Genome Assembly Pipeline

Complete workflow from sequencing reads to polished, quality-assessed genome assembly.

Workflow Overview

code

Reads (short and/or long)
    |
    v
[1. QC & Filtering] -----> fastp, NanoPlot
    |
    v
[2. Assembly] -----------> SPAdes (short) or Flye (long)
    |
    v
[3. Polishing] ----------> Pilon (short) or medaka (long)
    |
    v
[4. QC Assessment] ------> QUAST, BUSCO
    |
    v
Final polished assembly

Path A: Short-Read Assembly (SPAdes)

Step 1: QC

bash

fastp -i reads_R1.fastq.gz -I reads_R2.fastq.gz \
    -o trimmed_R1.fq.gz -O trimmed_R2.fq.gz \
    --detect_adapter_for_pe \
    --qualified_quality_phred 20 \
    --length_required 50 \
    --html qc_report.html

Step 2: Assembly with SPAdes

bash

# Standard bacterial assembly
spades.py \
    -1 trimmed_R1.fq.gz \
    -2 trimmed_R2.fq.gz \
    -o spades_output \
    --careful \
    -t 16 \
    -m 64

# For isolate genomes
spades.py --isolate \
    -1 trimmed_R1.fq.gz \
    -2 trimmed_R2.fq.gz \
    -o spades_output \
    -t 16

Step 3: Polishing with Pilon

bash

# Align reads to assembly
bwa index spades_output/scaffolds.fasta
bwa mem -t 16 spades_output/scaffolds.fasta \
    trimmed_R1.fq.gz trimmed_R2.fq.gz | \
    samtools sort -@ 4 -o aligned.bam
samtools index aligned.bam

# Polish
pilon --genome spades_output/scaffolds.fasta \
    --frags aligned.bam \
    --output polished \
    --threads 16

Path B: Long-Read Assembly (Flye)

Step 1: QC

bash

# NanoPlot for long-read QC
NanoPlot --fastq reads.fastq.gz \
    --outdir nanoplot_output \
    --threads 8

Step 2: Assembly with Flye

bash

# ONT raw reads
flye --nano-raw reads.fastq.gz \
    --out-dir flye_output \
    --threads 16 \
    --genome-size 5m

# ONT HQ reads (sup/dna_r10)
flye --nano-hq reads.fastq.gz \
    --out-dir flye_output \
    --threads 16 \
    --genome-size 5m

# PacBio HiFi
flye --pacbio-hifi reads.fastq.gz \
    --out-dir flye_output \
    --threads 16 \
    --genome-size 5m

Step 3: Polishing with medaka

bash

# Polish with medaka (for ONT)
medaka_consensus \
    -i reads.fastq.gz \
    -d flye_output/assembly.fasta \
    -o medaka_output \
    -t 16 \
    -m r1041_e82_400bps_sup_v4.3.0  # Match your basecalling model

Path C: Hybrid Assembly

bash

# Flye with long reads, then polish with short reads
flye --nano-hq long_reads.fastq.gz \
    --out-dir flye_output \
    --threads 16 \
    --genome-size 5m

# Polish with short reads using Pilon
bwa index flye_output/assembly.fasta
bwa mem -t 16 flye_output/assembly.fasta \
    short_R1.fq.gz short_R2.fq.gz | \
    samtools sort -@ 4 -o aligned.bam
samtools index aligned.bam

pilon --genome flye_output/assembly.fasta \
    --frags aligned.bam \
    --output hybrid_polished \
    --threads 16

Step 4: Quality Assessment

QUAST

bash

quast.py polished.fasta \
    -r reference.fasta \
    -g genes.gff \
    -o quast_output \
    -t 8

# Without reference
quast.py polished.fasta \
    -o quast_output \
    -t 8

BUSCO

bash

# Download lineage database
busco --download bacteria_odb10

# Run BUSCO
busco -i polished.fasta \
    -l bacteria_odb10 \
    -o busco_output \
    -m genome \
    -c 8

Parameter Recommendations

Tool	Parameter	Bacteria	Eukaryote
SPAdes	--careful	Yes	Optional
SPAdes	-m	64GB	256GB+
Flye	--genome-size	5m	Species-specific
Flye	--meta	If metagenome	No
BUSCO	-l	bacteria_odb10	eukaryota_odb10

Troubleshooting

Issue	Likely Cause	Solution
Fragmented assembly	Low coverage, repetitive genome	Increase coverage, use long reads
Low N50	Short reads only	Add long reads for scaffolding
Low BUSCO	Incomplete assembly, wrong lineage	Check coverage, try different lineage
Assembly too large	Contamination, heterozygosity	Filter reads, check for contamination

Complete Pipeline Script

bash

#!/bin/bash
set -e

THREADS=16
GENOME_SIZE="5m"
LONG_READS="long_reads.fastq.gz"
SHORT_R1="short_R1.fastq.gz"
SHORT_R2="short_R2.fastq.gz"
BUSCO_LINEAGE="bacteria_odb10"
OUTDIR="assembly_results"

mkdir -p ${OUTDIR}/{qc,assembly,polished,quast,busco}

# Step 1: QC
echo "=== QC ==="
NanoPlot --fastq ${LONG_READS} --outdir ${OUTDIR}/qc/nanoplot -t ${THREADS}
fastp -i ${SHORT_R1} -I ${SHORT_R2} \
    -o ${OUTDIR}/qc/short_R1.fq.gz -O ${OUTDIR}/qc/short_R2.fq.gz \
    --html ${OUTDIR}/qc/fastp.html

# Step 2: Assembly with Flye
echo "=== Assembly ==="
flye --nano-hq ${LONG_READS} \
    --out-dir ${OUTDIR}/assembly \
    --threads ${THREADS} \
    --genome-size ${GENOME_SIZE}

# Step 3: Polish with short reads
echo "=== Polishing ==="
bwa index ${OUTDIR}/assembly/assembly.fasta
bwa mem -t ${THREADS} ${OUTDIR}/assembly/assembly.fasta \
    ${OUTDIR}/qc/short_R1.fq.gz ${OUTDIR}/qc/short_R2.fq.gz | \
    samtools sort -@ 4 -o ${OUTDIR}/polished/aligned.bam
samtools index ${OUTDIR}/polished/aligned.bam

pilon --genome ${OUTDIR}/assembly/assembly.fasta \
    --frags ${OUTDIR}/polished/aligned.bam \
    --output ${OUTDIR}/polished/final \
    --threads ${THREADS}

# Step 4: QC
echo "=== Quality Assessment ==="
quast.py ${OUTDIR}/polished/final.fasta -o ${OUTDIR}/quast -t ${THREADS}
busco -i ${OUTDIR}/polished/final.fasta -l ${BUSCO_LINEAGE} \
    -o busco -m genome -c ${THREADS} --out_path ${OUTDIR}

echo "=== Assembly Complete ==="
echo "Final assembly: ${OUTDIR}/polished/final.fasta"
cat ${OUTDIR}/quast/report.txt

Related Skills

•genome-assembly/short-read-assembly - SPAdes details
•genome-assembly/long-read-assembly - Flye, Canu, Hifiasm
•genome-assembly/assembly-polishing - Pilon, medaka, Racon
•genome-assembly/assembly-qc - QUAST, BUSCO metrics