Abstract
This study reported the first telomere-to-telomere (T2T) genome assembly of Castanopsis orthacantha, a keystone tree species with significant ecological and economic values endemic to the subtropical evergreen forests of southwestern China. Using multi-platform sequencing data and high-throughput chromosome conformation capture (Hi-C) scaffolding, we successfully generated a chromosome-scale assembly. The final assembly spanned 893.28 Mb, with a contig N50 of 76.19 Mb, indicating a high degree of continuity. Remarkably, 97.94% of the genome was successfully anchored to 12 chromosomes. Terminal telomeric repeat sequences were identified at both ends of all of the chromosomes, and the assembly contained only a single unresolved gap. A total of 35,978 protein-coding genes were detected in the assembly, with an average coding sequence (CDS) length of 1,116.3 bp. Genomic analysis further revealed that repetitive elements comprised 59.28% of the genome. The generation of this near-complete reference genome of C. orthacantha provides a critical genomic resource for advancing evolutionary study within the Fagaceae family and supports conservation genomics strategies aimed at the ecological restoration of this species.
Subject terms: Genome, Genome evolution
Background & Summary
Castanopsis is one of the most species-rich genera within the Fagaceae family, comprising approximately 120 species predominantly found in the tropical and subtropical regions of Asia1,2. China serves as a key distribution center for Castanopsis, with approximately 70 species including 30 species that are endemic to the region3,4. One notable species, Castanopsis orthacantha is native to southwestern China and primarily inhabits the subtropical evergreen broad-leaved forests of the Yunnan plateau. It typically grows at elevations ranging from 1,700 to 2,500 m5. C. orthacantha holds considerable economic and ecological significance across multiple domains, such as timber production and food supply. Its hardwood is dense, highly durable, and naturally resistant to decay, making it a valuable resource for construction and furniture manufacturing. In addition to its timber value, the species produces edible nuts that have historically served as a crucial food source for local ethnic communities during periods of food scarcity. Beyond its utilitarian applications, C. orthacantha plays a vital role in supporting ecological balance and biodiversity within subtropical montane forest ecosystems6. As a dominant and foundational species, it significantly contributes to species diversity, soil and water conservation, carbon sequestration, and climate regulation across southwestern China5.
This study presents the first telomere-to-telomere (T2T) genome assembly of C. orthacantha, constructed using a comprehensive integration of Illumina short reads, PacBio high-fidelity (HiFi) sequencing data, Oxford Nanopore Technology (ONT) ultra-long reads, and high-throughput chromosome conformation capture (Hi-C) scaffolding. The final genome assembly spans 893.28 Mb, with a contig N50 of 76.19 Mb, demonstrating a high level of assembly continuity. A total of 874.83 Mb, accounting for 97.94% of the assembled genome, was successfully anchored to 12 pseudo-chromosomes. The assembly contained only a single gap, with telomeric repeat sequences detected at both termini of all the chromosomes. The consensus sequence accuracy, as indicated by the Merqury quality value (Qv), was 60.46. Completeness assessment using benchmarking universal single-copy orthologs (BUSCOs) revealed 99.9% of conserved eukaryotic genes from the embryophyta_odb12 dataset. This T2T genome provides a complete and authoritative reference for of C. orthacantha, enabling systematic investigation of complex, repeat-rich regions that are often unresolved in fragmented assemblies. It offers a crucial foundation for deciphering the genetic basis of the species’ adaptation to extreme habitats and for elucidating mechanisms of forest resilience and ecosystem stability under climate change, thereby supporting future functional and ecological research in this keystone species.
Methods
Sampling and DNA/RNA extraction
In 2024, a mature specimen of C. orthacantha was selected from Maxiong Mountain, located in Qujing City, Yunnan Province, China (25°54′13.6″ N, 103°55′55.3″ E) (Fig. 1a). Young leaves were harvested, immediately flash-frozen in liquid nitrogen, and subsequently stored in a freezer set at –80 °C until DNA extraction. Genomic DNA was isolated using a TIANGEN plant DNA kit (DP305, Tiangen Biotech, Beijing) in accordance with the manufacturer’s protocol. For transcriptome sequencing, total RNA was extracted from leaves, flowers, and young stems with a TIANGEN RNAprep Pure Plant Kit (DP441, Tiangen Biotech, Beijing). The integrity and purity of the extracted nucleic acids were evaluated through 1% agarose gel electrophoresis, complemented by quantification using the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham).
