Changes between Version 9 and Version 10 of SOP/PooledCRISPR

Timestamp:

05/09/19 10:53:08 (6 years ago)

Author:

gbell

Comment:

--

SOP/PooledCRISPR

@@ -65 +65 @@
 
 * Command to assign read counts for each sgRNA:
-* grep -v ^\@ foo.sam | awk -F"\t" '{ if($2==0 && $13=="MD:Z:20") print $3 }' |sort |uniq -c |awk '{ print $2"\t"$1 }' 
 
+{{{
+grep -v ^\@ foo.sam | awk -F"\t" '{ if($2==0 && $13=="MD:Z:20") print $3 }' | sort | uniq -c | awk '{ print $2"\t"$1 }'
+}}}
+ 
+
+==== MAGeCK ====
+
+  * [[https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0554-4 | Publication]]
+  * [[https://sourceforge.net/projects/mageck/ | MAGeCK Home/Download Page]]
+  * [[https://sourceforge.net/p/mageck/wiki/demo/ | Tutorial]]
+
+
+==== Test: compare two conditions ====
+
+  * Common usage to test, or compare, two conditions
+
+
+{{{
+mageck test -k count_matrix.txt -t top1,top2 -c bot1,bot2 -n mageck_out.txt
+# the options -t and -c specificity the treatment and control samples, respectively.
+}}}
+
+
+The input file, count_matrix.txt, column names must match arguments to -c and -t, e.g.
+
+
+{{{
+|sgRNA|gene|bot1|bot2|top1|top2|
+|sgACTL7A_2|ACTL7A|32|14|10|26|
+|sgACTL7A_3|ACTL7A|44|40|82|118|s
+|gACTL7A_4|ACTL7A|64|61|418|313|
+|sgACTL7A_5|ACTL7A|9|0|17|74|
+|sgACTL7A_6|ACTL7A|42|5|47|166|
+|sgACTL7A_7|ACTL7A|14|32|23|60|
+}}}
+
+
+The output files include,
+  - .summary.pdf file which summarizes (only) the top hits, and also includes a waterfall plot.
+  - .gene_summary.txt results summarized by gene (for all genes)
+  - .sgrna_summary.txt resuls by guide (for all guides); this file can be made into a matrix using a few UNIX commands, e.g. 
+
+
+{{{
+#get only the columns of interest: Gene, sgrna, control_mean, treat_mean
+cut -f 1,2,5,6 mageck.sgrna_summary.txt | awk '{print $2"\t"$1"\t"$3"\t"$4}' > CRISPR_score_sgRNA.txt
+
+#convert the single column into a (wide) matrix, each column is a guide and each row is a gene
+grep -v crispr_sgRNA.txt | sed 's/_/\t/' | sort -k 1,1 -k 2,2 -k 3,3n | awk -F '\t' '{print $1"\t"$2"_"$3"\t"$4"\t"$5}' | grep  -v INTERGENIC | grep -v CTRL0 | cut -f 1,4 | groupBy -g 1 -c 2 -o collapse |sed 's/,/\t/g' > CRISPR_score_sgRNA.txt
+}}}
+
+