sp. 2) here.

Huasp. 3, GZA-001A (Fig. 1E): Shell small, solid, ovate, with four whorls. Surface dark brown, smooth except for growth lines. Body whorl inflated. Apex blunt. Aperture ovate. The blast results of COI show low identity values (less than 89.04%), but 16S show high identity (over 99%) with unpublished species. Morphological features and molecular information cannot determine species. It might be a potential new species and is marked as an undetermined species (Hua sp. 3). Therefore, it is marked as an undetermined species (Hua sp. 3) here.

Hua wujiangensisL.-N. Du, J.-X. Yang & Chen, 2023, GZB-001A, (Fig. 1F): Shell small, thin, conical, with five whorls. Surface brown, with 3-4 dark brown bands on each whorl, smooth except for growth lines. Apex pointed. Aperture ovate. The blast results show high identity values (99.48% of COI and 98.49% of 16S) with H. wujiangensis^2,10.

Work-flow

A diagram of the workflow used in this study is presented in Fig. 2. Transcriptomes were de novo assembled from short-read sequences by Trinity. Each assembly was assessed using multiple indicators, and transcripts were annotated based on sequence similarity using Trinotate.

[See PDF for image]

Fig. 2

Flow chart of de novo assembly and annotation. The flow chart was created by the online Mermaid Live Editor (https://mermaid-js.github.io/mermaid-live-editor).

Total RNA extraction, library construction, RNA sequencing, and quality control

Total RNA extraction and sequencing were performed by Novogene Company (Beijing, China). The quality of RNA was detected by electrophoresis on BioAnalyzer (Agilent, Santa Clara CA). Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. Paired-end sequencing was performed on the DNBSEQ-T7 platform with 150 bp read length. The quality of raw data was assessed using FastQC version 0.11.8 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality reads (i.e. short reads which are <50 bp, reads with an average Phred score <30 or N > 3) and sequencing adaptors were removed using fastp (version 0.23.4)³².

De novo assembly and redundancy removal

De novo assembly was performed using Trinity software (version 2.8.5)^24,25,33 (default parameters). The assembled sequences were then clustered and de-duplicated with 95% sequence similarity using CD-HIT(v.4.8.1)³⁴. The completeness of each transcriptome was evaluated using Benchmarking Universal Single-Copy Orthologs (BUSCO) software³⁵ against the Mollusca database (BUSCO version 5.7.1, dataset: mollusca_odb10, 2024-01-08). Additionally, mitochondrial genes (13 protein-coding genes and two ribosomal RNAs) were annotated and extracted from the transcripts using MitoFinder (version 1.4).

Open reading frame prediction and transcriptome annotation

Functional annotations were performed using Trinotate (version 3.3.0). Open reading frames (ORFs) were identified using TransDecoder (version 5.7.1) (https://transdecoder.github.io/). The predicted ORFs were annotated against the UniProtKB/Swiss-Prot (a manually annotated and reviewed protein sequence database)³⁶ and Pfam database (classification of protein families)³⁷ using the Blastp tool from DIAMOND (version 2.1.8.162)^38,39 and hmmscan from HMMER (version 3.1b2)⁴⁰. Additionally, annotations were conducted against the KEGG (Kyoto Encyclopedia of Genes and Genomes) database⁴¹, Gene Ontology (GO)⁴², and eggNOG (Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups)⁴³ databases. The version of the eggNOG database used in this study is 4.5, which includes COG (Cluster of Orthologous Groups)⁴⁴ annotation information. Ultimately, all results were integrated to generate a summary spreadsheet.

Data Records

The raw data were deposited in NCBI under the BioProject accession number PRJNA1160414: BioSample accessions SAMN47530241, SAMN47526927, SAMN47526927, SAMN47523056, SAMN47521559, SAMN47521559; Sequence Read Archive (SRA) accessions SRR32814904⁴⁵, SRR32814972⁴⁶, SRR32835171⁴⁷, SRR32835202⁴⁸, SRR32834839⁴⁹, SRR32833658⁵⁰. The remaining information (i.e. 01.fastp&FastQC_results, 02.Trinity_results, 03.BUSCO_results, 04.Transcriptome_annotation_results, and 05.MitoFinder_results) was uploaded to Figshare (https://doi.org/10.6084/m9.figshare.28637714.v2)⁵¹.

Technical Validation

Accuracy of species identification

Species identifications were verified through integrative taxonomic approaches combining morphological and Sanger sequencing of partial mitochondrial genes (COI and 16S rRNA) data. The three undetermined species were recognized by the second author, Yuanzheng Meng, a taxonomic specialist in Semisulcospiridae. Therefore, we ensure reliable species identification for all specimens included in this study.

