Sample Submission Guidelines
What is T2T Genome Sequencing?
The T2T genome refers to a gap-free genome assembly that spans from telomere to telomere, with minor gaps only permitted in specific regions such as ribosomal DNA (rDNA), sex chromosomes, and centromeres. This genome assembly showcases exceptionally high quality (Q) values for accuracy and BUSCO scores for completeness, with minimal structural errors between raw data and the final assembly.
Since the publication of the complete human X chromosome sequence in Nature in 2020, the T2T Consortium has been working diligently to refine and perfect the human genome. In 2022, the consortium unveiled the first-ever complete, gapless human genome sequence. This landmark achievement revealed highly identical segmentally duplicated regions and their variations within the human genome, marking the first time these areas have been fully characterized. This milestone represents the beginning of the T2T era in genome assembly.
Why T2T? The Limitation of Traditional Assemblies
In the history of genomics, "reference quality" has always come with an asterisk. Even the benchmark human reference genome (GRCh38) left approximately 8% of the sequence unresolved due to technological limitations. These missing pieces are not "junk DNA"—they are biologically significant regions enriched with:
- Centromeres & Telomeres: Critical for chromosome stability and cell division.
- Segmental Duplications: Hotspots for rapid evolution and disease-associated rearrangements.
- Satellites & Repeats: Complex tandem repeats that short reads fail to map uniquely.
The release of the T2T-CHM13 human genome marked a paradigm shift, proving that gapless assembly is possible. At CD Genomics, we democratize this technology, applying the same rigorous strategies to your research species. By achieving a true T2T assembly, you gain access to the complete sequence from one chromosomal end to the other, enabling the discovery of novel structural variants (SVs) and providing a perfect foundation for Pan-genome studies.
Our Technology Strategy: The HiFi + Ultra-Long Synergy
Achieving a gapless genome requires a strategic combination of sequencing power. We utilize a "Hybrid Assembly" approach that balances accuracy with extreme read length.
1. PacBio HiFi Sequencing (The Accuracy Anchor)
Role: Provides high-fidelity long reads (15-20 kb) with >99.9% accuracy.
Benefit: Resolves most genome complexities while ensuring the base-level precision (QV score) of the final assembly is extremely high.
2. ONT Ultra-Long Sequencing (The Bridge Builder)
Role: Provides ultra-long reads (up to 100kb+ or even Mb scale).
Benefit: Spans the largest repetitive regions (such as ribosomal DNA arrays and centromeres) that even HiFi reads cannot bridge. This effectively "anchors" the contigs into complete chromosomes.
3. Hi-C / Bionano (The Scaffolding Architect)
Role: Captures long-range chromatin interactions.
Benefit: Orders and orients contigs into chromosome-scale scaffolds, correcting any structural misassemblies.
T2T assembly strategy combining PacBio HiFi accuracy with Oxford Nanopore ultra long reads to resolve centromeres and genomic gaps.
Service Portfolio: Tailored T2T Solutions
CD Genomics offers a variety of flexible T2T genome sequencing service types to cater to different research objectives and budget requirements:
- T2T Genome Sequencing
By integrating advanced technologies such as third-generation sequencing and Hi-C, a "0-gap" T2T assembly is achieved for one or more chromosomes.
- Haplotype-Resolved T2T Genome Sequencing
It incorporates haplotype-resolved genome assembly, which differentiates chromosomes inherited from each parent, thus obtaining the complete genetic information from both paternal and maternal sources.
1. Vertebrate T2T Genome Sequencing
Vertebrate genomes are large (Gb scale) and complex. We offer three tiers of assembly strategies to match your budget and continuity goals.
| Strategy Tier | Technology Combination | Ideal Application |
|---|---|---|
| Foundation T2T | PacBio HiFi only | High-quality assembly for less repetitive vertebrate genomes; excellent gene space completeness. |
| Advanced T2T | PacBio HiFi + ONT Ultra-long | Recommended for resolving complex repeats, centromeres, and achieving near-gapless continuity. |
| Platinum T2T | PacBio HiFi + ONT Ultra-long + Hi-C | The ultimate standard. Anchors sequences to chromosomes for a true Telomere-to-Telomere finish. |
2. Plant T2T Genome Sequencing
Plants present unique challenges: high ploidy, massive repetitive elements, and extreme heterozygosity. Our pipelines specifically address these hurdles.
Target: Complex crops, medicinal plants, and evolutionary models.
Strategy: We utilize high-depth HiFi sequencing to distinguish between haplotypes in polyploid species, combined with ultra-long reads to span massive transposon regions common in plant genomes.
