Question: What is best tool to call diferentially expressed isforms ?
1
gravatar for jack
3.8 years ago by
jack800
Germany
jack800 wrote:

Hi all,

I have raw count of isform expression levels and I would like to do differential isform expression analysis between two different conditions. Does any of you know about tool that do DE isoform calling?

genomics rna-seq R gene • 2.0k views
ADD COMMENTlink modified 3.8 years ago by EagleEye6.6k • written 3.8 years ago by jack800
4
gravatar for WouterDeCoster
3.8 years ago by
Belgium
WouterDeCoster42k wrote:

Isoform inference is not straight forward, but is implemented in the cufflinks package. It's rather hard to distinguish to which transcript reads belong with short reads. More straightforward is to count on the exon level, less ambiguous. This you can do using DEXSeq

ADD COMMENTlink written 3.8 years ago by WouterDeCoster42k
2
gravatar for andrew.j.skelton73
3.8 years ago by
London
andrew.j.skelton735.9k wrote:

Depends on how you obtained your "raw isoform counts". If you have a very simple experiment, and I mean 2 groups to compare against, then Kallisto -> Sleuth is the most straightforward was to achieve what you want. Sleuth is however an absolute pain if you have a more complex design than two groups, there seems to be no support for contrast matrices like you'd see in Limma, or ways of interrogating your model fit with flexibility, like DESeq2.

ADD COMMENTlink written 3.8 years ago by andrew.j.skelton735.9k

Does Sleuth do differential isoform expression? Or is it only differential expression at the isoform (as opposed to gene) level?

ADD REPLYlink written 3.8 years ago by igor9.5k
1

Sleuth is designed for differential transcript expression. Kallisto quantifies at the transcript level, however that data can be aggregated for the gene level and used in tools such as DESeq2 for gene level differential expression. What's the difference between "differential isoform expression", and "differential expression at the isoform (as opposed to gene) level"?

ADD REPLYlink written 3.8 years ago by andrew.j.skelton735.9k

The difference I was referring to is:

(1) geneA-iso1 is higher in group1 versus group2 because geneA is higher (would also show up in gene-level analysis)

(2) geneA-iso1 is higher in group1 versus group2 because of alternative splicing in group1 (would not necessarily show up in gene-level analysis)

ADD REPLYlink modified 3.8 years ago • written 3.8 years ago by igor9.5k

In that case yes, Sleuth would show differential transcript expression, which would show you effects due to alternative splicing that you could possibly miss at the gene level.

ADD REPLYlink written 3.8 years ago by andrew.j.skelton735.9k
1
gravatar for igor
3.8 years ago by
igor9.5k
United States
igor9.5k wrote:

This questions has been previously discussed at length here: Is There Any Other Method For Isoform-Level Differential Expression Analysis Except Cufflinks/Cuffdiff?

ADD COMMENTlink written 3.8 years ago by igor9.5k
0
gravatar for EagleEye
3.8 years ago by
EagleEye6.6k
Sweden
EagleEye6.6k wrote:

If your quantification technique worked well and you got the counts.

You can use the raw counts as input. There are many tools available for DE, example edgeR, DESeq2, DEXseq etc,

Or else there are specialized method to detect differential Isoforms. Like mentioned in this following post.

A: How to determine alternative splicing read counts

ADD COMMENTlink written 3.8 years ago by EagleEye6.6k
0
gravatar for fanli.gcb
3.8 years ago by
fanli.gcb690
Los Angeles, CA
fanli.gcb690 wrote:

To highlight a few points from recent benchmarking papers here, here, and here:

  • Quantification of isoform abundance is still pretty tricky, especially for low abundance transcripts
  • Differential expression is even harder, and even the best methods still perform poorly
  • RSEM or kallisto for quantification, DEXSeq for differential transcript usage
ADD COMMENTlink written 3.8 years ago by fanli.gcb690
0
gravatar for EagleEye
3.8 years ago by
EagleEye6.6k
Sweden
EagleEye6.6k wrote:

I agree fanli.gcb, there are no perfect method to perform isoform quantification and differential expression. But there are studies uses available methods to perform the task. There is also a recent study where they used similar approach as I described in previous post using MISO,

A: How to determine alternative splicing read counts

And here is the recent study,

http://www.cell.com/molecular-cell/fulltext/S1097-2765(16)00047-2

ADD COMMENTlink modified 3.8 years ago • written 3.8 years ago by EagleEye6.6k
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