no differentially expressed genes using cuffdiff nor HTSeq/DESeq2-single reads

[alyamahmoud]

I have two conditions, knock down and wild-type RNASeq strand specific single-end reads miseq reads. I tried tophat2 -> cuffdiff and tophat2 -> HTSeq -> DESeq2 and I am getting no significantly different genes (FDR < 0.1) in either cases (DESeq2 or Cuffdiff). The exact commands I used are written below. The knocked down gene is also not differentially-expressed.I have two replicates per condition (2*kd, 2*wt). The knock down has been confirmed by RT-qPCR apriori.

My question is: are there any parameters that I should have adjusted for single end reads that I have missed ? or what I am seeing is true ? are there any further steps I could possible make to make sure its not a technical error ?

Thanks a lot

# tuxedo suite
tophat2 -G genome.gff -o kd_thout kd.fastq
tophat2 -G genome.gff  -o wt_thout wt.fastq
cuffdiff -b genome.fa -u genome.gff -o diff_out --library-type ff-firststrand -L kd,wt ./kd_thout_1/accepted_hits.bam,./kd_thout_2/accepted_hits.bam ./wt_thout_1/accepted_hits.bam,./wt_thout_2/accepted_hits.bam

cuffdiff_output: 
gene_id sample_name     fpkm  conf_hi conf_lo quant_status
1 PBANKA_123320        cyc3  53.0098  78.4418 27.5779           OK
2 PBANKA_123320          wt 147.5740 206.9180 88.2291           OK

# HTSeq_DESeq2
samtools view -h -o out.sam accepted_hits.bam
python htseq-count -i ID --stranded=yes accepted_hits.sam genome.gff > wt_counts