count matrices Martin Reczko Thu, May 31, 2018 at 3:18 PM To: Margarita Chatzimike , Giorgos Giagkas Dear Margarita and Giorgos, first, please excuse the long session yesterday, for clarification just 15 min of quiet reading were necessary (see below). All (seq) results have been processed again with correct KO vs WT contrasts. The results are at: polysome gene in: /data/images/proton/DKlab/mr/Polysomes/reads/stringtie/per-gene/ edgeRgenes_featurecount-0h.csv edgeRgenes_featurecount-2h.csv edgeRgenes_featurecount-6h.csv edgeRgenes_featurecount-IFN.csv edgeRgenes_featurecount-IL4.csv edgeRgenes_featurecountExpr-0h.csv edgeRgenes_featurecountExpr-2h.csv edgeRgenes_featurecountExpr-6h.csv edgeRgenes_featurecountExpr-IFN.csv edgeRgenes_featurecountExpr-IL4.csv transcript in: /data/images/proton/DKlab/mr/Polysomes/reads/stringtie edgeRtranscripts3-0h.csv edgeRtranscripts3-2h.csv edgeRtranscripts3-6h.csv edgeRtranscripts3-IFN.csv edgeRtranscripts3-IL4.csv edgeRtranscripts3Expr-0h.csv edgeRtranscripts3Expr-2h.csv edgeRtranscripts3Expr-6h.csv edgeRtranscripts3Expr-IFN.csv edgeRtranscripts3Expr-IL4.csv RNAseq gene in: /data/images/proton/DKlab/mr/rnaseq/per-gene/ edgeRgenes_featurecount-0h.csv edgeRgenes_featurecount-2h.csv edgeRgenes_featurecount-6h.csv edgeRgenes_featurecount-IFN.csv edgeRgenes_featurecount-IL4.csv edgeRgenes_featurecountExpr-0h.csv edgeRgenes_featurecountExpr-2h.csv edgeRgenes_featurecountExpr-6h.csv edgeRgenes_featurecountExpr-IFN.csv edgeRgenes_featurecountExpr-IL4.csv transcript: in: /data/images/proton/DKlab/mr/rnaseq/stringtie edgeRtranscripts3-0h.csv edgeRtranscripts3-2h.csv edgeRtranscripts3-6h.csv edgeRtranscripts3-IFN.csv 9/14/2018 Biomedical Sciences Research Center Al. Fleming Mail - count matrices https://mail.google.com/mail/u/1/?ui=2&ik=f5ece6b682&jsver=K8SpjNhSmRc.en.&cbl=gmail_fe_180909.14_p3&view=pt&msg=… 2/2 edgeRtranscripts3-IL4.csv edgeRtranscripts3Expr-0h.csv edgeRtranscripts3Expr-2h.csv edgeRtranscripts3Expr-6h.csv edgeRtranscripts3Expr-IFN.csv edgeRtranscripts3Expr-IL4.csv The issue with edgeR was that we used a default paining in the exactTest. The doc for this is at: https://rdrr.io/bioc/edgeR/man/exactTest.html pair vector of length two, either numeric or character, providing the pair of groups to be compared; if a character vector, then should be the names of two groups (e.g. two levels of object$samples$group); if numeric, then groups to be compared are chosen by finding the levels of object$samples$group corresponding to those numeric values and using those levels as the groups to be compared; if NULL, then first two levels of object$samples$group (a factor) are used. ***Note that the first group listed in the pair is the baseline for the comparison*** —so if the pair is c("A","B") then the comparison is B - A, so genes with positive log-fold change are up-regulated in group B compared with group A (and vice versa for genes with negative log-fold change). exp_study$samples$group [1] WT WT KO KO Levels: KO WT 1 2 et = exactTest(exp_study) > topTags(et,n=5,adjust.method = "fdr") Comparison of groups: WT-KO logFC logCPM PValue FDR Xist -11.438030 9.283467 7.097269e-229 1.550327e-224 Ddx3y 11.062962 5.910218 4.047085e-163 4.420226e-159 Eif2s3y 11.076514 5.198332 4.966167e-127 3.616032e-123 Tsix -10.994961 4.853194 1.952988e-109 1.066527e-105 Uty 9.657196 4.492298 4.170986e-94 1.822220e-90 > et = exactTest(exp_study,pair=c("WT","KO")) > topTags(et,n=5,adjust.method = "fdr") Comparison of groups: KO-WT logFC logCPM PValue FDR Xist 11.438030 9.283467 7.097269e-229 1.550327e-224 Ddx3y -11.062962 5.910218 4.047085e-163 4.420226e-159 Eif2s3y -11.076514 5.198332 4.966167e-127 3.616032e-123 Tsix 10.994961 4.853194 1.952988e-109 1.066527e-105 Uty -9.657196 4.492298 4.170986e-94 1.822220e-90 The option pair=c("WT","KO") (giving KO vs WT) is used in all the results above. BW Martin