Quality control, de novo assembly, and transcript statistics

The RIN scores of all RNA were in the range of 7.40–8.50 (Table 3). The FastQC analysis of the cleaned reads from all six species revealed high quality, with the majority of reads (more than 95%) exhibiting Phred quality scores above 35. The GC content per sequence ranged from 45% to 47%, and no adapter contamination was detected. Sequencing yields, assembly statistics, and transcriptome completeness for the six datasets are summarized in Table 4. The cleaned reads retained exceeded 90%, ranging from 36.7 to 59.3 million. The number of transcripts ranged from 147,147 to 268,741. Average contig length ranged from 716.6 to 883.3 bp. Excluding Hua sp. 2, transcripts from the other five species had N50 values that exceeded 1,000 bp. For BUSCO analysis, the complete BUSCO ranged from 59.1% to 84.0%, with two species failing to attain 70%, and fragmented BUSCO from 3.1% to 6.6%. A total of 4,666 BUSCOs were identified across six species, and 978 of them were present in all species. Most of the mitochondrial genes (except tRNAs) were successfully assembled in all species (Table 5), and COI (1,515–1,536 bp) and 16S (1,322–1,338 bp) were used for species identification (Table 2).

Table 3. Summary of RNA concentration (ng/µL), total RNA amount (ng), and RIN scores for all six Hua species (SEM-A1, SEM-B1, SEM-C1, SEM-D1, GZA-001A, GZB-001A).

Metrics	Hua textrix (SEM-A1)	Hua yangi (SEM-B1)	Hua sp. 1 (SEM-C1)	Hua sp. 2 (SEM-D1)	Hua sp. 3 (GZA-001A)	Hua wujiangensis (GZB-001A)
RNA concentration (ng/µL)	60.0	33.0	83.0	42.0	6.0	17.0
Total RNA amount (ng)	2.100	1.155	2.905	1.470	0.210	1.645
RIN score	8.10	7.70	7.40	7.60	8.50	8.50

Each abbreviation corresponds to the species and collection information in Table 1.

Table 4. Summary of de novo assembly and annotation statistics for transcriptomes from six Hua species.

Metrics	Hua textrix (SEM-A1)	Hua yangi (SEM-B1)	Hua sp. 1 (SEM-C1)	Hua sp. 2 (SEM-D1)	Hua sp. 3 (GZA-001A)	Hua wujiangensis (GZB-001A)
Raw Reads (M)	43.4	47.2	40.6	39.5	55.9	62.0
Cleaned Reads (M)	41.3	45.1	38.9	36.7	53.4	59.3
Transcripts	147,147	166,059	176,784	158,952	252,938	268,741
Trinity “genes”	112,211	120,699	125,890	120,980	175,267	173,907
Median length (bp)	514	502	440	484	399	437
Average length (bp)	866.8	883.3	723.9	817.5	716.6	797.0
GC (%)	46.31	46.41	45.95	46.08	46.93	46.82
Contig N50	1,306	1,386	1,010	979	1,086	1,250
Complete BUSCOs	3,950 (74.6%)	4,019 (75.9%)	3,129 (59.1%)	3,637 (68.6%)	3,930 (74.2%)	4,447 (84.0%)
Fragmented BUSCOs	214 (4.0%)	232 (4.4%)	352 (6.6%)	271 (5.1%)	241 (4.6%)	162 (3.1%)
Missing BUSCOs	1,131 (21.4%)	1,044 (19.7%)	1,814 (34.3%)	1,387 (26.3%)	1,124 (21.2%)	686 (12.9%)
Transcripts with BLAST hits	18,069	18,926	20,327	18,281	29,884	24,433
Transcripts with GO terms	17,649	18,503	19,889	17,884	29,374	23,980
Transcripts with KEGG pathways	15,354	16,134	17,405	15,594	26,122	20,893
Transcripts with Pfam accession	8,142	8,724	8,867	8,200	10,591	10,753
Transcripts with COG functional categories	147	140	234	153	212	169

Table 5. Length of mitochondrial genes from six Hua species.

Mitochondrial genes	Hua textrix (SEM-A1)	Hua yangi (SEM-B1)	Hua sp. 1 (SEM-C1)	Hua sp. 2 (SEM-D1)	Hua sp. 3 (GZA-001A)	Hua wujiangensis (GZB-001A)
ATP6 (bp)	696	NA	696	696	429	696
ATP8 (bp)	162	NA	162	162	162	162
COX1 (COI, bp)	1,536	1,515	1,521	1,533	1,533	1,533
COX2 (bp)	NA	678	NA	NA	NA	678
COX3 (bp)	780	780	780	NA	780	780
CYTB (bp)	1,140	1,125	1,140	1,125	1,140	1,128
ND1 (bp)	939	939	939	939	939	939
ND2 (bp)	1,068	1,068	1,068	NA	1,068	1,068
ND3 (bp)	354	NA	354	198	354	354
ND4 (bp)	1,368	1,368	1,368	1,368	1,368	1,368
ND4L (bp)	291	291	291	285	291	291
ND5 (bp)	1,719	1,719	1,719	1,719	1,701	1,719
ND6 (bp)	549	507	552	NA	552	543
16S (bp)	1,338	1,325	1,329	1,322	1,338	1,327
12S (bp)	889	894	892	886	891	894

Mitochondrial genes that were not annotated were denoted as “NA”.

