Diffbind analysis of ATAC-seq data
1
0
Entering edit mode
young • 0
@225f54f3
Last seen 7 weeks ago
China

Hello,

I have ATAC sequencing data for two types of cells, each with three replicates. Get strange results by DiffBind analysis. Why is my volcano map only halfway？ I don't know where I went wrong. Is there a problem with the original data or with the processing results? Can anyone answer it. here is my SampleSheet.

last.BAM was compared to the genome.Mitochondrial duplication, PCR duplication and low-quality reads were removed. Paired-ended sequencing to the same chromosome

figure2

figure3

figure4

figure5

here is my code

       a <- dba(sampleSheet="diff.csv")
a <- dba.count(a,bUseSummarizeOverlaps = TRUE)

#warning:In serialize(data, node\$con) :
#'package:stats' may not be available when loading

dba.plotPCA(a,attributes=DBA_FACTOR,label=DBA_ID)
#figure2

plot(a)
#figure3

a <- dba.contrast(a,categories = DBA_FACTOR,minMembers = 2)
a <- dba.analyze(a,method=DBA_DESEQ2)
#warning: solve(): system is singular (rcond: 3.89123e-18); attempting approx solution

dba.show(a,bContrasts = T)
#  Factor Group Samples Group2 Samples2 DB.DESeq2
# 1 Factor    ab       3     aa        3         8

dba.plotMA(a)
#warning:In KernSmooth::bkde2D(x, bandwidth = bandwidth, gridsize = nbin,  :Binning grid too coarse for current (small) bandwidth: consider increasing 'gridsize'

#figure4

#This picture is weird.

dba.plotVolcano(a)

#figure5

sessionInfo( )

ATACSeq DiffBind • 127 views
ADD COMMENT
0
Entering edit mode
Rory Stark ★ 4.4k
@rory-stark-5741
Last seen 3 days ago
CRUK, Cambridge, UK

This is showing that all of the open chromatin changes are in the same "direction": sites that are open in the aa condition only. There are no sites open in ab that are not also open in aa, so the MA and volcano plots are highly asymmetric.

There are many biological conditions that lead to these types of asymmetric differences. Given how few sites there are, I'd suggest looking at them in a genome browser to see if the sequencing data show the gain in open chromatin in aa at these sites.

ADD COMMENT

Login before adding your answer.

Traffic: 220 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6