Animal/Plant Whole Genome De Novo Sequencing

CD Genomics offers an end-to-end animal and plant whole genome de novo sequencing service combining PacBio Revio HiFi and Oxford Nanopore PromethION long-read platforms with optional Hi-C scaffolding, delivering chromosome-level and telomere-to-telomere (T2T) assemblies without dependence on a reference genome. The resulting assemblies resolve centromeres, telomeres, rDNA arrays, segmental duplications, and repetitive regions that short-read approaches cannot assemble.

For species with no reference genome, complex polyploidy, or heterozygosity profiles that confound existing tools, our dual-platform strategy provides a flexible, independently validated assembly path from raw reads to annotated, publication-ready genome sequences.

Why researchers choose our de novo genome sequencing service

• PacBio Revio HiFi reads: ≥99.9% single-read accuracy, N50 typically 15–20 kb, enabling high-confidence SNV and structural variant calling
• ONT PromethION ultra-long reads: N50 routinely >50 kb, up to 2 Mb+ per read, spanning centromeric repeats and complex transposon arrays
• Hybrid HiFi + ONT + Hi-C strategy for complete T2T and haplotype-resolved assemblies
• Full bioinformatics pipeline: assembly, polishing, chromosome anchoring, gene annotation, repeat masking, and comparative genomics
• Applicable to any eukaryote — plants, animals, fungi — regardless of genome size or ploidy

Introduction to Animal/Plant Whole Genome De Novo Sequencing

De novo genome sequencing reconstructs a genome sequence directly from raw reads, without alignment to a pre-existing reference. It is the only approach capable of capturing genome-specific features such as novel chromosomal rearrangements, species-specific gene family expansions, and structural variants absent from any reference, making it the foundation of new genome projects and a necessity for species where reference genomes are incomplete, outdated, or do not exist.

Historically, the prohibitive cost and assembly complexity of long-read technologies — combined with the requirement for high-molecular-weight DNA — limited de novo assembly to a small number of model organisms. The introduction of PacBio Revio, which roughly quadruples the throughput of Sequel IIe at equivalent or lower per-base cost, and the scaling of Oxford Nanopore PromethION to routine ultra-long read lengths (N50 > 100 kb with optimal library preparation), has transformed de novo assembly into a cost-accessible, high-resolution service for any eukaryotic species.

Modern assemblies routinely achieve contig N50 values exceeding 20 Mb for plant and animal genomes, with chromosome-level scaffolding enabled by Hi-C chromatin conformation data. Where ultra-long ONT reads are combined with HiFi accuracy, gapless telomere-to-telomere assemblies are now achievable for most diploid and some polyploid species, resolving regions that all previous technologies left unassembled.

Assembly Strategy Selection

Not every genome requires the same data strategy. CD Genomics consults with clients on the biological question, genome characteristics (size, ploidy, repeat content, heterozygosity), and downstream use cases to select the most cost-efficient assembly path before sequencing begins.

For established species with reference genomes that need structural variant characterisation or resequencing, see our Animal/Plant Whole Genome Resequencing service. For pan-genome projects requiring multiple high-quality assemblies from the same species, CD Genomics offers scalable multi-accession de novo workflows.

Applications of Animal/Plant Whole Genome De Novo Sequencing

New Reference Genome Construction

• Establish the first high-quality reference genome for a species with no existing assembly.
• Replace fragmented short-read draft genomes with chromosome-level or T2T-quality sequences.
• Provide a community resource for population genetics, comparative genomics, and gene function studies.

Polyploid and Complex Plant Genome Resolution

• Resolve sub-genomes in allotetraploid or higher-ploidy crop species (wheat, cotton, sugarcane, coffee) where short reads collapse homeologous chromosomes.
• Characterise genome-specific repeat landscapes and gene family expansions in polyploids.
• Support molecular breeding programs by providing accurate gene model positions across all haplotypes.

Structural Variant Discovery and Comparative Genomics

• Detect large insertions, deletions, inversions, translocations, and copy number variants (>50 bp) that short reads miss.
• Compare genome architecture between ecotypes, populations, or related species to identify adaptive structural differences.
• Identify gene fusion events, chromosomal rearrangements, and transposable element activity shaping genome evolution.

Centromere, Telomere, and Repeat Region Biology

• Sequence and annotate centromeric satellite arrays and CENH3-binding domains for epigenomic studies.
• Map telomere lengths and sub-telomeric repeat organisation at chromosome resolution.
• Characterise active transposable element families and their insertion site distribution.