Fig. 1.
Morphological traits and genome features of Castanopsis orthacantha. (a) Tree, flower, and fruit of C. orthacantha. (b) Genome size estimation based on k-mer (21-mer) frequency distribution analysis. (c) Hi-C interaction heat map of C. orthacantha.
Illumina short-read sequencing and genome survey
Genomic DNA libraries with 350 bp insert sizes were constructed using Covaris sonication-based fragmentation. This step was followed by a series of enzymatic treatments, including end repair, A-tailing, adapter ligation, and subsequent PCR amplification. Paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq 6000 platform, producing 49.11 Gb of sequencing data (Table 1). Raw sequencing data were subjected to quality filtering using fastp (version 0.19.5)7 with the following criteria: (1) removal of reads containing adapter sequences; (2) exclusion of reads with >10% N bases; and (3) elimination of reads with >20% low-quality bases (Q-score < 5). Subsequently, the k-mer frequency distribution (k = 21) of the filtered genomic sequencing data was calculated using Jellyfish (version 2.2.7)8. Genomic features were assessed through statistical fitting algorithms implemented in GenomeScope (version 2.0)9. For C. orthacantha, the genome size was estimated at 849.45 Mb, with a heterozygosity rate of 1.75% and a repeat content of 51.30% (Fig. 1b).
Table 1.
Statistics of the sequencing data for C. orthacantha.
| libraries | Insert size (bp) | Total data (Gb) | Read length (bp) | Sequence coverage (×) |
|---|---|---|---|---|
| Illumina reads | 350 | 49.11 | 150 | 56.95 |
| PacBio HiFi reads | — | 64.57 | — | 74.88 |
| ONT ultra-long reads (>100 kb) | — | 35.56 | — | 41.24 |
| Hi-C | 350 | 96.17 | 150 | 111.52 |
| Total | — | 245.41 | — | 284.59 |
Long-read sequencing and Hi-C sequencing
A HiFi sequencing library, comprising insert sizes ranging from 15–18 kb, was prepared using a SMRTbell Express Template Prep Kit 3.0 (Pacific Biosciences). Sequencing was subsequently performed on the PacBio Revio platform. The resulting subreads were subjected to a length filter, retaining only those with a minimum length of 50 bases. HiFi reads were then processed using ccs10 software, applying stringent parameters of a minimum of three passes (min-passes = 3), and a minimum read quality threshold of 0.99 (min-rq = 0.99). This procedure ultimately yielded a total of 64.57 Gb PacBio HiFi sequencing data, corresponding to approximately 74-fold coverage of the target genome. Additionally, a one-dimensional (1D) ultra-long DNA library was constructed using the Ligation Sequencing 1D kit protocol provided by Oxford Nanopore Technology. This library was sequenced using the PromethION platform, generating a total of 35.56 GB of long-read data. The sequencing data demonstrated high-quality long reads, with an N50 read length of 100 kb and 93.3% of reads that achieved a quality score of Q10 or above. To anchor contigs to chromosomes, Hi-C libraries were prepared by crosslinking chromatin with formaldehyde, digesting the DNA with the MboI restriction enzyme, repairing the ends with biotinylation, and performing proximity ligation11. Sequencing was performed on the Illumina NovaSeq platform, yielding 96.16 Gb of paired-end data (Table 1). The raw Hi-C reads were processed using fastp7, applying the same quality filtering parameters as described previously.
