Cuffdiff2 skipped genes

[Zapages]

Hi everyone,

I have weird problem. I followed the Tuxedo pipeline it has been working well so far. I was conducting CummeRbund and in my heatmap visualization, I noticed one of the genes of interest was not present. I went back to all my Cufflinks and Cuffmerge folders and saw that the genes of interest or location of the gene was present there. But in cuffdiff2's gene_exp.diff file, it did not have gene loci present.

For example it went from:

Gene10355
Gene10357

Gene10356 was skipped.

What did I do wrong to have one of my gene of interest missing in my gene_exp.diff.

Here's my pipeline:

Trimmomatic > Tophat 2 with Ensembl GTF and Fasta > Cufflinks 2.0.2 with Ensembl Fasta (fragment bias) and GTF > Cuffmerge2 2.0.2 with Ensembl GTF > Cuffdiff2 2.0.2 with Ensembl Fasta and merged_w_refs.gtf file from Cuffmerge2 2.0.2_Fragment(False discovery rate of 0.01 and multiple hits correction) > CummeRbund for heatmap

Also I have 2 replicates for Organ A and 3 replicates for Organ B.

Thank you in advance.

EDIT: Looking at the Cufflinks files, the expression (FPKM) of the gene is high in Organ A and literally 0 in Organ B. Would this lead to this problem?

EDIT2: I still not get the skipped gene even when I take out multi hit correction or taking away the fasta file.

EDIT: In the Cuffmerge file: The first 2 matches are Cuff (Organ A) and the second 2 matches are the actual gene location (Organ B). Could that cause it not work properly for Cuffdiff2?

This is very strange and I did not have this occur before. Could someone kindly help me on this. Thank you. I would really appreciate it.

-Zapages