Pan-Genome and Population Reference Projects

• Generate multiple high-quality assemblies across accessions or individuals to build a species pan-genome.
• Capture dispensable genome content (presence/absence variants) relevant to stress tolerance or disease resistance in crop species.
• Anchor population resequencing studies to a more representative reference than any single individual assembly.

Genome-Assisted Breeding and Conservation Genetics

• Identify genes underlying agronomic traits through QTL mapping anchored to a chromosome-level assembly.
• Assess genetic diversity and population structure in endangered species for conservation planning.
• Support marker-assisted selection by providing accurate physical maps of target genomic regions.

Bioinformatics Analysis

CD Genomics provides a structured bioinformatics pipeline covering assembly, quality evaluation, annotation, and comparative analysis. Analysis scope is agreed before project start and documented in the final delivery report.

Sample Requirements

High-molecular-weight, high-purity DNA is the single most critical determinant of assembly quality in long-read de novo sequencing. Samples that fail to meet minimum integrity thresholds are typically flagged at QC before library preparation at no additional charge.

For plant samples with high polysaccharide or secondary metabolite content (e.g., conifers, citrus, legumes), CD Genomics offers in-house HMW DNA extraction optimised for difficult plant matrices. Contact our team before sample preparation to confirm the recommended extraction protocol for your species. For long-amplicon target sequencing rather than whole genome, see our Animal/Plant Long Amplicon Sequencing service.

Case Study: Haplotype-Resolved T2T Genome Assembly of Populus nigra NL-1976 Using PacBio Revio and ONT PromethION

Liu F, Wang Y, Geng T, et al. Haplotype-resolved T2T genome assembly of the Populus nigra NL-1976. Scientific Data 2025. DOI: 10.1038/s41597-025-06361-2.

1. Background

Populus nigra (black poplar) is an ecologically and economically important forest tree whose genome had not been assembled to full haplotype-resolved, telomere-to-telomere quality. The species' high heterozygosity and complex repeat landscape made it an ideal test case for evaluating the combined capacity of PacBio Revio HiFi, ONT PromethION ultra-long, and Hi-C sequencing for a challenging woody plant genome.

2. Methods

High-molecular-weight genomic DNA was extracted from Populus nigra NL-1976 and subjected to three sequencing strategies run in parallel:

• PacBio Revio HiFi: 53.14 Gb generated (approximately 124× haploid genome coverage), HiFi read N50 = 18.84 kb, 2,852,700 reads total
• ONT PromethION: 47.82 Gb generated, 464,052 reads, average read length 103,053.2 bp, N50 = 100 kb, N90 = 78,120 bp — confirmed ultra-long library performance
• Hi-C: Generated for chromosome-scale scaffolding of the two haplotype assemblies

Assembly was performed with Hifiasm in HiFi+ONT+Hi-C mode, producing two phased haplotype contig sets that were scaffolded separately to chromosome level using Hi-C contact data and verified for telomere and centromere completeness.

3. Results

Both haplotype assemblies were delivered at near-T2T quality:

• nigraHap1: total length 385.18 Mb, 19 chromosomes, contig N50 = 22.31 Mb, BUSCO completeness = 98.7% (embryophyta_odb10), QV = 41.57
• nigraHap2: total length 390.48 Mb, 19 chromosomes, contig N50 = 22.05 Mb, BUSCO completeness = 98.8%, QV = 40.70
• Gene annotation: 49,077 genes (nigraHap1) and 50,129 genes (nigraHap2)
• Repeat content: 46.35% (nigraHap1) and 47.35% (nigraHap2)
• Telomere and centromere sequences were resolved on all 19 chromosomes in both haplotypes

Figure 4 from Liu et al. (2025). Circos plot of the two haplotype-resolved Populus nigra assemblies (nigraHap1 and nigraHap2) showing chromosome-level features including gene density, repeat distribution, and inter-haplotype synteny. Reprinted under CC BY 4.0.

4. Conclusions

This study demonstrates that PacBio Revio HiFi combined with ONT PromethION ultra-long reads and Hi-C scaffolding can deliver fully haplotype-resolved, chromosome-level T2T assemblies for a complex, heterozygous woody plant genome at QV >40. The approach provides a reproducible template directly applicable to other forest trees, crop species, and animal genomes of comparable complexity.

Demo

1. Assembly Continuity Plot — Contig N50 comparison across assembly strategies (HiFi-only vs HiFi+ONT hybrid) for a representative plant genome

2. BUSCO Completeness Bar Chart — Genome completeness assessment against embryophyta_odb10 database showing C, D, F, M proportions for both haplotypes

3. Contig Length vs QV Scatter Plot — Per-contig quality value (Merqury) plotted against contig length for the final polished assembly

For Research Use Only. Not for use in diagnostic procedures.