Long-Read Assembly

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

Tool Comparison

Tool	Speed	Memory	Best For
Flye	Fast	Moderate	General purpose, bacteria, ONT
Canu	Slow	High	High accuracy, complex genomes
Wtdbg2	Very fast	Low	Draft assemblies

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

Flye

Installation

bash

conda install -c bioconda flye

Basic Usage

bash

# 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

Option	Read Type
`--nano-raw`	ONT regular reads
`--nano-corr`	ONT corrected reads
`--nano-hq`	ONT Q20+ reads (Guppy 5+)
`--pacbio-raw`	PacBio CLR
`--pacbio-corr`	PacBio corrected
`--pacbio-hifi`	PacBio HiFi/CCS

Key Options

Option	Description
`--out-dir`	Output directory
`--threads`	Number of threads
`--genome-size`	Estimated genome size (e.g., 5m, 100m)
`--iterations`	Polishing iterations (default: 1)
`--meta`	Metagenome mode
`--plasmids`	Recover plasmids
`--keep-haplotypes`	Don't collapse haplotypes
`--scaffold`	Enable scaffolding

Genome Size Estimation

bash

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

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

Output Files

code

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

Bacterial Assembly

bash

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

Metagenome Assembly

bash

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

With Plasmid Recovery

bash

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

Canu

Installation

bash

conda install -c bioconda canu

Basic Usage

bash

# 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

Option	Description
`-p`	Assembly prefix
`-d`	Output directory
`genomeSize=`	Estimated size (required)
`maxThreads=`	Max threads
`maxMemory=`	Max memory (e.g., 64g)
`useGrid=false`	Disable grid execution
`correctedErrorRate=`	Expected error rate

Read Type Options

Option	Read Type
`-nanopore`	ONT reads
`-nanopore-raw`	ONT raw (deprecated)
`-pacbio`	PacBio CLR
`-pacbio-hifi`	PacBio HiFi/CCS

Fast Mode

bash

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

High-Quality Mode (PacBio HiFi)

bash

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

Output Files

code

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

Wtdbg2 (Fast Draft)

Installation

bash

conda install -c bioconda wtdbg

Basic Usage

bash

# 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

Preset	Platform
`-x ont`	ONT R9
`-x ccs`	PacBio HiFi
`-x rs`	PacBio CLR
`-x sq`	ONT R10

Complete Workflows

ONT Bacterial Assembly

bash

#!/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)

bash

#!/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

Metric	Bacterial	Eukaryotic
Contigs	1-10	100-1000+
N50	>1 Mb	Variable
Complete chromosomes	Often	Rare

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