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--- ploidy_analysis_using_ploidyngs [2022/06/24 15:23] – 134.190.232.29
+++ ploidy_analysis_using_ploidyngs [2022/06/24 16:01] (current) – [UPDATE (June 2022)] 134.190.232.29
@@ Line 31: / Line 31: @@
-One of the options you will be interested in is
+One of the options you will be interested in is ''-d'' or ''--max_depth'', which is set to 100 by default.
--d 100 (default), --max_depth 100 (default)
-    Max number of reads kepth at each position in the
-    reference genome (integer, default: 100)
-    If you have low coverage you will need to change this otherwise you won't have
-    many sites that qualify for examination.
+<code>
+Max number of reads kepth at each position in the reference genome (integer, default: 100)
+</code>
+This sets the maximum read depth ''ploidyNGS.py'' will read until it moves on to the next alignment position. For example, if you have it set to a 100, but at position 123 you have actually 500 reads mapped, it will only load the basecalls of the first 100 reads to calculate allele frequencies from. I (Joran) found that it is pretty safe to increase this value to say 2000, to use as much information as possible.
 Examine the tab output file. In particular, if the lines for Third and Fourth contain
@@ Line 45: / Line 45: @@
 To see the sorted content of the Third and Fourth lines
-grep Fourth myoutput.tab |cut -f4 |sort -n\\
+<code>
-grep Third myoutput.tab |cut -f4 |sort -n
+$ grep Fourth myoutput.tab | cut -f4 | sort -n
+$ grep Third  myoutput.tab | cut -f4 | sort -n
+</code>
+To remove lines containing "Third" and "Fourth" from the .tab file:
+<code>
+$ grep -vP "Third|Fourth" myoutput.tab > mytabforhistogram.tab
+</code>
-To remove Third and Fourth lines from the tab
+Once satisfied with the tab you need to make a PDF (it is suppose to generate it automatically
+but it doesn't)
-grep -v Fourth  myoutput.tab |grep -v Third > mytabforhistogram.tab
+On the command line execute
+<code>
+$ Rscript --vanilla /scratch2/software/ploidyNGS/ploidyNGS_generateHistogram.R mytabforhistogram.tab
+</code>
-Once satisfied with the tab you need to make a pdf (it is suppose to generate it automatically
+This will generate a PDF called ''NA'' which you can transfer to your home computer to look at.
-but it doesn't)
+==== UPDATE (June 2022) ====
+I (Joran) have updated the ''ploidyNGS.py'' and ''ploidyNGS_generateHistogram.R'' scripts. They are available in Perun under the new names ''ploidyNGS_minCov.py'' and ''ploidyNGS_generateHistogram_minCov.R'', respectively.
+The main new feature is the ''--min_cov'' option, which is set to 0 by default. It allows the user to ignore positions that have a lower coverage than the specified value in its allele frequency calculations. This can be useful if you have mysterious 50%/50% or 33%/67% peaks in your histogram which is otherwise strongly hinting haploidy. In my experience these mysterious peaks can stem from low coverage regions with sequencing errors. For example, if you have a position that is covered twice, and one of them is an error, it will yield a 50%/50% peak even though there is no diploidy going on here.
+Other updates:
+  * Positions reported in the .tab file are now 1-indexed (i.e. starting at 1) instead of 0-indexed (i.e. starting at 0)
+  * If you have ''/scratch2/software/ploidyNGS/'' in your ''$PATH'', the updated Python script will properly execute the histogram R script without any issues. The PDF file will have a proper file name ending in .pdf, instead of the mysterious ''NA''
+  * The axis titles in the histogram will be a bit easier to understand
+  * The script will now report the total number of heteromorphic positions per contig and over all contigs
+To call it, use a Perun submission script like this one
+<code>
+#!/bin/bash
+#$ -S /bin/bash
+. /etc/profile
+#$ -cwd
+#$ -q 256G-batch
+source activate ploidyNGS-dependencies
+export PATH="/scratch2/software/ploidyNGS:$PATH"
+BAMFILE='dnaseq_vs_canu_ergo_assembly.sorted.bam'
+MINCOV=10
+MAXDEPTH=2000
+OUTBASE='dnaseq_vs_canu_ergo_assembly'
+ploidyNGS_minCov.py \
+    --out $OUTBASE \
+    --bam $BAMFILE \
+    --min_cov $MINCOV \
+    --max_depth $MAXDEPTH
-On the commandline
+conda deactivate
+</code>
-   Rscript --vanilla /scratch2/software/ploidyNGS/ploidyNGS_generateHistogram.R mytabforhistogram.tab
+A more elaborate script is available on the GitHub repository of the Roger lab: https://github.com/RogerLab/gospel_of_andrew.
-This will generate a pdf called NA which you can transfer to your home computer to look at.
+The updated scripts are also available on the original GitHub repository https://github.com/diriano/ploidyNGS under the original script names ''ploidyNGS.py'' and ''ploidyNGS_generateHistogram.R''