Question: TSS profiles ChIP seq
1
gravatar for Lila M
2.2 years ago by
Lila M 460
UK
Lila M 460 wrote:

Hi everyone! I'm performing some ChIP seq experiments and I want to read depth coverage over the region -500 to 3000pb of the TSS of all Ensemble protein coding transcript.

I have different approach, but as I am totally naive, I appreciate very much your advises.

a)First of all I've normalized the bam files using bamCoverage (deepTools), the output is a bedgraph, then I will do the anntotaion peak over TSS using ChIPseeker (R)

b)Also, I can do the peakannot over the bed file (after bam file conversion) and I will normalize the peakannot when I will plot it.

Other questions is, Is necessary make the peakcalling to calculate the read depth over TSS?

I'm looking forward to hear from you

Than you!!!

chip-seq • 1.2k views
ADD COMMENTlink modified 2.2 years ago • written 2.2 years ago by Lila M 460
2
gravatar for Carlo Yague
2.2 years ago by
Carlo Yague4.4k
Belgium
Carlo Yague4.4k wrote:

a) looks good to me.

b) I don't really understand what you want to achieve here. What is 'doing a peakannot' ? Do you refer to the annotatePeak function of the ChIPseeker R package ?

It is not necessary to make a peakcalling to calculate read depth over TSS. You only need genome coverage and TSS annotation.

ADD COMMENTlink written 2.2 years ago by Carlo Yague4.4k

Hi again, As ChIPseeker gives me some error, you know any other way in which I can get the read depth coverage over TSS onces I have the normalized file ? Thanks!!

ADD REPLYlink written 2.2 years ago by Lila M 460
1

There are many many tools to do that. Since you are already familiar with deeptools, you could try their computeMatrix tool.

ADD REPLYlink written 2.2 years ago by Carlo Yague4.4k

Thank you, Do you know what file can I use as TSS.bed in computeMatrix? I downloaded one from USC but the program gives to me an error message

Warning: chrY:9748406-9748406 is an invalid BED interval! Ignoring it.

Thanks!

ADD REPLYlink written 2.2 years ago by Lila M 460
1

Could it be that your ChIP-seq data is from a female and that there is no Y ? This is a warning, not an error, and it could be ok to proceed if the non-Y coordinates are considered correct by the script.

More generally, check that the chromosome names in the bed file are the same as in the bedgraph file.

Carlo

ADD REPLYlink modified 2.2 years ago • written 2.2 years ago by Carlo Yague4.4k
1
gravatar for Lila M
2.2 years ago by
Lila M 460
UK
Lila M 460 wrote:

Thank you very much, Other question that I have, for bamCoverage I have two options, --nomralizeto1x --normalizeUsingRPKN

Is there any way in which I could specify that I want a normalization equal to 25 milliom reads?

Thanks!

ADD COMMENTlink written 2.2 years ago by Lila M 460
1

a normalization equal to 25 million reads

Do you mean that you want to normalize as if you had 25 million reads mapped ? If so, you need to use a third option : --scaleFactor set to 25000000/number_of_reads_mapped. To calculate the number of reads mapped, you can just use samtools flagstat on the .bam file.

ADD REPLYlink modified 2.2 years ago • written 2.2 years ago by Carlo Yague4.4k
1

One more question, as I can't introduce in scale factor 2500000/31567890, the argument should be: --sacaleFactor 25000000 or --scaleFactor 0.64?

Thanks!

ADD REPLYlink written 2.2 years ago by Lila M 460
1

25000000/31567890 = 0.7919 : this is your scaleFactor for this particular bam file :)

ADD REPLYlink written 2.2 years ago by Carlo Yague4.4k

last question, when I've tried to run reads <- import.bed(con="NGS974Norm")

NGSNorm is the bedgraph file, 4 cols and size ~ 400MB

cov <- coverage(reads) peakAnno <- annotatePeak(reads, tssRegion=c(-500, 2000), TxDb=TxDb.Hsapiens.UCSC.hg19.knownGene, annoDb="org.Hs.eg.db")

I have the next error Error in rsqlite_send_query(conn@ptr, statement) : out of memory

any ideas?

ADD REPLYlink modified 2.2 years ago • written 2.2 years ago by Lila M 460

Thank you! I'm working on that :)

ADD REPLYlink written 2.2 years ago by Lila M 460
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