De novo genome assembly
The HiFi sequencing reads were assembled de novo into contigs using Hifiasm (version 0.19.9)12 with default parameters, producing contigs with an N50 of 63.30 Mb. To construct chromosome-level scaffolds, Hi-C data were integrated using ALLHiC (version 0.9.8)13, which enabled the accurate grouping and orientation of contigs. The ALLHiC pipeline was executed with the following parameter settings: “–minREs 50,–maxlinkdensity 3, and–NonInformativeRatio 2”. The initial automated scaffolding output was then manually curated in Juicebox (version 1.11.08)14 to correct mis-assemblies and optimize the assembly. This step involved breaking chimeric joins and adjusting the order and orientation of contigs or scaffolds where the Hi-C interaction matrix indicated conflicting or weak support, thereby resolving local ambiguities primarily caused by repetitive regions. The final chromosome-scale assembly comprised 12 pseudomolecules, each corresponding to a chromosome, along with 131 unanchored scaffolds. The measured genome size was 893.20 Mb, with only five gaps remaining. Remarkably, 97.94% of the genome (874.83 Mb) was successfully anchored to chromosomes (Fig. 1c). To enhance the completeness of the genome assembly, the Hi-C scaffolded genome assembly was further processed using TGS-GapCloser (version 1.2.1)15. This process incorporated ONT ultra-long reads, featuring an N50 value >100 kb, applied under the software’s default parameters. Subsequently, the draft assembly was polished by Racon (version 1.4.20)16, using both PacBio HiFi and Illumina short-read datasets. The finalized chromosome-level assembly of C. orthacantha demonstrated superior continuity, with a 20.3% increase in contig N50, improving from 63.30 to 76.19 Mb. Additionally, the assembly achieved near-complete gap closure, reducing gaps from five to one (Fig. 2a, Table 2). To assess potential contamination, we performed a blastn search of the 131 unanchored scaffolds against the NCBI core-nt database. Alignments were predominantly to species within the Fagaceae family. Only two contigs produced matches to taxa outside Fagaceae: Contig 92 matched Betula, Gossypium and Vitis; Contig 129 matched Vitis (Table S1). No alignments to non-plant taxa were detected, confirming the absence of detectable exogenous contamination in the final assembly.
Fig. 2.
Visualization of the T2T genome assembly of C. orthacantha. (a) Gene density and telomere distribution across the 12 chromosomes. The purple rectangle indicates assembly gap, and blue triangles denote telomeres. Gene density is represented using a sliding window of 100 kb, with blue and red colors indicating 0–37 genes per window. (b) Genomic map of C. orthacantha: (i) The 12 chromosomes; (ii) GC content density; (iii) Gene density; (iv) Tandem repeat density; (v) LTR Copia density; (vi) LTR Gypsy density; and (vii) Intragenomic synteny information. All of the above statistics were calculated using a 100 kb sliding window.
Table 2.
Chromosome-level genome assembly statistics for C. orthacantha.
| Type | Length (bp) | Number | ||
|---|---|---|---|---|
| Contig | Scaffold | Contig | Scaffold | |
| Total | 893,276,527 | 893,276,627 | 144 | 143 |
| Max | 102,159,305 | 102,159,305 | — | — |
| N50 | 76,186,574 | 76,186,574 | 5 | 5 |
| N60 | 66,176,003 | 66,176,003 | 7 | 7 |
| N70 | 64,179,113 | 64,179,113 | 8 | 8 |
| N80 | 60,132,393 | 60,132,393 | 10 | 10 |
| Total Gap | 1 | |||
Genome annotation
A comprehensive annotation framework integrating both homology-based and de novo methodologies was applied to characterize repetitive sequences within the C. orthacantha genome. For homology-driven prediction, the RepBase17 database was leveraged in conjunction with RepeatMasker (version 4.1.2-p1)18 and RepeatProteinMask18, using default parameters. In parallel, de novo prediction was performed using RepeatModeler (v5.8.8)19, which generated an initial repeat library. From the generated library, sequences >100 bp containing <5% ambiguous bases (N) were selected to construct a preliminary TE database. This de novo library was then integrated with the RepBase database and clustered using UCLUST to generate a non-redundant, custom repeat library. The resulting library was subsequently employed as input for RepeatMasker to facilitate the identification of repetitive DNA elements. Tandem repeat sequences were detected using Tandem Repeats Finder (TRF, version 4.09)20. The results revealed that repetitive sequences constitute 59.28% of the C. orthacantha genome. These repeats include short interspersed nuclear elements (578.85 Kb), long interspersed nuclear elements (42.74 Mb), long terminal repeats (304 Mb), DNA transposons (159.68 Mb), and other unclassified elements (14.29 Mb) (Table 3).
Table 3.
