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Old 03-20-2013, 02:19 AM   #1
Daniel1977
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Default No Replicates Cuffdiff; How does the 'variability model good fit' apply? and other...

Hi,

I want to clarify (in my head of course) how the system works so that I can make the proper choices when I use it.

I have RNAseq data for several conditions, cell line with or without a virus (and deletion mutants) and/or different treatments (no biological/technical replicates, and yes I understand what this means in terms of the results).

So, what Cuffdiff will do is ‘‘pool the conditions together to derive a dispersion model’’, which assumes that most genes won´t be differentially expressed then describes overall gene variances based on ‘expression’ levels (roughly; e.g. my gene’s fpkm is 500, I don´t know its variance as I do not have replicates, but most genes around that expression have a variance of 620, hence I will assign a variance of 620 to my gene for testing).

Q1-Right?

Next,’’ Cuffdiff assumes that gene's and transcripts with similar expression levels have similar variances in those expression levels. However, that's not always the case - some genes have unusually high variability, for biologically meaningful reasonss. Thus, Cuffdiff checks that the variability model is good fit before performing any signficance testing. This works as follows: Cuffdiff first calculates the expression level of the gene or transcript in each condition begin compared. It does so by pooling the data for the replicates of a condition together. The model gives an expression and variance estimate for each condition, along with confidence intervals around the expression estimates. Then, Cuffdiff calculates the expression level of the gene in each replicate independently. If the expression level of one or more replicates lies outside the confidence interval generated by the model, Cuffdiff flags the transcript as poorly fit by the model, and no signficance testing is performed.’’

Q2-How does this apply to my setting (no replicates)?

Q3- What exactly are the ‘NOTEST’ , ‘HIGHDATA’ and ‘FAIL’ flags?

Q4- How does the –min-outlier-p option apply to the settings (no replicates)?

Q5- Should I run Cuffdiff with all my conditions at once so that the variance model benefits from all the possible data instead of doing pairwise Cuffdiff runs?

Q6- In case I want to use the expression values with tools not included in Cuffdiff (PCAs, correlations etc.) is the FPKM value alone the one to use?

Any comments etc. would be greatly appreciated.
D
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Old 03-21-2013, 06:26 AM   #2
Daniel1977
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Any comments at least for Q4 and Q5?
cheers
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Old 03-21-2013, 09:43 AM   #3
Krish_143
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Incase if i do not have replicates,
1) I will run cufdiff for 2 samples (ctl,sample1)(ctl,sample2)... do it for all and then compare all together.
or
a) I will preffer cufflinks 1st step (all samples), so i will have fpkm value for all the genes to all the samples and then based on gene of interest using R i will analyze the data.

choose what you like.
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Last edited by Krish_143; 03-21-2013 at 09:46 AM.
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Old 03-22-2013, 12:52 AM   #4
Daniel1977
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Thanks Kris_143.

Yes one can always use the (a) option.

But if you want to use cuffdiff it seems like a better option to run cuffdiff with all the conditions so that the variance model is more informed, hence you will probably get more precise results.

Also regarding Q4 I´ve tried 0.01, 0.05 and 0.99 and got:

P =0.99
Performed 0 isoform-level transcription difference tests
Performed 0 tss-level transcription difference tests
Performed 38023 gene-level transcription difference tests
Performed 0 CDS-level transcription difference tests

P=0.05
Performed 45266 isoform-level transcription difference tests
Performed 40546 tss-level transcription difference tests
Performed 38023 gene-level transcription difference tests
Performed 39875 CDS-level transcription difference tests

P=0.01
Performed 45266 isoform-level transcription difference tests
Performed 40546 tss-level transcription difference tests
Performed 38025 gene-level transcription difference tests
Performed 39875 CDS-level transcription difference tests

It seems that it isn´t really doing anything (also from other comments in the post), but it puzzles me that the 0.99 option is devoid of all tests but the genes' .
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Old 05-11-2013, 07:16 AM   #5
pengchy
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For the experiment without any replicates, the following threads give suggestions:
How to detect alternative splicing when there is no replicate
DESeq, experimental design
DESeq without replicates

the document of DESeq also gives a pipeline for experiments without any replicates.

Last edited by pengchy; 05-11-2013 at 07:09 PM.
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Old 05-12-2013, 11:39 PM   #6
Daniel1977
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thanks for that, I´ll look into it.
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Old 12-02-2013, 03:57 PM   #7
IBseq
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Hi Daniel77,
did you get any answer on q1 and q2?
I am also running cuffdiff without replicates and would like to understand how it does this

thanks
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