/bio-genome-assembly-assembly-polishing

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--- name: bio-genome-assembly-assembly-polishing description: Polish genome assemblies to reduce errors using short reads (Pilon), long reads (Racon), or ONT-specific tools (medaka). Essential for improving long-read assembly accuracy. Use when improving assembly accuracy with polishing tools. tool_type: cli primary_tool: Pilon measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:

• read_file
• run_shell_command

---

Assembly Polishing

Improve assembly accuracy by correcting errors using additional sequencing data.

Polishing Strategies

| Tool | Input Reads | Best For | |------|-------------|----------| | Pilon | Illumina | Final polishing | | medaka | ONT | ONT assemblies | | Racon | Long reads | Quick polishing | | NextPolish | Both | Combined approach |

Recommended Workflows

ONT Assembly

1. Racon (2-3 rounds with ONT) 2. medaka (1 round) 3. Pilon (2-3 rounds with Illumina)

PacBio CLR Assembly

1. Racon (2-3 rounds) 2. Pilon (2-3 rounds with Illumina)

PacBio HiFi Assembly

• Often no polishing needed (>99% accuracy)
• Optional Pilon if Illumina available

Pilon (Illumina Polishing)

Installation

conda install -c bioconda pilon

Basic Usage

# Map short reads to assembly
bwa index assembly.fasta
bwa mem -t 16 assembly.fasta R1.fq.gz R2.fq.gz | samtools sort -o aligned.bam
samtools index aligned.bam

# Run Pilon
pilon --genome assembly.fasta --frags aligned.bam --output polished

Key Options

| Option | Description | |--------|-------------| | `--genome` | Input assembly | | `--frags` | Paired-end BAM | | `--output` | Output prefix | | `--changes` | Write changes file | | `--vcf` | Write VCF of changes | | `--fix` | What to fix (snps, indels, gaps, all) | | `--threads` | Threads for alignment | | `--mindepth` | Min depth for correction |

Multiple Rounds

#!/bin/bash
ASSEMBLY=$1
R1=$2
R2=$3
ROUNDS=${4:-3}

current=$ASSEMBLY

for i in $(seq 1 $ROUNDS); do
    echo "=== Pilon round $i ==="
    bwa index $current
    bwa mem -t 16 $current $R1 $R2 | samtools sort -o round${i}.bam
    samtools index round${i}.bam

    pilon --genome $current --frags round${i}.bam --output pilon_round${i} --changes

    current=pilon_round${i}.fasta

    changes=$(wc -l < pilon_round${i}.changes)
    echo "Changes made: $changes"

    if [ $changes -eq 0 ]; then
        echo "No more changes, stopping"
        break
    fi
done

cp $current final_polished.fasta

Fix Specific Issues

# Only fix SNPs and small indels
pilon --genome assembly.fa --frags aligned.bam --output polished --fix snps,indels

# Only fill gaps
pilon --genome assembly.fa --frags aligned.bam --output polished --fix gaps

medaka (ONT Polishing)

Installation

conda install -c bioconda medaka

Basic Usage

medaka_consensus -i reads.fastq.gz -d assembly.fasta -o medaka_output -t 8

Key Options

| Option | Description | |--------|-------------| | `-i` | Input reads | | `-d` | Draft assembly | | `-o` | Output directory | | `-t` | Threads | | `-m` | Model name |

Model Selection

# List available models
medaka tools list_models

# Use specific model (match your basecaller)
medaka_consensus -i reads.fq.gz -d assembly.fa -o output -m r1041_e82_400bps_sup_v5.1.0

Models for Common Chemistries

| Chemistry | Model | |-----------|-------| | R10.4.1 + SUP | r1041_e82_400bps_sup_* | | R10.4.1 + HAC | r1041_e82_400bps_hac_* | | R9.4.1 + SUP | r941_sup_* |

Output

medaka_output/
├── consensus.fasta    # Polished assembly
├── calls_to_draft.bam # Alignments
└── *.hdf              # Intermediate files

Racon (Long-Read Polishing)

Installation

conda install -c bioconda racon

Basic Usage

# Map reads to assembly
minimap2 -ax map-ont assembly.fasta reads.fastq.gz > aligned.sam

# Polish
racon -t 16 reads.fastq.gz aligned.sam assembly.fasta > polished.fasta

Multiple Rounds

#!/bin/bash
ASSEMBLY=$1
READS=$2
ROUNDS=${3:-3}

current=$ASSEMBLY

for i in $(seq 1 $ROUNDS); do
    echo "=== Racon round $i ==="
    minimap2 -ax map-ont $current $READS > round${i}.sam
    racon -t 16 $READS round${i}.sam $current > racon_round${i}.fasta
    current=racon_round${i}.fasta
done

cp $current racon_polished.fasta

Key Options

| Option | Description | |--------|-------------| | `-t` | Threads | | `-m` | Match score (default: 3) | | `-x` | Mismatch score (default: -5) | | `-g` | Gap penalty (default: -4) | | `-w` | Window size (default: 500) |

Complete Polishing Workflow

ONT Assembly Polishing

#!/bin/bash
set -euo pipefail

ASSEMBLY=$1      # Flye assembly
ONT_READS=$2     # ONT reads
ILLUMINA_R1=$3   # Illumina R1
ILLUMINA_R2=$4   # Illumina R2
OUTDIR=$5

mkdir -p $OUTDIR

# Step 1: Racon polishing (2 rounds)
echo "=== Racon Polishing ==="
current=$ASSEMBLY
for i in 1 2; do
    minimap2 -ax map-ont $current $ONT_READS > ${OUTDIR}/racon_${i}.sam
    racon -t 16 $ONT_READS ${OUTDIR}/racon_${i}.sam $current > ${OUTDIR}/racon_${i}.fasta
    current=${OUTDIR}/racon_${i}.fasta
done

# Step 2: medaka polishing
echo "=== medaka Polishing ==="
medaka_consensus -i $ONT_READS -d $current -o ${OUTDIR}/medaka -t 8
current=${OUTDIR}/medaka/consensus.fasta

# Step 3: Pilon polishing (2 rounds)
echo "=== Pilon Polishing ==="
for i in 1 2; do
    bwa index $current
    bwa mem -t 16 $current $ILLUMINA_R1 $ILLUMINA_R2 | samtools sort -o ${OUTDIR}/pilon_${i}.bam
    samtools index ${OUTDIR}/pilon_${i}.bam
    pilon --genome $current --frags ${OUTDIR}/pilo