Transcriptome annotation

The annotation of the transcripts against the UniProtKB/Swiss-Prot database identified over 18,000 significant hits for each species (Table 4), with the highest of over 29,000 hits. Most of the transcripts (>95%) with BLAST hits were also annotated with GO terms and fewer with KEGG pathways and COG functional categories (Table 4, Fig. 3). The annotation against the Pfam database identified over 8,000 Pfam accessions of each species with the highest of 10,700 in H. wujiangensis (Table 4).

[See PDF for image]

Fig. 3

Distribution of Gene Ontology terms (GO). The stacked bar shows the number of transcripts of each GO term for six species.

Usage Notes

Given that Hua species are endemic to Southwest China and threatened by human activities, these transcriptomic resources support conservation efforts by providing genetic markers for phylogenetics, population studies to assess genetic diversity, and identify evolutionarily significant units. The functional annotations will serve as valuable references for future single- and multi-species analyses. The homologous genes across the transcriptomes of these six species will enhance our understanding of evolutionary and functional relationships in Hua. Furthermore, the sequence assemblies can be utilized to design species-specific primers for biomarkers. The dataset complements the limited genomic resources for Semisulcospiridae, creating a more comprehensive molecular framework for family-wide comparative studies and phylogenetic analyses. With its potential for extensive analysis, this dataset is a valuable resource for research in Semisulcospiridae genomic diversity.

Acknowledgements

We thank Mr. Baogang Liu and Xiaobo Qian for their help in sample collection. This work is financially supported by the Monitoring of the ecological restoration status of mangroves in the first phase of Xiatanwei Mangrove Park, Xiamen City (No. 20233160A0406).

Author contributions

D.Y.Y., Y.J.Z. and S.C.Y. conceived the study design; Y.Z.M. and S.Z. identified specimens, Z.Y.L., S.Z., D.Y.Y. and Y.J.Z. analyzed the data and interpreted the statistical results; S.Z., Y.Z.M., Z.Y.L., D.Y.Y., Y.J.Z. and S.C.Y. wrote and reviewed the manuscript. All authors were involved in editing the manuscript and approved the final version.

Code availability

Parameters for the software tools involved are described below:

(1) FastQC: version 0.11.8, default parameters;

(2) fastp: version 0.23.4, parameters: -q 30 -l 50 -n 3;

(3) Trinity: version 2.8.5, parameters: --seqType fq --max_memory 200 G --CPU 64 --samples_file sample.txt;

(4) CD-HIT: version 4.8.1, parameters: cd-hit-est -M 16000 -T 96 -i Trinity.fasta -o Trinity_cd-hit.fa -c 0.95 -aS 0.8 -d 0;

(5) BUSCO: version 5.7.1, dataset: mollusca_odb10(2024-01-08), default parameters;

(6) MitoFinder: version 1.4, -j Sample_Trinity_cd-hit_filter -a Sample_Trinity_cd-hit_filter.fa -r.gb -t trnascan -o 5 -p 16 -m 50–adjust-direction;

(7) TransDecoder: version 5.7.1, default parameters (--max-target-seqs. 1 --outfmt 6 --evalue 1e-5);

(8) DIAMOND: version 2.1.8.162, parameters: diamond blastx --query $transcripts --db $uniprot_sprot_db --threads 64 --max-target-seqs. 1 --outfmt 6, diamond blastp --query $proteins --db $uniprot_sprot_db --threads 64 --max-target-seqs. 1 --outfmt 6;

(9) Hmmer2: version 3.1b2, parameters: hmmscan --cpu 64 --domtblout TrinotatePFAM.out $Pfam $proteins > pfam.log;

(10) Trinotate:version 3.3.0. parameters:

#init

Trinotate --db Trinotate.sqlite --init --gene_trans_map $gene_trans_map --transcript_fasta $transcripts --transdecoder_pep

$proteins_transdecoder;

#load protein hits

Trinotate --db Trinotate.sqlite --LOAD_swissprot_blastp $blastp_outfmt6;

#load transcript hits

Trinotate --db Trinotate.sqlite --LOAD_swissprot_blastx $blastx_outfmt6;

#load pfam hits

Trinotate --db Trinotate.sqlite --LOAD_pfam $pfam_out;

#report

Trinotate --db Trinotate.sqlite --report > trinotate_annotation_report.xls.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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