Hi!

I'm trying to analyse my RNA-seq data using the WGCNA package for R. The package manual says that you need a minimum of 15 samples, although i've been reading other posts and the FAQs and one should be able to produce an analysis for less than 15 samples.

In my case, I have gene counts data from RNA-seq for two conditions, from which I have three bioreplicates for each group. I wanted to produce a WGCNA for both my WT and experimental samples. I used DESeq2 to produce the DGE, but also i obtained the normalised counts for each condition. At the end, my input data for WGCNA has this format:

        gene1       gene2       gene3       …   genen
Sample1

Sample2

Sample3

My problem comes when i want to run the function goodSamplesGenes, which returns this error in R:

gsg = goodSamplesGenes(datExpr0, verbose = 3) Flagging genes and samples with too many missing values... ..step 1 Error in goodGenes(datExpr, goodSamples, goodGenes, minFraction = minFraction, : Too few genes with valid expression levels in the required number of samples.

Also, if I try to look at the pickSoftThreshold, the results i obtain for the SFT R2 are really low:

Power SFT.R.sq    slope truncated.R.sq mean.k. median.k. max.k.

1       1 3.55e-01  3.06000          0.935   17400     18400  19500

2      2 2.44e-01  1.60000          0.927   14100     15300  16900

3      3 1.74e-01  1.08000          0.921   12200     13300  15300

4      4 1.24e-01  0.80000          0.920   11000     12000  14100

5      5 9.11e-02  0.62800          0.920   10000     10900  13200

6      6 6.64e-02  0.50100          0.919    9320     10100  12500

7      7 4.64e-02  0.39800          0.918    8730      9450  11900

8      8 3.45e-02  0.32800          0.915    8250      8890  11400

9      9 2.45e-02  0.26800          0.918    7840      8420  11000

10    10 1.71e-02  0.21900          0.919    7490      8000  10600

11    12 7.63e-03  0.14000          0.914    6900      7330   9900

12    14 2.77e-03  0.08140          0.919    6440      6790   9360

13    16 4.23e-04  0.03090          0.913    6060      6350   8900

14    18 3.52e-06 -0.00277          0.914    5740      5980   8500

15    20 6.56e-04 -0.03730          0.916    5470      5670   8160

Am I just being too ambitious trying to use WGCNA with my number of samples, or am I missing some kind of data processing that I should include for my analysis?

Many thanks

Dan