3. Bacterial T2T Genome Sequencing
For microbiology, "T2T" means achieving a single, closed circular chromosome and fully assembled plasmids without gaps.
Methodology: Hybrid approach using PacBio/ONT long reads for assembly + Illumina short reads (optional) for Polishing (Pilon correction).
Deliverable: 0 Gaps, complete plasmid assembly, methylation patterns.
T2T Sequencing Service Workflow
At CD Genomics, we offer a seamless, end-to-end whole genome sequencing service designed to ensure consistent, high-quality results. Our standardized workflow—from sample submission to data delivery—is built to support reproducibility, streamline research, and accelerate discovery across all types of studies.
- Sample Prep: Extraction of High Molecular Weight (HMW) DNA is critical. We ensure fragments are long enough for Ultra-long library construction.
- Library Construction: Parallel preparation of SMRTbell libraries (PacBio) and LSK/ULK libraries (ONT).
- Sequencing: High-depth sequencing on PacBio Revio/Sequel IIe and Oxford Nanopore PromethION platforms.
- Assembly: Utilization of T2T-specific assemblers (e.g., Hifiasm, Verkko) to integrate data types.
- Polishing & QC: Error correction and rigorous evaluation using QV scores and Busco.
- Annotation: Gene prediction and functional annotation of the newly revealed regions.
Figure 1. The CD Genomics T2T Workflow.
T2T Genome Sequencing Bioinformatics Analysis
CD Genomics offers comprehensive and flexible bioinformatics analysis services, ranging from basic data processing to advanced customized analyses.
- Assembly Graph Evaluation: Inspecting the graph for connectivity and resolving tangles.
- Continuity Metrics: Contig N50 and Scaffold N50. In successful T2T projects, Contig N50 often approaches the size of the chromosome itself.
- Completeness (BUSCO): Assessing gene content to ensure essential genes are present (>98% target).
- Accuracy (QV & Merqury): K-mer based evaluation to ensure base-level accuracy (aiming for Q50-Q70).
- Structural Consistency: Mapping Hi-C data to confirm correct orientation and order of contigs.
T2T genome sequencing Applications
It empowers researchers to resolve previously inaccessible structural variations, novel genes, and functional elements—driving breakthroughs in agriculture, medicine, and evolutionary biology.
Discovery of New Genes and Variations:
T2T sequencing enables the identification of previously unknown genes and structural variations, enhancing our understanding of genetic diversity and disease mechanisms.
Chromosomal and Telomere-End Region Investigation:
T2T sequencing reveals the genetic makeup of telomeres and centromeres, improving the understanding of chromosomal abnormalities and inheritance patterns.
Repetitive and Complex Genomic Sequences:
T2T sequencing solves the challenges of mapping repetitive and complex genomic regions, providing accurate data on difficult-to-sequence areas like segmental duplications.
Plant and Agricultural Research:
T2T sequencing supports plant genomics by mapping genetic variations related to growth, stress resistance, and disease, benefiting crop breeding and genetic engineering.
Molecular and Functional Genomics:
The technique aids in understanding the functional roles of genes involved in essential biological processes like immune response and development.
Sample Requirements
| Sample Type | Amount Required | Concentration | Purity / Integrity |
|---|---|---|---|
| Genomic DNA | ≥15μg | ≥60ng/μL | OD260/280 = 1.8-2.0; No RNA/Protein contamination. |
| Tissue Samples | Plant: >5 g Animal: >5 gCell: 1×108 |
N/A | Fresh or Liquid Nitrogen frozen. Avoid repeated freeze-thaw. |
| Blood | ≥10mL | N/A | Fresh, with EDTA anticoagulant (no Heparin). |
Note: For HMW DNA extraction, fresh tissue is always preferred over stored DNA to maximize fragment length.
What You'll Receive
- Raw data files (FASTQ)
- Alignment files (BAM) and variation files (VCF)
- Statistical and annotation reports (PDF + Excel)
- Graphical analysis results
- Project documentation and usage guidance
Why Choose CD Genomics for T2T Genome Sequencing?
From advanced sequencing platforms to high-quality data delivery, CD Genomics offers an efficient, end-to-end solution tailored to diverse research needs. Whether exploring non-model organisms or pioneering novel research areas. our team ensures reliable results with flexible support.
- Advanced Technology: We use the latest third-generation sequencing and Hi-C technologies to deliver the most accurate and complete genome assemblies.
- High Accuracy and Completeness: Our T2T sequencing provides a gap-free, error-free genome with high accuracy and completeness.
