Question: Calculate non-expressing genes from RNA-seq data
gravatar for Dexter
11 weeks ago by
Dexter10 wrote:

Is it possible to calculate genes which are not expressing from RNA-seq data? One obvious way is to look at gene with 0 TPM and FKPM values. For sake of simplicity, ignore false-negatives from this analysis.

However, there will be some genes which will be false positive in RNA-seq analysis and can report some low level of TPM. Is there some crude way in which I can define TPM/FKPM cut off below which I can assume these gene are not expressing? I found one reference in (Mortazavi A et al 2008) where they suggest

Transcript detection was robust at 1.0 RPKM and above for a typical 2-kilo-base (kb) mRNA

However, they had spiked their samples with standard foreign mRNA to estimate levels later. How can I get non-expressing genes information from general RNA-seq data? Any crude method should be fine as far as it is logical. One method I though was to just plot the histogram of TPM and use 0.1 quantile as cutoff.

Note: I am fine if low expressing transcripts are not included.

rna-seq expression gene • 111 views
ADD COMMENTlink modified 11 weeks ago by ATpoint24k • written 11 weeks ago by Dexter10
gravatar for ATpoint
11 weeks ago by
ATpoint24k wrote:

Check the zFPKM framework at BioC ( and Github ( This framework aims to define data-driven thresholds for expressed genes that are not as arbitrary as simply defining something like FPKM > 1.

ADD COMMENTlink modified 11 weeks ago • written 11 weeks ago by ATpoint24k

Hi. I have read Hart T et al paper. In the paper author used -15 (of log2(FPKM))as cut off for not expressing genes. However, It was not mentioned how the cut off was selected. In addition, I am looking for some logical reason to select cut off. Or at least some statistical method to be unbiased. I tried to fit the Gaussian to my histogram and calculate zFPKM as described in the paper but it poses same question about selecting cut off.

ADD REPLYlink written 11 weeks ago by Dexter10
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