Hexaploid Sweet Potato Assembly & Scaffolding (Pore-C Mode)¶

This tutorial demonstrates the assembly and scaffolding workflow for the hexaploid sweet potato (Ipomoea batatas, 2n = 6x = 90) utilizing PacBio HiFi and Pore-C reads.

1. Haplotype-Resolved Contig Assembly using Hifiasm¶

Step 1.1: Generate Initial Unitigs¶

First, run hifiasm in default mode using PacBio HiFi reads to generate the primary unitigs, and convert the GFA graph structure to FASTA format:

~/software/hifiasm-0.25.0/hifiasm -t 100 \
    -o hifi.asm \
    hifi.fastq.gz

gfatools gfa2fa hifi.asm.p_utg.gfa > hifi.asm.p_utg.fasta

Step 1.2: Map Pore-C Reads to Unitigs¶

Map your Pore-C reads back to the generated unitigs:

cphasing mapper hifi.asm.p_utg.fasta porec_reads.fastq.gz -t 100

Step 1.3: Convert Pore-C to Pseudo-Hi-C Reads¶

Convert the multi-contact Pore-C alignments into virtual paired-end "pseudo-Hi-C" reads to make them compatible with Hifiasm's phasing algorithm:

cphasing-rs porec2 porec_reads.porec.gz -l 0 -o porec2hic

Step 1.4: Resolve Assembly with Hifiasm (Hi-C Mode)¶

Run hifiasm in Hi-C mode using the PacBio HiFi reads along with the newly generated pseudo-Hi-C paired-end reads to obtain highly contiguous, phased contigs:

Note

Executing this command in the same directory as Step 1.1 will reuse the cached files, allowing hifiasm to bypass the time-consuming HiFi read correction and overlapping phases.

~/software/hifiasm-0.25.0/hifiasm -t 100 \
    -o hifi.asm \
    hifi.fastq.gz \
    --h1 porec2hic_R1.fa.gz \
    --h2 porec2hic_R2.fa.gz

Step 1.5: Merge the Haplotype Assemblies¶

Merge the assemblies and assembly graphs from both haplotypes (hap1 and hap2) to prepare them for downstream scaffolding:

# Merge FASTA files
cat hifi.asm.hic.hap1.p_ctg.fasta hifi.asm.hic.hap2.p_ctg.fasta > haps.p_ctg.fasta 

# Merge GFA files (without sequence)
cat hifi.asm.hic.hap1.p_ctg.noseq.gfa hifi.asm.hic.hap2.p_ctg.noseq.gfa > haps.p_ctg.noseq.gfa

2. Scaffolding with C-Phasing¶

Scaffold the merged haplotype assembly using the cphasing pipeline.

Since sweet potato is a hexaploid organism with a basic chromosome number of 15 ($2n = 6x = 90$), the expected number of final chromosome-level groups is 90. We set the phasing parameters to -n 15:6 (15 homology groups, each with 6 haplotypes), adjust the Pore-C resolution thresholds, and enable the --collapsed-rescue module to resolve collapsed regions:

cphasing pipeline \
    -f haps.p_ctg.fasta \
    -pcd porec_reads.fastq.gz \
    -t 100 \
    -n 15:6 \
    -e 0 --split-length 2m \
    --collapsed-rescue \
    --gfa haps.p_ctg.noseq.gfa \
    -hcr \
    -p GATC \
    -o cphasing_output