Alternative splicing analysis, rna-seq

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10.2 years ago

marina-orlova ▴ 90

Hello

I took rna-seq data (d. melanogaster, SAM fles) from modencode for two samples (empbryos 0-4 hrs and 4-8 hrs), put it to cufflinks (cuffdiff) and got output with isoforms' RPKM in both samples and genes' RPKM.

I compared the data I got about genes' RPKM to data on flybase.org and found out that they are quite different.

For example, for gene Act79B I got RPKM_1 392.168 and RPKM_2 1184.06 but data on flybase shows that less than 3 for both samples.

What am I doing wrong? Or this comparison is incorrect?

RNA-Seq alternative-splicing cufflinks • 2.8k views

ADD COMMENT • link updated 2.9 years ago by Ram 44k • written 10.2 years ago by marina-orlova ▴ 90

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I wouldn't assume that the values are comparable. There's more than one way to get at an RPKM value (e.g., do you use raw or normalized counts). Even using different versions of cufflinks will lead to changes in RPKM values.

ADD REPLY • link updated 2.9 years ago by Ram 44k • written 10.2 years ago by Devon Ryan 104k

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To add on to Devon, here is the paper on the problem of RPKM and previous discussion on biostar.

ADD REPLY • link updated 2.9 years ago by Ram 44k • written 10.2 years ago by Sam ★ 4.8k

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So, does it mean that I can't use it for alternative splicing analysis or not?

ADD REPLY • link updated 2.9 years ago by Ram 44k • written 10.2 years ago by marina-orlova ▴ 90

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Thank you for the reply!

ADD REPLY • link updated 2.9 years ago by Ram 44k • written 10.2 years ago by marina-orlova ▴ 90

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