Call pilon
In the next step, we call pilon with the mappings to polish our assembly:
cd ~/workdir/
mkdir pilon
java -Xmx32G -jar ~/pilon-1.24.jar --genome ~/workdir/assembly/assembly.fasta --fix all --changes --frags ~/workdir/illumina_mapping/mapping.sorted.bam --output ~/workdir/pilon/pilon_round1 | tee ~/workdir/pilon/round1.pilon
You can repeat this for several rounds like this:
Round2:
bwa index ~/workdir/pilon/pilon_round1.fasta
bwa mem -t 14 ~/workdir/pilon/pilon_round1.fasta ~/workdir/data/illumina/Illumina_R1.fastq.gz ~/workdir/data/illumina/Illumina_R2.fastq.gz | samtools view - -Sb | samtools sort - -@14 -o ~/workdir/illumina_mapping/mapping_pilon1.sorted.bam
samtools index ~/workdir/illumina_mapping/mapping_pilon1.sorted.bam
java -Xmx32G -jar ~/pilon-1.24.jar --genome ~/workdir/pilon/pilon_round1.fasta --fix all --changes --frags ~/workdir/illumina_mapping/mapping_pilon1.sorted.bam --output ~/workdir/pilon/pilon_round2 | tee ~/workdir/pilon/round2.pilon
Round3:
bwa index ~/workdir/pilon/pilon_round2.fasta
bwa mem -t 14 ~/workdir/pilon/pilon_round2.fasta ~/workdir/data/illumina/Illumina_R1.fastq.gz ~/workdir/data/illumina/Illumina_R2.fastq.gz | samtools view - -Sb | samtools sort - -@14 -o ~/workdir/illumina_mapping/mapping_pilon2.sorted.bam
samtools index ~/workdir/illumina_mapping/mapping_pilon2.sorted.bam
java -Xmx32G -jar ~/pilon-1.24.jar --genome ~/workdir/pilon/pilon_round2.fasta --fix all --changes --frags ~/workdir/illumina_mapping/mapping_pilon2.sorted.bam --output ~/workdir/pilon/pilon_round3 | tee ~/workdir/pilon/round3.pilon
Round4:
bwa index ~/workdir/pilon/pilon_round3.fasta
bwa mem -t 14 ~/workdir/pilon/pilon_round3.fasta ~/workdir/data/illumina/Illumina_R1.fastq.gz ~/workdir/data/illumina/Illumina_R2.fastq.gz | samtools view - -Sb | samtools sort - -@14 -o ~/workdir/illumina_mapping/mapping_pilon3.sorted.bam
samtools index ~/workdir/illumina_mapping/mapping_pilon3.sorted.bam
java -Xmx32G -jar ~/pilon-1.24.jar --genome ~/workdir/pilon/pilon_round3.fasta --fix all --changes --frags ~/workdir/illumina_mapping/mapping_pilon3.sorted.bam --output ~/workdir/pilon/pilon_round4 | tee ~/workdir/pilon/round4.pilon
You can inspect the Pilon_roundX.changes file to see if there are changes to the previous round.
In case, you don’t want to do these steps for yourself, we have precomputed some pilon rounds for you. You can copy those rounds from the precomputed Result directory to your workdir:
cp -r ~/workdir/results/pilon/pilon_round{2..4}* ~/workdir/pilon/.