Bio Genome Assembly Long Read Assembly

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

conda install -c bioconda flye

Basic Usage

# 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

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

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

Output Files

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

Bacterial Assembly

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

Metagenome Assembly

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

With Plasmid Recovery

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

Canu

Installation

conda install -c bioconda canu

Basic Usage

# 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

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

High-Quality Mode (PacBio HiFi)

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

Output Files

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

Wtdbg2 (Fast Draft)

Installation

conda install -c bioconda wtdbg

Basic Usage

# 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

#!/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)

#!/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