Classification of the repeats in the C. orthacantha genome assembly.
| Type | Number | Length (bp) | Percentage (%) |
|---|---|---|---|
| SINE | 1,875 | 578,852 | 0.06 |
| LINE | 125,846 | 42,738,536 | 4.78 |
| LTR | 416,679 | 304,603,065 | 34.10 |
| DNA | 859,130 | 159,677,554 | 17.88 |
| Unknown | 72,137 | 14,287,656 | 1.60 |
| Total | 529,564,860 | 59.28 |
SINE, short interspersed nuclear elements; LINE, long interspersed nuclear elements; LTR, long terminal repeat; DNA, DNA transposons.
The structural annotation of genes was conducted through an integrative approach, combining ab initio prediction, homology-based prediction, and RNA-seq-assisted evidence. Ab initio gene predictions were generated using Augustus (version 3.5)21 and SNAP (version 2013.11.29)22, which identify putative gene models based on inherent statistical signatures. The specific command lines employed for Augustus and SNAP were: “--species =pasa1 --uniqueGeneId =TRUE --noInFrameStop =TRUE --GFF3=on --genemodel=complete --strand=both” and “-gff pasa1.hmm”, respectively. For homology-based annotation, protein sequences from Castanopsis hystri, Castanopsis tibetana, and Arabidopsis thaliana were aligned against the genome assembly using tblastn (version 2.2.26) with an e-value cutoff of ≤1 × 10−5. Subsequently, GeneWise (version 2.4.1)23 was employed to perform precise spliced alignments to accurately infer gene structures. In parallel, RNA-seq data obtained from multiple tissues, such as leaves, flowers, and young stems, were assembled using Trinity (version 2.8.5)24. The assembled transcripts were subsequently mapped to the genome using Hisat2 (version 2.2.1)25, and further processed using StringTie (version 2.2.1)26 to reconstruct transcript isoforms and refine exon-intron boundaries. The final gene annotation set was integrated using EvidenceModeler (EVM, version 1.1.1)27, employing the parameters “--species=pasa1 --uniqueGeneId=TRUE --noInFrameStop=TRUE --GFF3=on --genemodel=complete --strand=both”. This step consolidated gene predictions from multiple sources. The integrated models were then refined using PASA (version 2.3.3)28 to incorporate RNA-seq splice-junction evidence and concurrently mask transposable elements. To obtain a high-confidence annotation, stringent filters were applied: removing models with ≥ 20% overlap with TEs, those containing premature stop codons or ambiguous bases (Ns), retaining only expressed de novo predictions (RPKM ≥ 1), and requiring additional support (homology, transcript, or expression) for single-exon genes. The final annotated gene set comprised 35,978 protein-coding genes, with an average CDS length of 1,116.3 bp and a mean of 4.61 exons per gene (Table 4). Intragenomic synteny blocks were identified through an all-against-all BLASTP search of the protein sequences, followed by collinearity analysis using MCScanX (version 1.0.0)29. Furthermore, chromosome-level characteristics—including GC content, gene density, and repetitive element distribution—were systematically quantified and visualized on the genome map (Fig. 2b).
Table 4.
Predicted gene sets in the C. orthacantha genome assembly.
