Question: Copy Number Variation from paired end RNA-Seq data
0
gravatar for ChIP
4.4 years ago by
ChIP490
Netherlands
ChIP490 wrote:

Hi!

I want to know, is it possible to identify copy number variations from paired end RNA-seq data? if yes , how? and which tools to use?

What I have done till now is:

a) Executed mpileup on the bam file of paired end RNA-seq file.

b) Next I wanted to use varscan copynumber, but it requires  normal_pileup and tumour pile-up. I only have pileup on my experimental data. Hence I cannot move forward or am I missing a trick here?

 

Please help

Thank you
 

rna-seq cnv snp • 5.9k views
ADD COMMENTlink modified 4.4 years ago by Christian2.8k • written 4.4 years ago by ChIP490
3
gravatar for Christian
4.4 years ago by
Christian2.8k
Cambridge, US
Christian2.8k wrote:

I have seen papers calling CNVs from RNA-seq data, albeit only the very large ones (chromosome arm-sized).

For example, have a look at Figure 1C and accompanying methods in this paper:

Patel et al. (2014)
Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma

 

ADD COMMENTlink written 4.4 years ago by Christian2.8k
1
gravatar for Devon Ryan
4.4 years ago by
Devon Ryan89k
Freiburg, Germany
Devon Ryan89k wrote:

The only way to see CNVs with RNAseq is if some of the copies result in fusion events with other genes, thus resulting in one read in a pair aligning to gene A and the other to gene B. Aside from that, one can't look at CNVs with RNAseq.

Even with a matched control sample, you still can't use the method you outlined to call copy number. It's impossible to discriminate between differential expression and an underlying copy number variation with RNAseq data. This is a fundamental limitation. If you want  to look at CNVs, you need to sequence DNA.
 

ADD COMMENTlink written 4.4 years ago by Devon Ryan89k
1

I wouldn't say it is impossible, just limited to very large CNVs due to the high coverage variability introduced by differential expression (see my answer and the referenced paper below).

ADD REPLYlink written 4.4 years ago by Christian2.8k

True, if the CNV is absolutely huge like that then it can work.

ADD REPLYlink written 4.4 years ago by Devon Ryan89k

Will WGBS can help in detecting CNVs?


 

ADD REPLYlink written 4.4 years ago by ChIP490

Yes, you can call CNVs from WGBS data (I've never seen anyone do it, but there's no reason you couldn't). You could use the mpileup->varscan copy number method with that.

ADD REPLYlink written 4.4 years ago by Devon Ryan89k

I take that back, while you can use WGBS data to call CNVs, varscan still needs a control dataset. You might be able to find something close enough in a public dataset, if you're lucky. Otherwise, there ends up being no great way to call CNVs. If one were to use the naive method of just going by depth, then any bias introduced by mappability, library construction, etc. could seriously skew the results. Having said that, if you're going to validate things anyway and you can tolerate a moderately high false-positive rate, then perhaps this can suffice.

ADD REPLYlink written 4.4 years ago by Devon Ryan89k

Thank's, will keep your points in mind.
 

ADD REPLYlink written 4.4 years ago by ChIP490

Best of luck with this. Feel encouraged to leave a comment when you have the results. I'm curious to hear how well this works in practice.

ADD REPLYlink written 4.4 years ago by Devon Ryan89k

Thank's again. I have no clue how well this will work as I do not have control datasets, but we will validate some of our calls so lets see. Will keep you posted.
 

ADD REPLYlink written 4.4 years ago by ChIP490
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