- Customizable Services: We offer flexible sequencing options tailored to meet your specific research needs.
- Expert Bioinformatics: Our bioinformatics team ensures your data is analyzed using the best tools, delivering clear and actionable insights.
- Proven Track Record: CD Genomics has years of experience in providing reliable, high-quality genomic data across various fields
Frequently Asked Questions (FAQ)
Q1: What is the main difference between "Reference Grade" and "T2T Grade" genomes?
A: Standard "Reference Grade" often refers to high-quality assemblies that still contain gaps (scaffolds). T2T Grade aims for "Zero Gaps" across the entire chromosome, including the difficult centromeric and telomeric regions. T2T provides the highest possible resolution of the genome.
Q2: Why do you combine PacBio HiFi and ONT Ultra-long data?
A: This is the "Best of Both Worlds" approach. PacBio HiFi provides the necessary accuracy to correct errors and resolve small repeats, while ONT Ultra-long reads provide the length needed to span massive repetitive regions that exceed the length of HiFi reads. This combination is currently the most effective method for gapless assembly.
Q3: Can T2T sequencing be applied to polyploid plants?
A: Yes. T2T strategies are particularly powerful for polyploids. The high accuracy of HiFi reads allows us to separate haplotypes (phasing) effectively, ensuring that sub-genomes are assembled distinctly rather than collapsed into a mosaic sequence.
Q4: Do I always need Hi-C data?
A: For Vertebrate and Plant T2T projects, Hi-C is strongly recommended. While HiFi+ONT can create massive contigs, Hi-C provides the physical linkage information to order these contigs into chromosome-scale scaffolds definitively. For bacteria, Hi-C is usually not necessary.
Case Study: Gap-Free T2T Genome Assembly of Octoploid Strawberry (Fragaria × ananassa)
Source: Adapted from Ran, K., et al. (2024). Horticulture Research.https://academic.oup.com/hr/article/11/1/uhad252/7452862
1. Background
Cultivated strawberry (Fragaria × ananassa) is an allo-octoploid species (2n=8x=56) with a highly complex genome involving four subgenomes. Traditional reference assemblies were fragmented due to high heterozygosity and repetitive elements, making it difficult to distinguish between subgenomes. The researchers aimed to construct a haplotype-resolved, gap-free T2T genome to decode the genetic divergence among these subgenomes.
2. Methods
The study utilized a state-of-the-art hybrid sequencing strategy, perfectly matching the "Platinum T2T" workflow:
- PacBio HiFi Sequencing: Generated high-fidelity long reads (~38 Gb, 46× coverage) to build the primary contig backbone.
- ONT Ultra-Long Sequencing: Produced ultra-long reads (N50 > 50 kb) to bridge large repetitive gaps.
- Hi-C Technology: Employed Hi-C data to anchor sequences into 56 distinct chromosomes and phase the haplotypes.
3. Results
The resulting assembly achieved a milestone in polyploid genomics:
- Gapless Assembly: All 56 chromosomes were assembled from telomere to telomere with 0 gaps.
- Haplotype Resolution: Successfully phased the four subgenomes (A, B, C, D), revealing that Subgenome A retains the most genes and highest expression levels.
- Epigenetic Discovery: The gap-free assembly allowed for precise mapping of DNA methylation patterns, uncovering divergence in transposable element (TE) silencing between subgenomes.
Gap-free T2T genome assembly of octoploid strawberry (Fragaria × ananassa cv. Benihoppe). The Circos plot illustrates the 56 chromosomes resolved into four subgenomes with zero gaps.
4. Conclusions
This study proves that the combination of PacBio HiFi and ONT ultra-long reads can resolve even the most complex polyploid genomes. The T2T assembly provides a foundational resource for strawberry molecular breeding, particularly for identifying genes related to fruit development and disease resistance.
References:
- Miga, Karen H., et al. "Telomere-to-telomere assembly of a complete human X chromosome." Nature 585.7823 (2020): 79-84. https://doi.org/10.1038/s41586-020-2547-7
- Nurk, Sergey, et al. "The complete sequence of a human genome." Science 376.6588 (2022): 44-53. DOI: 10.1126/science.abj6987
- Wang, Xiaoxuan, et al. "Complete genome assembly provides insights into the centromere architecture of pumpkin (Cucurbita maxima)." Molecular Plant 17.4 (2024): 773-786. https://doi.org/10.1016/j.xplc.2024.100935
- Hu, Fengkun, et al. "The telomere-to-telomere gapless genome of grass carp provides insights for genetic improvement." Science China Life Sciences (2025). doi: 10.1093/gigascience/giaf059
