--- 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 ```bash conda install -c bioconda pilon ``` ### Basic Usage ```bash # 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 ```bash #!/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 ```bash # 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 ```bash conda install -c bioconda medaka ``` ### Basic Usage ```bash 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 ```bash # 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 ```bash conda install -c bioconda racon ``` ### Basic Usage ```bash # 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 ```bash #!/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 ```bash #!/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 ${OUTDIR}/pilon_${i}.bam --output ${OUTDIR}/pilon_${i} current=${OUTDIR}/pilon_${i}.fasta done cp $current ${OUTDIR}/final_polished.fasta echo "Done: ${OUTDIR}/final_polished.fasta" ``` ## NextPolish (Combined Approach) ### Installation ```bash conda install -c bioconda nextpolish ``` ### Usage ```bash # Create config file cat > run.cfg << EOF [General] job_type = local job_prefix = nextPolish task = best rewrite = yes rerun = 3 parallel_jobs = 2 multithread_jobs = 8 genome = assembly.fasta genome_size = auto workdir = ./01_rundir [lgs_option] lgs_fofn = lgs.fofn lgs_options = -min_read_len 1k -max_depth 100 lgs_minimap2_options = -x map-ont [sgs_option] sgs_fofn = sgs.fofn sgs_options = -max_depth 100 EOF # File of filenames ls reads.fastq.gz > lgs.fofn ls R1.fq.gz R2.fq.gz > sgs.fofn # Run nextPolish run.cfg ``` ## Quality Assessment After polishing, assess improvement: ```bash # Compare to reference (if available) quast.py -r reference.fa original.fa polished.fa -o quast_comparison # Check error rate minimap2 -ax map-ont polished.fa reads.fq.gz | samtools stats | grep "error rate" ``` ## Related Skills - long-read-assembly - Initial assembly - short-read-assembly - Source of polishing reads - assembly-qc - Assess polishing improvement - long-read-sequencing - medaka variant calling