hi
I have 4 samples, A1,A2,B1,B2.
two conditions:A,B. each conditions have two replicates.
when I feed the reads count to the DESeq with replicates and without any replicates, the results vary greatly.
1.With replicates(A1A2-VS-B1B2), I get about 12000 DEGs(padj<0.1).
2.Without any replicates(A1-VS-B1), I get about only 200 DEGs(padj<0.1).
Both of the MA-plot are attached.
When working without any replicates, it sames that many informations are lossed, even the fold change is very high. Such as the gene "23416":
how to explain this?
PS:
I have 4 samples, A1,A2,B1,B2.
two conditions:A,B. each conditions have two replicates.
when I feed the reads count to the DESeq with replicates and without any replicates, the results vary greatly.
1.With replicates(A1A2-VS-B1B2), I get about 12000 DEGs(padj<0.1).
2.Without any replicates(A1-VS-B1), I get about only 200 DEGs(padj<0.1).
Both of the MA-plot are attached.
When working without any replicates, it sames that many informations are lossed, even the fold change is very high. Such as the gene "23416":
HTML Code:
"id" "baseMean" "baseMeanA" "baseMeanB" "foldChange" "log2FoldChange" "pval" "padj" "without replicates" 292.791254092328 9.3250242940964 576.25748389056 61.7968882135121 5.94946228783961 0.0039474188223427 0.179640885315504 "with replicates" 285.324742802128 12.0227636114328 558.626721992823 46.4640859661922 5.53804412255897 5.31049903576043e-59 6.44978249349437e-58
PS:
HTML Code:
head(countsTable) gene A1 A2 B1 B2 57573 104 67 233 224 8563 357 346 110 104 8434 88 94 94 120 10309 33 23 11 14 7652 17 16 1 0
HTML Code:
#With replicates
countsTable <- read.delim("A1A2B1B2",header=TRUE, stringsAsFactors=TRUE, row.names="gene")
conds <- factor(c( "A", "A","B","B"))
cds <- newCountDataSet( countsTable, conds )
cds <- estimateSizeFactors( cds, locfunc=shorth )
cds <- estimateDispersions( cds )
res <- nbinomTest( cds, "A", "B" )
HTML Code:
#Without any replicates
cds11 <- cds[,c("A1","B1")]
cds11 <- estimateDispersions(cds,method="blind", fitType ="local",sharingMode="fit-only") #is right here?
res112 <- nbinomTest(cds112,"A","B")
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