Hi-C tutorial: haplotype-aware scaffolding from hifiasm hap assemblies (tetraploid example)¶

This tutorial shows how to scaffold pre-separated haplotypes produced by hifiasm using C-Phasing in haplotype-aware mode (--mode hapaware) with Hi-C data.

The goal is to keep haplotypes consistent while producing chromosome-scale scaffolds.

0. Prerequisites¶

Input data¶

• hifiasm haplotype assemblies (FASTA), for example:
  • hap1.fa
  • hap2.fa
  • hap3.fa
  • hap4.fa
• Hi-C read pairs (FASTQ), for example:
  • hic_R1.fastq.gz
  • hic_R2.fastq.gz

Notes - This tutorial assumes you already have haplotype-separated assemblies (one FASTA per haplotype). - If you have hifiasm *.hap*.p_ctg.fasta outputs, convert/select the hap FASTA files you want to scaffold.

1. Prepare a combined FASTA (recommended)¶

C-Phasing expects a single assembly FASTA as the contig set to scaffold.
For hap-aware scaffolding, concatenate the hap assemblies into one FASTA.

cat hap1.fa hap2.fa hap3.fa hap4.fa > haps.concat.fa

Important: unique contig names¶

Ensure contig IDs are unique across haplotypes. If hifiasm already prefixes contig names per hap, you are fine. Otherwise, rename contigs to avoid collisions.

A simple pattern is to add a prefix per haplotype, e.g. h1, h2, h3, h4.

2. Provide an initial haplotype clustering file (-fc)¶

--mode hapaware is designed for cases where haplotypes are already separated.
Providing an initial cluster file helps C-Phasing keep haplotypes consistent.

cphasing utils fa2cluster  haps.concat.fa -o haps.clusters.txt 

Create a haps.clusters.txt with two columns:

1. group_id
2. contig_id

Example (illustrative):

h1   h1tg000001l
h1   h1tg000002l
h2   h2tg000001l
h2   h2tg000002l
h3   h3tg000001l
h4   h4tg000001l

Tip: In hap-aware mode, -fc first.clusters.txt is the key input that encodes haplotype priors.

3. Run C-Phasing (Hi-C + hapaware)¶

Run the main pipeline with Hi-C read pairs and --mode hapaware.

cphasing pipeline \
  -f haps.concat.fa \
  -hic1 hic_R1.fastq.gz \
  -hic2 hic_R2.fastq.gz \
  --mode hapaware \
  -fc haps.clusters.txt \
  -o cphasing_hapaware_out \
  -t 32 -hcr -p AAGCTT