Question: Highly biased experiment showing funny histogram of p values for all tests. Suggestions?
0
Mozart140 wrote:

Dear all, I am struggling (as usual) with a really important/expensive experiment we manage to complete. Having done this in different days, plus given the fact we are not expecting a strong phenotype (we are treating cells with a molecule that shouldn't change these ones that much), I would imagine not so many genes differentially expressed. Perfect. I initially corrected the experiment for batch effect. Indeed, I added an extra column to my model matrix in this way:

``````Table <- data.frame(condition = factor(c("A","A","A","B","B","B","C","C","C","A","B","C","D","D","D","D")))

Table\$batch = factor(c('day1','day2','day3','day1','day2','day3','day1','day2','day3','day4','day4','day4','day4','day1','day2','day3'))
``````

table(sampleTable)

Thus, having:

``````> table(Table)
batch
condition day1 day2 day3 day4
A      1    1    1    1
B      1    1    1    1
C      1    1    1    1
D      1    1    1    1
``````

Then:

``````dds <- DESeqDataSetFromTximport(txi.kallisto.tsv, Table, ~batch+condition)
dds <- DESeq(dds)
nrow(dds) **# 29654**
res = results(dds)
``````

Say I want to compare A(treated) vs B(untreated):

``````dds\$condition <- relevel(dds\$condition, ref = "B")
dds <- DESeq(dds)

results_A_vs_B<-results(dds, c("condition", "A", "B"))
``````

If I plot the histogram of p values I get something like this: Which is pretty odd I guess, given the fact I get a lot of unfiltered values in my final experiment that are around pval=1.

then, I decided to remove low counts by arbitrarily choosing the following filtering parameter:

``````idx <- rowSums( counts(dds, normalized=TRUE) >= 8 ) >= 6
dds <- dds[idx,]
res = results(dds)
``````

then,

``````dds\$condition <- relevel(dds\$condition, ref = "B")
dds <- DESeq(dds)

results_A_vs_B<-results(dds, c("condition", "A", "B"))
``````

it's even worse, I guess: So there's no doubt there's something wrong here but not sure how to get rid of it.

Do you have any ideas?

Thanks

filtering rna-seq deseq2 • 119 views
written 4 weeks ago by Mozart140

I cannot see the figures, is it me or something went wrong?