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This post summarizes essential terms and techniques used in Telomere-to-Telomere (T2T) genome assembly and pangenome research, with concise definitions and reference illustrations

What are the T2T assembly and Pangenome?

T2T (Telomere-to-Telomere) assembly refers to a complete, gapless genome assembly from one telomere to the other, including previously unresolved regions like centromeres and rDNA.

Pangenome is a comprehensive reference model that captures the full range of genomic diversity across individuals, populations, or species—going beyond a single linear reference.

🖥️ Sequencing & Assembly Techniques in T2T & Pangenomics

Technique Definition Purpose / Strengths
HiFi (PacBio High-Fidelity) Long-read sequencing using circular consensus sequencing (CCS) to achieve high per-base accuracy (~Q30) with read lengths of ~15–25 kb. Long reads with high accuracy; ideal for resolving repeats and phasing haplotypes.
ONT (Oxford Nanopore) Long-read sequencing using nanopores that measure changes in electrical current as DNA passes through. Ultra-long reads (100 kb~Mb); great for spanning entire repeats, centromeres, telomeres.
Bionano Optical Mapping Maps ultra-long DNA molecules (100 kb–Mb) by fluorescently labeling specific motifs and imaging them in nanochannels. Scaffolding assemblies, detecting large structural variants (SVs), validating contiguity.
Hi-C (Chromatin conformation capture) Captures 3D spatial proximity between DNA regions using proximity ligation and sequencing. Helps in chromosome-scale scaffolding; anchors contigs based on nuclear organization.
Strand-seq Single-cell sequencing technique that sequences only one strand of DNA (template strand). Detects inversions, sister chromatid exchange; critical for resolving orientation and phasing in T2T.
Phasing Determining which variants are inherited together on the same chromosome. Enables haplotype-resolved genome assemblies; critical for understanding allele-specific variation.
Trio-binning Method for separating reads by parental origin using sequencing data from both parents. Produces haplotype-resolved assemblies from diploid individuals.
k-mer Substrings of length k from a nucleotide sequence (used in assembly, mapping). Fundamental unit for genome assembly, error correction, repeat detection, and sequence comparison.

Comparison of Genomic Technologies

Technology Read Length Accuracy Ideal for
HiFi 15–25 kb High (~Q30+) Resolving repeats, phasing
ONT 100 kb–1 Mb Moderate Centromeres, telomeres
Bionano Optical Mapping 100 kb–Mb (molecule length) N/A SV detection, scaffolding
Hi-C N/A (proximity-based) N/A Chromosome scaffolding

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🧬 Key Genomic Regions and Concepts in T2T and Pangenome Research

Term Description Why It Was Difficult to Assemble
rDNA repeats Repeated ribosomal RNA gene clusters; essential for protein synthesis. Highly repetitive sequences made alignment and assembly ambiguous.
Acrocentric DNA DNA on short arms of acrocentric chromosomes (13, 14, 15, 21, 22) containing rDNA and satellite DNA. Dense with similar repeats; often collapsed in assemblies.
Segmental Duplications (SDs) Large blocks (1–200 kb) of duplicated DNA with high sequence similarity. Difficult to distinguish between duplicated regions.
Centromeres Central chromosome regions critical for proper segregation during cell division; rich in alpha-satellite repeats. Extremely repetitive and megabase-sized; unreadable with short reads.
Telomeres Chromosome ends composed of repetitive TTAGGG sequences; protect against genomic instability. Simple and extremely repetitive; hard to resolve start/end.
VNTRs (Variable Number Tandem Repeats) Short sequence motifs repeated in tandem with variable counts among individuals. Hard to accurately estimate repeat number using short reads.
Heterochromatin Densely packed, gene-poor chromatin regions, often transcriptionally silent. Largely inaccessible and repetitive; often excluded from reference genomes.
Satellite DNA Tandemly repeated sequences found in centromeres and heterochromatic regions. Similar to alpha-satellite; hard to sequence and map due to size and uniformity.
Polymorphic Structural Variants (SVs) Large-scale genome changes (insertions, deletions, inversions, duplications) that vary among individuals. Often missed or mischaracterized by short-read sequencing.
Inversion toggling Regions that repeatedly flip orientation between species or populations. Hard to detect with short reads; can be misassembled.
Haplotypes / Haplotype-resolved assembly Distinct sets of variants inherited together on a chromosome. Phasing across long distances needs long reads and complex algorithms.
Haplotype false duplication Error where heterozygous variants are misrepresented as duplicated regions in assembly. Caused by failure to collapse allelic variation; often occurs in diploid genomes assembled without phasing.
Microchromosomes Tiny, gene-rich chromosomes seen in birds, reptiles, etc. Numerous, small, GC-rich, and repeat-dense; often fragmented or misassembled due to their short length and limited long-range scaffolding signals.

🐒 Structural & Evolutionary Genomics Concepts

Term Definition
Hybridization Breeding between genetically distinct lineages.
Introgression Gene flow between species/populations via hybrid backcrossing.
Synteny Conservation of blocks of genes across species, not necessarily in the same order.
Collinearity Preservation of gene order across species.

🧩 Pangenome Graph Concepts

Term Definition
Bidirected graph Graph representing both DNA strands (forward/reverse).
Node DNA sequence unit in the graph; may be shared across multiple haplotypes.
Edge Connectors between nodes, indicating how they form complete sequences.
Path A concatenation of nodes representing a specific haplotype.
Bubble Region where paths diverge and reconverge, reflecting sequence variation.
Snarl A generalized bubble with complex internal connections.

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Reference

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