Dear all,
I'm running this edgeR-robust code to analyse my miRNA-seq DE sequences. But I receive only highly/lowly expressed miRNAs with logFC values higher than 4/lower than -4. But nothing in between... Should I change some parametres to get the whole logFC spectrum???
Thank you, Marek D. Koter
My code:
rm(list = ls())
library(edgeR)
y <- read.delim("3DPI_All_bez_K2_I2.txt", row.names="seq", header = T)
group <- factor(c(1,1,1,2,2,2))
keep<-rowSums(cpm(y)>5) >=1
y<-y[keep, ]
y <- DGEList(counts=y,group=group)
design <- model.matrix(~group)
y <- calcNormFactors(y)
y <- estimateGLMRobustDisp(y, design)
fit <- glmFit(y,design)
lrt <- glmLRT(fit,coef=2)
topTags(lrt, n = 50)
Fragment of my results:
logFC | logCPM | LR | PValue | FDR | |
CTACCCCTGAACTTATTTTAT | -5,28003 | 2,902426 | 4,407249 | 0,035787 | 0,362601 |
TTTCGTTACTCTGAGGCACA | -4,83913 | 2,484342 | 3,876514 | 0,048966 | 0,362601 |
AAAGAGACATAACTTTGGGCT | 4,173604 | 1,771509 | 3,934848 | 0,047295 | 0,362601 |
AACAGGACACTCTAAATCGGG | 4,173604 | 1,771509 | 3,934848 | 0,047295 | 0,362601 |
> sessionInfo() R version 3.2.2 (2015-08-14) Platform: x86_64-w64-mingw32/x64 (64-bit) Running under: Windows 8 x64 (build 9200) locale: [1] LC_COLLATE=Polish_Poland.1250 LC_CTYPE=Polish_Poland.1250 [3] LC_MONETARY=Polish_Poland.1250 LC_NUMERIC=C [5] LC_TIME=Polish_Poland.1250 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] edgeR_3.10.5 limma_3.24.15 loaded via a namespace (and not attached): [1] tools_3.2.2
You're using an old version of Bioconductor. I suggest you upgrade to BioC 3.2 and
edgeR
3.12.0 and use that for your analyses, to make sure this behaviour isn't present in the latest version.