Hi Simon,

First of all, many thanks for DESeq. I have found it wonderfully easy to work with, even with my beginner's knowledge of R. My understanding of statistics is limited to a couple of graduate-level courses, so I would really appreciate your input on the following questions, which arise from using DESeq on my data. My project comprises 14 individuals (7 case-control pairs) and we have ~40 million Illumina transcriptome reads from each of these people. I used BedTools to overlap these reads with the UCSC knownGene annotations, and ran DESeq on the count data.

Question 1:

The ECDF curves lie above the diagonal in both cases, so I used a correction factor of 0.7, which brings them closer. I have attached both the uncorrected and corrected curves below, as well as the plots of the fit of the variance functions. Can you tell me if, in your opinion, the correction factor seems adequate?

Question 2:

On page 10 of the manual, you mention that any hit with a very large value in any one of the last two columns in the differential expression output file should be checked carefully to exclude false hits due to “variance outliers”. Do you have a ballpark figure in mind when you say "very large value"? In my data, the values range from 0.003 - 214, so I am confused what to use as a cutoff.

Any advice you have to offer would be very welcome.

Thanks,

Shurjo

First of all, many thanks for DESeq. I have found it wonderfully easy to work with, even with my beginner's knowledge of R. My understanding of statistics is limited to a couple of graduate-level courses, so I would really appreciate your input on the following questions, which arise from using DESeq on my data. My project comprises 14 individuals (7 case-control pairs) and we have ~40 million Illumina transcriptome reads from each of these people. I used BedTools to overlap these reads with the UCSC knownGene annotations, and ran DESeq on the count data.

Question 1:

The ECDF curves lie above the diagonal in both cases, so I used a correction factor of 0.7, which brings them closer. I have attached both the uncorrected and corrected curves below, as well as the plots of the fit of the variance functions. Can you tell me if, in your opinion, the correction factor seems adequate?

Question 2:

On page 10 of the manual, you mention that any hit with a very large value in any one of the last two columns in the differential expression output file should be checked carefully to exclude false hits due to “variance outliers”. Do you have a ballpark figure in mind when you say "very large value"? In my data, the values range from 0.003 - 214, so I am confused what to use as a cutoff.

Any advice you have to offer would be very welcome.

Thanks,

Shurjo

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