Moderator: Ryan C. Thompson

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Posts by Ryan C. Thompson

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Comment: C: Making sense out of makeContrasts and using coefficents for contrasts
... I'll assume you meant `T5.LD.FW` instead of `T5.SP`. If so, you are testing the T6 difference against the mean of the T5 and T4 differences. That's not quite the same as what you're after. In any case, I find that these "difference of differences" contrasts are difficult to interpret without the con ...
written 7 months ago by Ryan C. Thompson7.4k
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Answer: A: Random Effect in DESeq2
... To my knowledge, none of the negative binomial GLM-based packages (e.g. edgeR & DESeq2) support models with random effects. Limma is the only package I know of that does support RNA-seq analysis with random effects. It supports a model with any number of fixed effects and one factor as a random ...
written 7 months ago by Ryan C. Thompson7.4k
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Comment: C: Making sense out of makeContrasts and using coefficents for contrasts
... What hypothesis are you attempting to test with this contrast? We can't tell you if your contrast is testing what you want to test unless you say what you want to test. In any case, the contrast you have written appears to be subtracting two terms from one term, which usually yields a nonsense con ...
written 7 months ago by Ryan C. Thompson7.4k
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Answer: A: edgeR /DESeq2 normalization for differential expression in RNA-seq blood samples
... Aaron's answer adequately covers the theoretical reasons that the normalizations used in edgeR and DESeq2 are appropriate for data with variations in globin content, so I will just add that empirically, I have actually *used* edgeR on such a data set. Specifically, we were testing a custom globin bl ...
written 8 months ago by Ryan C. Thompson7.4k
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Comment: C: Appropriate design formula for DESeq2 from principles
... This isn't an answer to your main question, but for an in-depth discussion of how design matrices are constructed from factors, you should have a read through the [vignette for the codingMatrices package](https://cran.r-project.org/web/packages/codingMatrices/vignettes/codingMatrices.pdf). You gener ...
written 8 months ago by Ryan C. Thompson7.4k
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Comment: C: Order of contrasts.fit call (before or after eBayes)
... You can read the code of the functions to find out exactly what they are doing. I did that a while ago but I don't recall the exact details. I just remember that the rule I came up with was "always run `eBayes` on every fit", and I haven't had any trouble since. ...
written 8 months ago by Ryan C. Thompson7.4k
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Comment: C: Order of contrasts.fit call (before or after eBayes)
... It's only necessary to call `eBayes` if you need the statistics that it calculates. This will almost always be the case for the contrast fit (since you want to call `topTable` on it), but sometimes you also want it for the regular fit as well. It normally only takes a second or two to run it, so I j ...
written 8 months ago by Ryan C. Thompson7.4k
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Answer: A: Order of contrasts.fit call (before or after eBayes)
... I recommend saving the result of `contrasts.fit` as a separate object, so that you still have access to the original `lmFit` result as well. They both have their uses, as you have noted. Run `eBayes` on both of them. My typical workflow looks something like this: ``` fit <- lmFit(mat_data, desig ...
written 8 months ago by Ryan C. Thompson7.4k
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Answer: A: LIMMA dupcor() function time issue.
... Looking at the code of duplicateCorrelation, the main time sink is the loop that runs mixedModel2Fit on each row of the input. So the time required should indeed be a linear function of the number of rows. However, the linear trend might break down if you are bumping up against hardware limits. The ...
written 8 months ago by Ryan C. Thompson7.4k
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Comment: C: Use of pseudoalignment estimated counts as input to edgeR and Limma-Voom
... If you already have an alignment pipeline you're happy with, I wouldn't bother switching to another aligner. It's generally not going to make much of a difference. Just use featureCounts with your existing aligned BAM files. ...
written 8 months ago by Ryan C. Thompson7.4k

Latest awards to Ryan C. Thompson

Teacher 9 months ago, created an answer with at least 3 up-votes. For A: Correct assumptions of using limma moderated t-test
Scholar 9 months ago, created an answer that has been accepted. For A: Limma validity for only hundreds of genes/metabolites
Commentator 9 months ago, created a comment with at least 3 up-votes. For C: limma lmFit coefficients and p-values
Popular Question 9 months ago, created a question with more than 1,000 views. For Importing a BED file with floating point scores?
Teacher 9 months ago, created an answer with at least 3 up-votes. For A: Voom transformation from counts and normalization to negative values
Scholar 9 months ago, created an answer that has been accepted. For A: Finding DE genes from RNA-seq data
Popular Question 9 months ago, created a question with more than 1,000 views. For frma vectors for GPL13158 (Affymetrix HT HG-U133+ PM Array Plate)?
Popular Question 9 months ago, created a question with more than 1,000 views. For Low-count filtering with uneven sequencing depth between samples?
Appreciated 9 months ago, created a post with more than 5 votes. For A: Correct assumptions of using limma moderated t-test
Teacher 9 months ago, created an answer with at least 3 up-votes. For A: What's the relationship between "design" and "contrasts"?
Teacher 9 months ago, created an answer with at least 3 up-votes. For A: [Limma] Testing continuous variable with interaction
Teacher 9 months ago, created an answer with at least 3 up-votes. For A: Finding DE genes from RNA-seq data
Good Answer 10 months ago, created an answer that was upvoted at least 5 times. For A: Batch correction in DESeq2
Popular Question 11 months ago, created a question with more than 1,000 views. For summarizeOverlaps using GRanges or bed file as reads?
Appreciated 11 months ago, created a post with more than 5 votes. For A: Finding DE genes from RNA-seq data
Teacher 12 months ago, created an answer with at least 3 up-votes. For A: Voom transformation from counts and normalization to negative values
Scholar 12 months ago, created an answer that has been accepted. For A: Limma validity for only hundreds of genes/metabolites
Teacher 13 months ago, created an answer with at least 3 up-votes. For A: Voom transformation from counts and normalization to negative values
Teacher 14 months ago, created an answer with at least 3 up-votes. For A: What's the relationship between "design" and "contrasts"?
Teacher 16 months ago, created an answer with at least 3 up-votes. For A: RNA-seq Normalisation - normalise all samples in experiment or only the ones use
Popular Question 16 months ago, created a question with more than 1,000 views. For edgeR Quasi-likelihood with tagwise dispersion?
Scholar 16 months ago, created an answer that has been accepted. For A: Limma validity for only hundreds of genes/metabolites
Teacher 16 months ago, created an answer with at least 3 up-votes. For A: What's the relationship between "design" and "contrasts"?
Popular Question 16 months ago, created a question with more than 1,000 views. For summarizeOverlaps using GRanges or bed file as reads?

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