Question about setting threshold using filtering by expression on genes in edgeR
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@mohammedtoufiq91-17679
Last seen 1 day ago
United States

Hi,

I am working with RNA-Seq transcriptomics data, and have a question about setting thresholds using filtering by expression on genes in edgeR. I like this function, and by default, I set the below for my experiment set-up:

keep <- filterByExpr(y, group=Treatment_Timepoint)  ## Test case 1
table(keep)

In the help section of filterByExpr, I used the below code and obtained the same number of genes passing like test case 1.

keep.test <- filterByExpr(y, group=Treatment_Timepoint,
             min.count = 10, min.total.count = 15, large.n = 10, min.prop = 0.7)    ## Test case 2
table(keep.test)

From the test case 1, I would like to know apart from function utilizing y and group=Treatment_Timepoint, is the filterByExpr using any other thresholds or cut-off or it is by default considers arguments below when I run this function?

min.count = 10 
min.total.count = 15
large.n = 10
min.prop = 0.7
dput(Sample.info)

#>           Donor Treatment Timepoint Treatment_Timepoint
#> Sample.1     P1   Control       6hr         Control_6hr
#> Sample.2     P2   Control       6hr         Control_6hr
#> Sample.3     P3   Control       6hr         Control_6hr
#> Sample.4     P4   Control       6hr         Control_6hr
#> Sample.5     P1      High       6hr            High_6hr
#> Sample.6     P2      High       6hr            High_6hr
#> Sample.7     P3      High       6hr            High_6hr
#> Sample.8     P4      High       6hr            High_6hr
#> Sample.9     P1   Control      24hr        Control_24hr
#> Sample.10    P2   Control      24hr        Control_24hr
#> Sample.11    P3   Control      24hr        Control_24hr
#> Sample.12    P4   Control      24hr        Control_24hr
#> Sample.13    P1      High      24hr           High_24hr
#> Sample.14    P2      High      24hr           High_24hr
#> Sample.15    P3      High      24hr           High_24hr
#> Sample.16    P4      High      24hr           High_24hr
#> Sample.17    P1   Control      48hr        Control_48hr
#> Sample.18    P2   Control      48hr        Control_48hr
#> Sample.19    P3   Control      48hr        Control_48hr
#> Sample.20    P4   Control      48hr        Control_48hr
#> Sample.21    P1      High      48hr           High_48hr
#> Sample.22    P2      High      48hr           High_48hr
#> Sample.23    P3      High      48hr           High_48hr
#> Sample.24    P4      High      48hr           High_48hr

Best Regards,

Mohammed

filtering RNA-Seq CPM edgeR filterByExpr • 1.6k views
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2
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@james-w-macdonald-5106
Last seen 2 hours ago
United States

This information is provided in the help page.

Usage:

     ## S3 method for class 'DGEList'
     filterByExpr(y, design = NULL, group = NULL, lib.size = NULL, ...)
     ## S3 method for class 'SummarizedExperiment'
     filterByExpr(y, design = NULL, group = NULL, lib.size = NULL, ...)
     ## Default S3 method:
     filterByExpr(y, design = NULL, group = NULL, lib.size = NULL,
                  min.count = 10, min.total.count = 15, large.n = 10, min.prop = 0.7, ...)

The part where it says Default S3 method shows the default values that are used if you do not specify something different.

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Thank you James W. MacDonald

I frequently use test case 1 for analysis. I believe the arguments by defaults are already set to optimal values for differential expression analyses so I do not need to change them at all.

Are the already set arguments are right for my analysis set-up? If not, what are the best argument values according my sample information?

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1
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Yes, your test case 1 code is correct. You do not need to change any preset arguments.

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Gordon Smyth perfect, thank you very much. I will use the below:

keep <- filterByExpr(y, group=Treatment_Timepoint)  ## Test case 1
table(keep)

Additionally, referring the help("filterByExpr") min.count and min.total.count concept is clear, however, I am bit confused what actually, large.n=10 and min.prop = 0.7 argument does? Is it considering genes detected in at least 70% of the samples? How shall I relate to my sample information, is it considering the smallest group from the Treatment_Timepoint, anyways all groups have 4 sample each. Meaning to ensure at least 4 samples with a count of 10 or more, where 4 can be chosen as the sample size of the smallest group of samples.

In case, if smaller group with only 2 samples existed in the Treatment_Timepoint, then it would be at least 2 samples with a count of 10 or more, where 2 can be chosen as the sample size of the smallest group of samples.

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1
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As the help page explains, the min.prop=7 cutoff only comes into play when all the groups are larger than large.n=10 in size. The help page says:

If all the group sizes are larger than large.n, then this is relaxed slightly, but with n always greater than min.prop of the smallest group size (70% by default).

Your groups are all of size 4, and 4 is less than 10. Hence these arguments do not affect your experiment at all.

For your experiment, filterByExpr will simply keep genes that are expressed in at least 4 samples.

If all your groups were of size 20 intead of 4, then filterByExpr would keep genes expressed in at least 17 samples (where 17 is equal to 100% of the first 10 plus 70% of the extra 10). If all your groups were of size 100, then filterByExpr would keep genes expressed in at least 73 samples. In large sample situations, a gene is usually still of interest if it is expressed in most of the samples for at least one group.

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Gordon Smyth For larger groups it is approximately 70%, in my case filterByExpr will simply keep genes that are expressed in at least 4 samples. If I want to express this in terms of percentage, how shall I calculate?

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1
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The filtering is not based on a percentage. It makes no sense to try to express it that way.

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