What is best tool to call diferentially expressed isforms ?
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6.1 years ago
jack ▴ 930

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?

RNA-Seq genomics R gene • 3.2k views
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6.1 years ago

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

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6.1 years ago

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.

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

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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"?

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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)

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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.

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6.1 years ago
igor 12k

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

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EagleEye 7.3k

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

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6.1 years ago
fanli.gcb ▴ 730

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
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EagleEye 7.3k

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

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