Question

DESeq2 alternative hypyothesis: testing for genes NOT significantly downregulated

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casco ▴ 10

@2f2d9aca

Last seen 10 weeks ago

United States

Hi all,

I'm interested in testing which genes are not significantly downregulated. While I'm able to get genes significantly upregulated with the following code:

resUp <- results(dds, lfcThreshold=0, altHypothesis="greater")

And genes significantly unregulated with the following code:

resLA <- results(dds, lfcThreshold=1, altHypothesis="lessAbs")

I'm unable to define genes significantly NOT downregulated using altHypothesis = "greater" and lfcThreshold = -1 as the lfcThreshold must be a non-negative value. Is there another way to directly test this?

DESeq2 • 873 views

ADD COMMENT • link 8 months ago casco ▴ 10

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couldn't you do that by test for log2fc >1 or log2fc <-1 and add significance, ex padjust < 0.05?

ADD REPLY • link 8 months ago Theo ▴ 10

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That's essentially the standard analysis looking for significantly up- or down-regulated genes, isn't it? I'm trying to identify genes not downregulated. The best I've been able to achieve is looking for genes that do not have a log2fc < 0 and a p.adj < 0.05. The issue with this is that I'm still testing for genes that are downregulated, not the alternative which would be genes not downregulated.

ADD REPLY • link 8 months ago casco ▴ 10

score 0 · Answer 1 · 2023-08-25

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James W. MacDonald 65k

@james-w-macdonald-5106

Last seen 1 hour ago

United States

There is a help page for results that you can read to answer this sort of question. It says

altHypothesis: character which specifies the alternative hypothesis,
          i.e. those values of log2 fold change which the user is
          interested in finding. The complement of this set of values
          is the null hypothesis which will be tested. If the log2 fold
          change specified by 'name' or by 'contrast' is written as
          beta , then the possible values for 'altHypothesis' represent
          the following alternate hypotheses:

            * greaterAbs: |beta| > lfcThreshold , and p-values are
              two-tailed

            * lessAbs: |beta| < lfcThreshold , p-values are the maximum
              of the upper and lower tests. The Wald statistic given is
              positive, an SE-scaled distance from the closest boundary

            * greater: beta > lfcThreshold

            * less: beta < -lfcThreshold

Which seems to answer your question?

ADD COMMENT • link 8 months ago James W. MacDonald 65k

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Ideally, testing for the "greater" altHypothesis but with a lfcThreshold > -1. However, negative values are not allowed as input for the lfcThreshold.

ADD REPLY • link 8 months ago casco ▴ 10

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If I interpret your goal correctly, 'Not significantly down-regulated' means all genes that are not significant in one direction. Which can be easily found by taking the complement. In other words, it's all genes tested that do not fulfill the test using 'less' as the argument. So you can do that test and then select all the genes that are not in the set.

ADD REPLY • link 8 months ago James W. MacDonald 65k

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The test you suggest (greater with an lfcThreshold > -1) isn't what you said you wanted. That's asking for genes that are significantly different but restricted to a log fold change of -1 or greater. There could be any number of genes with a logFC < 0 that are significantly different in that test, and would be (by definition) significantly down-regulated.