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HiFi Assembly

Basic Assembly

bash
# Primary assembly (single haplotype consensus)
hifiasm -o output_prefix -t 32 reads.hifi.fastq.gz

# Output files:
# output_prefix.bp.p_ctg.gfa  - Primary contigs
# output_prefix.bp.a_ctg.gfa  - Alternate contigs
# output_prefix.bp.hap1.p_ctg.gfa - Haplotype 1 (if phased)
# output_prefix.bp.hap2.p_ctg.gfa - Haplotype 2 (if phased)

# Convert GFA to FASTA
awk '/^S/{print ">"$2;print $3}' output_prefix.bp.p_ctg.gfa > assembly.fasta

Trio-Binned Phasing

bash
# With parental short reads for trio binning
hifiasm -o trio_asm -t 32 \
    -1 paternal.yak \
    -2 maternal.yak \
    child.hifi.fastq.gz

# Create yak databases from parental Illumina reads first
yak count -b37 -t16 -o paternal.yak paternal_R1.fq.gz paternal_R2.fq.gz
yak count -b37 -t16 -o maternal.yak maternal_R1.fq.gz maternal_R2.fq.gz

Hi-C Phasing

bash
# Use Hi-C reads for phasing (no parents needed)
hifiasm -o hic_asm -t 32 \
    --h1 hic_R1.fastq.gz \
    --h2 hic_R2.fastq.gz \
    reads.hifi.fastq.gz

# Produces fully phased hap1 and hap2 assemblies

Key Parameters

| Parameter | Default | Description | |-----------|---------|-------------| | -t | 1 | Threads | | -l | 0 | Purge level (0=none, 1=light, 2=aggressive) | | -s | 0.55 | Similarity threshold for duplicate detection | | --primary | - | Output primary contigs only (no alternates) | | --n-hap | 2 | Expected number of haplotypes | | -D | 5.0 | Drop reads with depth > Daverage | | -N | 100 | Consider up to N overlaps for each read |

Purge Duplicates

bash
# Aggressive purging for high heterozygosity
hifiasm -o asm -t 32 -l 2 reads.hifi.fastq.gz

# Minimal purging for inbred samples
hifiasm -o asm -t 32 -l 0 reads.hifi.fastq.gz

Ultra-Long ONT Integration

bash
# Combine HiFi accuracy with ONT length
hifiasm -o hybrid_asm -t 32 \
    --ul ont_ultralong.fastq.gz \
    hifi_reads.fastq.gz

# UL reads help span complex repeats

Assembly Stats

bash
# Quick stats with seqkit
seqkit stats assembly.fasta

# Detailed with assembly-stats
assembly-stats assembly.fasta

# QUAST assessment
quast.py -o quast_output assembly.fasta

# BUSCO completeness
busco -i assembly.fasta -l mammalia_odb10 -o busco_out -m genome

Memory and Runtime

| Genome Size | HiFi Coverage | RAM | Time (32 cores) | |-------------|---------------|-----|-----------------| | 3 Gb | 30x | ~200 GB | 12-24 hours | | 3 Gb | 60x | ~400 GB | 24-48 hours | | 500 Mb | 40x | ~64 GB | 2-4 hours |

Python Wrapper

python
import subprocess
from pathlib import Path

def run_hifiasm(hifi_reads, output_prefix, threads=32, purge_level=0,
                hic_r1=None, hic_r2=None, ul_reads=None):
    cmd = ['hifiasm', '-o', output_prefix, '-t', str(threads), '-l', str(purge_level)]

    if hic_r1 and hic_r2:
        cmd.extend(['--h1', hic_r1, '--h2', hic_r2])

    if ul_reads:
        cmd.extend(['--ul', ul_reads])

    cmd.append(hifi_reads)
    subprocess.run(cmd, check=True)

    gfa = Path(f'{output_prefix}.bp.p_ctg.gfa')
    fasta = Path(f'{output_prefix}.fasta')

    with open(fasta, 'w') as out:
        with open(gfa) as f:
            for line in f:
                if line.startswith('S'):
                    parts = line.strip().split('\t')
                    out.write(f'>{parts[1]}\n{parts[2]}\n')

    return fasta

# Example
assembly = run_hifiasm('sample.hifi.fq.gz', 'sample_asm', threads=48, hic_r1='hic_R1.fq.gz', hic_r2='hic_R2.fq.gz')

Troubleshooting

| Issue | Solution | |-------|----------| | High duplication | Increase purge level (-l 2) | | Missing haplotypes | Add Hi-C or trio data for phasing | | Memory errors | Reduce -D parameter or downsample reads | | Fragmented assembly | Check read quality; consider UL ONT addition |

Related Skills

• genome-assembly/assembly-qc - QUAST and BUSCO
• genome-assembly/scaffolding - YaHS Hi-C scaffolding
• genome-assembly/contamination-detection - CheckM2 decontamination
• long-read-sequencing/read-qc - HiFi quality control

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