Bio Workflows Genome Assembly Pipeline

Version Compatibility

Reference examples tested with: BUSCO 5.5+, BWA 0.7.17+, Flye 2.9+, QUAST 5.2+, SPAdes 3.15+, fastp 0.23+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Genome Assembly Pipeline

"Assemble and polish a genome from my sequencing reads" → Orchestrate read QC, assembly (hifiasm, Flye, SPAdes), polishing (Medaka, Pilon), scaffolding, contamination detection (BlobToolKit), and quality assessment (QUAST, BUSCO).

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

Workflow Overview

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

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

# 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

# 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

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

Step 2: Assembly with Flye

# 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

# 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

# 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

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

# 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

Troubleshooting

Complete Pipeline Script

#!/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

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