| Type | Gene Set | Number | Average transcript length (bp) | Average CDS length (bp) | Average exons per gene | Average exon length (bp) | Average intron length (bp) |
|---|---|---|---|---|---|---|---|
| De novo | Augustus | 50,026 | 3,530.64 | 967.32 | 3.93 | 246.11 | 874.72 |
| SNAP | 32,112 | 8,975.83 | 528.71 | 3.21 | 164.95 | 3,830.29 | |
| Homolog | Castanopsis hystri | 43,101 | 3,831.25 | 888.53 | 3.79 | 234.73 | 1,056.51 |
| Arabidopsis thaliana | 22,066 | 4,856.58 | 1,130.95 | 4.6 | 245.71 | 1,034.09 | |
| Castanopsis tibetana | 40,300 | 4,000.57 | 961.83 | 4.16 | 231.05 | 960.76 | |
| RNAseq | transcripts | 38,760 | 11,931.86 | 2,085.87 | 6.84 | 305.13 | 1,687.13 |
| PASA | 26,927 | 4,614.51 | 1,025.95 | 4.21 | 243.41 | 1,116.25 | |
| EVM | EVM | 48,531 | 4,094.13 | 976.08 | 3.99 | 244.69 | 1,043.18 |
| PASA-update | PASA-update | 48,436 | 4,055.01 | 980.81 | 3.98 | 246.26 | 1,030.66 |
| Final set | Final | 35,978 | 4,792.09 | 1,116.31 | 4.61 | 242.11 | 1,018.00 |
The tRNAs within the genome were predicted using tRNAscan-SE (version 1.4)30 with default parameter settings. A comparative genomics approach was implemented by aligning the genome sequence against known rRNA sequences from closely related species using blastn, with a stringent e-value threshold set at <1 × 10−10. Additional non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and small nuclear RNAs (snRNAs), were systematically identified through homology-based searches against the Rfam database (release 14.1). This analysis was performed using Infernal (version 1.1.4)31, which employs covariance models for the sensitive detection of RNA families. The search parameters included an e-value cutoff of 0.01, in addition to bit score thresholds specific to each RNA family, as defined in the Rfam database. As a result, the genome was found to contain 957 miRNAs, 1,175 tRNAs, 7,706 rRNAs, and 654 snRNAs (Table 5).
Table 5.
Summary of ncRNA statistics in the C. orthacantha genome.
| Type | Copy number | Average·length (bp) | Total·length (bp) | Percentage of genome (%) | |
|---|---|---|---|---|---|
| miRNA | miRNA | 957 | 124.184 | 118,844 | 0.013 |
| tRNA | tRNA | 1,175 | 75.39 | 88,583 | 0.01 |
| rRNA | rRNA | 7,706 | 341.205 | 2,629,322 | 0.294 |
| 18S | 1,008 | 1,703.73 | 1,717,360 | 0.192 | |
| 28S | 3,737 | 142.996 | 534,376 | 0.06 | |
| 5.8S | 952 | 161.172 | 153,436 | 0.017 | |
| 5S | 2,009 | 111.573 | 224,150 | 0.025 | |
| snRNA | snRNA | 654 | 130.404 | 85,284 | 0.01 |
| CD-box | 353 | 110.731 | 39,088 | 0.004 | |
| HACA-box | 102 | 159.225 | 16,241 | 0.002 | |
| splicing | 191 | 149.147 | 28,487 | 0.003 | |
| scaRNA | 8 | 183.5 | 1,468 | 0 | |
| Unknown | 0 | 0 | 0 | 0 |
Gene functional annotation
Gene function annotation was performed by aligning the predicted protein sequences against the Swiss-Prot32 and non-redundant (NR)33 protein databases using blastp (version 2.2.26) with an e-value threshold of ≤1 × 10−5. Protein domains and conserved motifs were systematically annotated using InterProScan (version 5.39)34, with the following parameters: -cpu 20 -format tsv –appl ProDom,SMART,ProSiteProfiles,PRINTS,Pfam,Panther –iprlookup -dp –goterms. This analysis incorporated multiple databases, including InterPro and Pfam, to ensure comprehensive domain annotation. Gene Ontology (GO) terms were subsequently assigned using eggNOG-mapper (version 2.1.5)35. Pathway analysis was conducted by mapping the gene set to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/kaas/), where the most appropriate pathway for each gene was identified. Incorporating all of the above analyses, a total of 35,573 genes, representing 98.87% of the predicted gene set in the C. orthacantha genome, were successfully annotated with potential functions (Table 6).
Table 6.
Gene functional annotation of the C. orthacantha genome.
| Type | Number | Percent (%) |
|---|---|---|
| Total | 35,978 | 98.87 |
| Nr | 35,349 | 98.25 |
| Swissport | 25,617 | 71.2 |
| KEGG | 26,016 | 72.31 |
| InterPro | 33,077 | 91.94 |
| Pfam | 23,748 | 66.01 |
| GO | 19,889 | 55.28 |
| Annotated | 35,573 | 98.87 |
| Unannotated | 405 | 1.13 |
Data Records
The genomic datasets generated and analysed during the current study are available in the following repositories. The raw sequencing data for C. orthacantha, encompassing Illumina short reads (accession CRR2038529), PacBio HiFi reads (CRR2038533), ONT ultra-long reads (CRR2038535), Hi-C reads (CRR2038534), and RNA-seq data from leaf (CRR2038530), flower (CRR2038531), and stem (CRR2038532) materials, have been deposited in the National Genomics Data Center (NGDC; https://ngdc.cncb.ac.cn/)36,37 under the BioProject accession number PRJCA04417538. The assembled genome sequence has been deposited in the NCBI GenBank database under accession JBSYXC00000000039. The assembled genome assembly is also available in the NGDC genome warehouse under the accession number GWHGGEP00000000.140. Additionally, the genome annotation dataset, comprising the GFF files (for genes and repeat elements), CDS, and protein sequences, have been deposited in figshare41.
