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--- nanopore_tools_for_polishing [2020/06/17 13:04] – 24.138.68.92
+++ nanopore_tools_for_polishing [2024/08/07 13:01] (current) – 134.190.232.164
@@ Line 1: / Line 1: @@
 ====== Polishing your MinION assembly ======
 Documentation by Jon Jerlström Hultqvist and Shelby Williams
+(updates by Joran Martijn)
 **Be aware that some scripts and commands might not be working any longer on Perun due to the switch to the new conda-environment system. Sections will be progressively updated to reflect this.**
@@ Line 81: / Line 82: @@
 First, make a BWA index of the assembly you wish to map onto by using the following command:
 <code>
 bwa index assembly_to_polish.fasta
 </code>
 Next, use the meteora_bwa.sh script to map the short reads onto your assembly. This will create a sorted.bam file. In this example, two paired-end read files will be mapped:
 <code>
-bwa mem -t 16 assembly_to_polish.fasta /scratch2/user/path/to/trimmedreads_1_PairNtrim.fastq.gz /scratch2/user/path/to/trimmedreads_2_PairNtrim.fastq.gz | samtools view -Sb - | samtools sort >  piloninput.sorted.bam
+bwa mem \
+    -t 16 \
+    assembly_to_polish.fasta \
+    /scratch2/user/path/to/trimmedreads_1_PairNtrim.fastq.gz \
+    /scratch2/user/path/to/trimmedreads_2_PairNtrim.fastq.gz | \
+        samtools sort --threads 16 -o piloninput.sorted.bam
 </code>
+UPDATE: You can now run bwa-mem2, which is an optimized version of bwa mem. It generates the exact same output, but is 2-4x faster:
+<code>
+bwa-mem2 mem \
+    -t 16 \
+    assembly_to_polish.fasta \
+    /scratch2/user/path/to/trimmedreads_1_PairNtrim.fastq.gz \
+    /scratch2/user/path/to/trimmedreads_2_PairNtrim.fastq.gz | \
+        samtools sort --threads 16 -o piloninput.sorted.bam
+</code>
 Once this is finished, use Pilon.sh to make changes in the assembly and generate a new consensus sequence. Pilon.sh can be formatted like so:
 <code>
-java -Xmx16G -jar /scratch2/software/pilon/pilon-1.22.jar --genome assembly_to_polish.fasta --frags piloninput.sorted.bam --output P2x --outdir Pilon2x --threads 16
+java -Xmx16G -jar /scratch2/software/pilon/pilon-1.22.jar \
+    --genome assembly_to_polish.fasta \
+    --frags piloninput.sorted.bam \
+    --output P2x \
+    --outdir Pilon2x \
+    --threads 16
 </code>
+UPDATE: The --threads option is as of v1.24 no longer maintained. It seems Pilon doesn't use more than 200-300% CPU (i.e. 3 threads) at most, so setting --threads to 4 orso should be sufficient.
 You may run into an error where Pilon does not recognize the bam file created from the previous step as being indexed. To fix this, run:
 <code>
 samtools index /path/to_bam_file
 </code>
 This will return a .bam.bai file. This file needs to be in the same folder as Pilon.sh, but does not need to be placed in the script.
@@ Line 102: / Line 134: @@
 Shell script:
 {{ :unicyclersh.docx |}}
+<code>
+#!/bin/bash
+#$ -S /bin/bash
+. /etc/profile
+#$ -cwd
+#$ -pe threaded 16
+#cd /scratch2/jon/MinION/BMAN/assemblies/Unicycler_polish/
+echo "Starting"
+unset PYTHONPATH
+export PATH=/scratch2/software/gcc-6.3.0/bin:/scratch2/software/Python-3.6.0/bin:$PATH
+export LD_LIBRARY_PATH=/scratch2/software/gcc-6.3.0/lib64:/scratch2/software/Python-3.6.0/lib:$LD_LIBRARY_PATH
+/scratch2/software/Python-3.6.0/bin/unicycler_polish -1 /scratch2/shelbyw/RCL_Unicycler/RCL_1_PairNtrim.fq -2 /scratch2/shelbyw/RCL_Unicycler/RCL_2_PairNtrim.fq --long_reads RCL_MinION.CutAdapt75.3000.chop.fastq.gz -a RCL_unclean_AB_assembly_fix_Racon2_Pilon3.fasta --pilon=/scratch2/software/pilon/pilon-1.22.jar --samtools=/opt/perun/bin/samtools --threads 16
+echo "Done!"
+</code>
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