---

name: bio-genome-assembly-long-read-assembly description: De novo genome assembly from Oxford Nanopore or PacBio long reads using Flye and Canu. Produces highly contiguous assemblies suitable for complete bacterial genomes and resolving complex regions. Use when assembling genomes from ONT or PacBio reads. tool_type: cli primary_tool: Flye

Long-Read Assembly

Assemble genomes from Oxford Nanopore (ONT) or PacBio long reads for highly contiguous assemblies.

Tool Comparison

Note: For PacBio HiFi data, see the dedicated hifi-assembly skill which covers hifiasm.

Flye

Installation

Copyconda install -c bioconda flye

Basic Usage

Copy# Oxford Nanopore
flye --nano-raw reads.fastq.gz --out-dir flye_output --threads 16

# PacBio CLR
flye --pacbio-raw reads.fastq.gz --out-dir flye_output --threads 16

# PacBio HiFi
flye --pacbio-hifi reads.fastq.gz --out-dir flye_output --threads 16

Read Type Options

Key Options

Genome Size Estimation

Copy# Estimate if unknown
flye --nano-raw reads.fq.gz --out-dir output --genome-size 5m

# Size formats: 1000, 1k, 1m, 1g

Output Files

Copyflye_output/
├── assembly.fasta       # Final assembly
├── assembly_graph.gfa   # Assembly graph
├── assembly_info.txt    # Contig statistics
└── flye.log             # Log file

Bacterial Assembly

Copyflye \
    --nano-raw bacteria.fastq.gz \
    --out-dir bacteria_assembly \
    --genome-size 5m \
    --threads 16

Metagenome Assembly

Copyflye \
    --nano-raw metagenome.fastq.gz \
    --out-dir meta_assembly \
    --meta \
    --threads 32

With Plasmid Recovery

Copyflye \
    --nano-raw isolate.fastq.gz \
    --out-dir assembly \
    --plasmids \
    --threads 16

Canu

Installation

Copyconda install -c bioconda canu

Basic Usage

Copy# ONT reads
canu -p assembly -d canu_output genomeSize=5m -nanopore reads.fastq.gz

# PacBio HiFi
canu -p assembly -d canu_output genomeSize=5m -pacbio-hifi reads.fastq.gz

Key Options

Read Type Options

Fast Mode

Copycanu -p asm -d output genomeSize=5m \
    -nanopore reads.fq.gz \
    useGrid=false \
    maxThreads=16 \
    maxMemory=32g

High-Quality Mode (PacBio HiFi)

Copycanu -p asm -d output genomeSize=5m \
    -pacbio-hifi reads.fq.gz \
    correctedErrorRate=0.01

Output Files

Copycanu_output/
├── assembly.contigs.fasta   # Contigs
├── assembly.unassembled.fasta
├── assembly.report
└── assembly.seqStore/

Wtdbg2 (Fast Draft)

Installation

Copyconda install -c bioconda wtdbg

Basic Usage

Copy# Assemble
wtdbg2 -x ont -g 5m -t 16 -i reads.fq.gz -o draft

# Consensus
wtpoa-cns -t 16 -i draft.ctg.lay.gz -o draft.ctg.fa

Platform Presets

Complete Workflows

ONT Bacterial Assembly

Copy#!/bin/bash
set -euo pipefail

READS=$1
OUTDIR=$2
SIZE=${3:-5m}

echo "=== ONT Bacterial Assembly ==="

# Flye assembly
flye \
    --nano-raw $READS \
    --out-dir ${OUTDIR}/flye \
    --genome-size $SIZE \
    --threads 16

# Stats
echo "Assembly statistics:"
cat ${OUTDIR}/flye/assembly_info.txt

echo "Assembly: ${OUTDIR}/flye/assembly.fasta"

Hybrid Assembly (Long + Short)

Copy#!/bin/bash
set -euo pipefail

LONG=$1
SHORT_R1=$2
SHORT_R2=$3
OUTDIR=$4

# 1. Long-read assembly with Flye
flye --nano-raw $LONG --out-dir ${OUTDIR}/flye --genome-size 5m --threads 16

# 2. Polish with short reads (Pilon)
# See assembly-polishing skill

Quality Expectations

Troubleshooting

Low Contiguity

• Check coverage (need >30x)
• Try increasing iterations in Flye
• Consider supplementing with short reads

Memory Issues

• Use Flye (more memory efficient)
• Reduce threads
• Filter reads by length/quality

Misassemblies

• Polish with Pilon/medaka
• Validate with short reads
• Check for contamination

Related Skills

• hifi-assembly - PacBio HiFi assembly with hifiasm
• assembly-polishing - Polish long-read assemblies
• assembly-qc - QUAST and BUSCO assessment
• short-read-assembly - Hybrid with Illumina
• long-read-sequencing - Read QC and alignment