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/.


References
^^^^^^^^^^

**pilon** https://github.com/broadinstitute/pilon/wiki