Hi all,
Regarding my question I have found another threads but it could not help and I really need your help because my work deadline is approaching soon.
I have an rna-seq dataset (6 samples for each labels) and I should announce differentially expressed genes.
Since my read sequence length is very short (read length = 36), we decided to use tophat version 1 instead of version 2 or the newest one (now I am not sure whether my decision was true or not?)
Then I applied cuffdiff over the bam output file from tophat (I did not run cufflinks since in the manual it is mentioned that there is no need to run it if I only need the differentially expressed genes without any intentions to find new genes). First I used cuffdiff version 2 but I got very few numbers of DEgenes so my college offered to apply cuffdiff version 2 which he believes is less restrict. Below are the results in gene_exp.diff file:
with cufflinks/2.0.2 :
30 DE genes (those which have pvalue <0.01)
status:
fail 116
lowdata 122
notest 10789
ok 12228
with cuflinks cufflinks/1.3.0 and additive parameters: cuffdiff -c 0 -u (I used this version plus these parameters to make cuffdiff less restrict):
74 DE genes
fail 4050
lowdata 104
notest 2618
ok 16513
Now first I want to know if you think it was a correct decision to use tophat version 1 due to the short reads and second do you have any suggestion to which cuffdiff result should I announce or if you have any advice to get more reliable result.
Thanks a lot in advance.
Regarding my question I have found another threads but it could not help and I really need your help because my work deadline is approaching soon.
I have an rna-seq dataset (6 samples for each labels) and I should announce differentially expressed genes.
Since my read sequence length is very short (read length = 36), we decided to use tophat version 1 instead of version 2 or the newest one (now I am not sure whether my decision was true or not?)
Then I applied cuffdiff over the bam output file from tophat (I did not run cufflinks since in the manual it is mentioned that there is no need to run it if I only need the differentially expressed genes without any intentions to find new genes). First I used cuffdiff version 2 but I got very few numbers of DEgenes so my college offered to apply cuffdiff version 2 which he believes is less restrict. Below are the results in gene_exp.diff file:
with cufflinks/2.0.2 :
30 DE genes (those which have pvalue <0.01)
status:
fail 116
lowdata 122
notest 10789
ok 12228
with cuflinks cufflinks/1.3.0 and additive parameters: cuffdiff -c 0 -u (I used this version plus these parameters to make cuffdiff less restrict):
74 DE genes
fail 4050
lowdata 104
notest 2618
ok 16513
Now first I want to know if you think it was a correct decision to use tophat version 1 due to the short reads and second do you have any suggestion to which cuffdiff result should I announce or if you have any advice to get more reliable result.
Thanks a lot in advance.