Technical Validation
To evaluate the accuracy of the genome assembly, short-insert library reads were aligned to the assembled genome using BWA (version 0.7.8)42. The alignment rate of the reads was approximately 98.94%, with the genome coverage reaching 99.98%. Additionally, the PacBio HiFi long reads were mapped to the genome assembly using minimap2 (version 2.24-r1122)43, resulting in 99.93% of the reads being successfully mapped and achieving a genome coverage of 99.97% (Table 7). BUSCO (version 6.0.0)44 analysis demonstrated that the C. orthacantha genome assembly encompassed 99.9% of the complete BUSCO genes (embryophyta_odb v12; a total of 2026 genes), including 1933 single-copy (95.4%) and 90 duplicated genes (4.4%). The genome Qv, as assessed by Merqury (version 1.4.1)45, was 60.46. Furthermore, the LTR Assembly Index (LAI)46 calculated using LTR_retriever (version 2.9.0)47 was 20.72. Collectively, these results indicate that the C. orthacantha genome assembly is of high quality and meets reference-grade standards.
Table 7.
Mapping rate and coverage of Illumina short-read and HiFi long-read sequencing datasets.
| Features | Illumina short reads | HiFi reads |
|---|---|---|
| Mapping rate (%) | 98.94 | 99.93 |
| Coverage rate (%) | 99.98 | 99.97 |
| Coverage at least 4 × (%) | 99.90 | 99.83 |
| Coverage at least 10 × (%) | 99.43 | 99.70 |
| Coverage at least 20 × (%) | 95.65 | 98.84 |
Supplementary information
Acknowledgements
This study was supported by the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities (202401BA070001-126), the Project for Science Research in Department of Education in Yunnan (2024J0937 and 2026J0935), the funds of the Yunnan Revitalization Talents Support Plan (YNQR-QNRC-2018-127), and the Program of Innovation Research Team from Qujing Normal University.
Author contributions
Y.G. designed the study framework, and supervised the project. S.Y. and H.W. were responsible for sample collection and processing. S.Y., H.W., H.C., Y.Z., C.L. and Y.X. analyzed the data. S.Y. drafted the manuscript, and Y.G. revised it. All of the authors have read and approved the final version of the manuscript.
Data availability
All data generated in this study are openly available under the following accession numbers: raw sequencing reads at NGDC GSA (PRJCA044175, CRR2038529-35), assembled genome at NGDC GWH (GWHGGEP00000000.1) and NCBI GenBank (JBSYXC000000000), and annotation files at figshare (10.6084/m9.figshare.29938532).
Code availability
No custom code was used in this study. All analyses were performed with publicly available software following the standard protocols recommended by their developers. The specific versions and parameters used are described in detail in the Methods section.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
The online version contains supplementary material available at 10.1038/s41597-026-06787-2.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- Yin, S. et al. A telomere-to-telomere genome assembly of Castanopsis orthacantha (Fagaceae). figshare10.6084/m9.figshare.29938532 (2025). [DOI] [PMC free article] [PubMed]
Supplementary Materials
Data Availability Statement
All data generated in this study are openly available under the following accession numbers: raw sequencing reads at NGDC GSA (PRJCA044175, CRR2038529-35), assembled genome at NGDC GWH (GWHGGEP00000000.1) and NCBI GenBank (JBSYXC000000000), and annotation files at figshare (10.6084/m9.figshare.29938532).
No custom code was used in this study. All analyses were performed with publicly available software following the standard protocols recommended by their developers. The specific versions and parameters used are described in detail in the Methods section.