Hi Davis/Group:
I have a question when I was trying edgeR for the common.lib.size and the
normalized counts for each library. My RNA-seq data consists of seven
conditions with two replicates each. As the user's guide indicates the
common.lib.size is expected for each library (approximately!), but what I got
are quite different from each other.
countsTable <- read.delim("Deep_seq_data.csv", header=T, stringsAsFactors=T)
d<-countsTable[, -1] # Remove the first column which is FeatureID as AGI
number
conditions<-rep(c("Zygote", "Octant", "Globular", "Heart", "Torpedo", "Bent",
"Mature"), each=2)
d<-DGEList(counts=countsTable[,-1], group=conditions)
dim(d) #check the counts
# Filter genes with >=1 counts per million, in at least 2 samples
d<-d[rowSums(1e+06*d$counts/expandAsMatrix(d$samples$lib.size, dim(d))>=1)>=2,]
dim(d) #get the gene number satisfying the above condition
d<- calcNormFactors(d)
d$samples$lib.size
# "Zygote", "Zygote", "Octant", "Octant", "Globular", "Globular", "Heart",
"Heart", "Torpedo", "Torpedo", "Bent", "Bent", "Mature", "Mature"
# 21012147 19924212 26002900 9660245 17139388 7649319 16430105
20101956 12920266 6306742 44241095 20094409 15166090 23203758
d$common.lib.size
[1] 16554344.47
colSums(d$pseudo.alt)
# Zygote1 Zygote2 Octant1 Octant2 Globular1 Globular2
Heart1 Heart2 Torpedo1 Torpedo2
# 21523774.62 21638415.63 14533481.82 12046955.46 18920316.62 18439528.30
11754608.30 12759230.11 11248245.52 11410667.92
# Bent1 Bent2 Mature1 Mature2
# 16101723.65 17980670.24 26785396.02 27067289.80
It looks within each condition (i.e. between replicates), the normalized counts
are quite similar, but not between the conditions.
I have tried different normalizations (TMM, quantile and RLE), the results are
quite similar. Also I tried to only include two conditions with 2 replicates
each, say the Zygote and Octant, the result are the same. Not sure if I missed
anything, and I appreciate if you could have a look at my situation.
Thanks a lot!
Yifang
Yifang Tan
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