Hi all,I have some questions about how Cufffdiff does the statistical analysis.
I am looking for DE genes in two sample groups ( 3 replicate per group). In the Cuffdiff;s gene_exp.diff, I found many genes that have very large RPKM fold-change between two groups (with p value < or > 0.05) but still NO significant. Something like this:
test_id |
gene_id |
gene |
locus |
sample_1 |
sample_2 |
status |
value_1 |
value_2 |
log2(fold_change) |
test_stat |
p_value |
q_value |
significant |
| ENSMUSG00000047139 |
ENSMUSG00000047139 |
Cd24a |
10:43579168-43584262 |
q1 |
q2 |
OK |
96.2585 |
2700.55 |
4.8102 |
1.6486 |
0.03995 |
0.078237 |
no |
| ENSMUSG00000066975 |
ENSMUSG00000066975 |
Cryba4 |
5:112246492-112252518 |
q1 |
q2 |
OK |
424.582 |
46190.2 |
6.7654 |
0.598327 |
0.3408 |
0.442128 |
no |
Then I checked the READ_GROUP_TRACKING file for those genes to check the RPKM value for each replicate:
| tracking_id |
condition |
replicate |
raw_frags |
internal_scaled_frags |
external_scaled_frags |
FPKM |
effective_length |
status |
| ENSMUSG00000047139 |
q1 |
1 |
11256 |
5876.82 |
5876.82 |
125.915 |
- |
OK |
| ENSMUSG00000047139 |
q1 |
0 |
3783 |
4343.44 |
4343.44 |
42.0316 |
- |
OK |
| ENSMUSG00000047139 |
q1 |
2 |
10051 |
5639.48 |
5639.48 |
120.829 |
- |
OK |
| ENSMUSG00000047139 |
q2 |
1 |
76771 |
156059 |
156059 |
3343.66 |
- |
OK |
| ENSMUSG00000047139 |
q2 |
0 |
82394 |
162172 |
162172 |
1420.33 |
- |
OK |
| ENSMUSG00000066975 |
q1 |
1 |
12825 |
6696 |
6696 |
407.899 |
- |
OK |
| ENSMUSG00000066975 |
q1 |
0 |
3694 |
4241.26 |
4241.26 |
375.211 |
- |
OK |
| ENSMUSG00000066975 |
q1 |
2 |
14397 |
8077.95 |
8077.95 |
490.636 |
- |
OK |
| ENSMUSG00000066975 |
q2 |
1 |
348103 |
707619 |
707619 |
42455.1 |
- |
OK |
| ENSMUSG00000066975 |
q2 |
0 |
420896 |
828430 |
828430 |
48920.6 |
- |
OK |
| ENSMUSG00000066975 |
q2 |
2 |
331098 |
767405 |
767405 |
47195 |
- |
OK |
Would not I expect these DE genes are significant? Could anyone explain why Cufflinks show this result?
Best regards
Thanh