Assembly • Telomere-to-Telomere
Authority: T2T Consortium / Earth BioGenome Project
Production Blueprint v4.512. High-C & HiFi Telomere-to-Telomere (T2T) De Novo Genome Assembly
Gapless chromosome-scale phased diploid genome assembly combining ultra-accurate PacBio HiFi reads with chromosome conformation capture (Hi-C) scaffolding using Hifiasm and YaHS.
| Metric / Requirement | Specification | Target Configuration |
|---|---|---|
| Core Engine / Framework | Hifiasm + YaHS + Merqury + BUSCO v5 | Nextflow / Snakemake Compatible |
| Benchmark Runtime | ~12 – 20 hours (30× HiFi + 30× Hi-C) | 32 CPU cores / 128 GB RAM |
| Standard Container | quay.io/biocontainers/hifiasm:0.19.8--h43eeafb_0 | Docker • Singularity • Conda |
| Input → Deliverable | PacBio HiFi FASTQ + Hi-C FASTQ → Phased Chromosome FASTA, Merqury & BUSCO QC | |
Workflow Chain: PacBio HiFi + Hi-C → Hifiasm (Phased contigs) → YaHS (Hi-C scaffolding) → Inspector / Merqury QV → BUSCO v5
⚡
Step 1: Phased Telomere-to-Telomere Contig Assembly with Hifiasm
Hifiasm v0.19+
Generates telomere-to-telomere phased contigs using dual-haplotype graph resolving algorithms.
hifiasm -o genome_asm -t 32 \
--h1 hic_R1.fastq.gz --h2 hic_R2.fastq.gz \
hifi_reads.fastq.gz
# Extract Primary and Phased Alternate Haplotypes to FASTA
awk '/^S/{print ">"$2"\n"$3}' genome_asm.hic.hap1.p_ctg.gfa > hap1_contigs.fa
awk '/^S/{print ">"$2"\n"$3}' genome_asm.hic.hap2.p_ctg.gfa > hap2_contigs.fa
Key Flags & Notes:
--h1/--h2 incorporates cross-chromosomal contact frequencies for phased diploid assembly.
⚡
Step 2: Map Hi-C Reads to Contigs for Scaffolding
BWA-MEM2 • Samtools
Aligns Hi-C paired reads to haploid contigs, retaining 5-prime ligation junctions.
bwa-mem2 index hap1_contigs.fa
bwa-mem2 mem -5SP -t 16 hap1_contigs.fa hic_R1.fastq.gz hic_R2.fastq.gz | \
samtools view -bS - | samtools sort -@ 8 -n -o hic_mapped_namesort.bam -
Key Flags & Notes:
-5SP handles Hi-C chimeric ligation junctions properly without spurious mapping flags.
⚡
Step 3: Chromosome-Scale Scaffolding with YaHS
YaHS (Yet Another Hi-C Scaffolder)
Orders and orients contigs into whole chromosomes using contact matrix contact density.
yahs hap1_contigs.fa hic_mapped_namesort.bam -o yahs_scaffolds
Key Flags & Notes: YaHS delivers gapless chromosome scaffolds spanning complex centromeric satellite repeats.
⚡
Step 4: Gene Space Completeness Audit with BUSCO v5
BUSCO v5
Evaluates universal single-copy ortholog recovery to benchmark genome biological completeness.
busco -i yahs_scaffolds_scaffolds_final.fa -l embryophyta_odb10 -o busco_qc -m genome --cpu 16
Key Flags & Notes: Ensures complete assembly of protein-coding loci (>98% single-copy ortholog recovery target).
🚀 Automated Nextflow (nf-core) Production Execution
Execute this complete workflow across any SLURM, AWS Batch, or Google Cloud HPC cluster using the verified community container pipeline:
nextflow run nf-core/genomeassembly -r 1.0.0 \
--input samplesheet.csv \
--outdir ./results_t2t/ \
-profile docker