I have Human RNA-Seq data downloaded from NCBI, for which I am performing data analysis. I aligned the reads using tophat2 by providing the premade indices from GRCh38.fa and GRCh38.chr.gtf. After aligning and assembling with Cufflinks when I checked the transcripts.gtf file; most FPKM, Coverage, showed 0.0000 values while very few showed values greater than 0.00.. But at the same time, when i ran the assembly using Cufflinks and without providing '-g' option which points towards GRCh38.chr.gtf, the FPKM, Coverage values showed up in all sequences.
Could anyone explain me what might be the probable reasons for such difference? Should I proceed with the analysis?
Also, when I checked the quality of read distribution, using the command read_distribution.py from RSeQC it showed not tags counted. The output is pasted below:
Total Reads 1914074 Total Tags 2097688 Total Assigned Tags 0
Group Total_bases Tag_count Tags/Kb
CDS_Exons 103371993 0 0.00
5'UTR_Exons 5217678 0 0.00
3'UTR_Exons 29324747 0 0.00
Introns 1500197093 0 0.00
TSS_up_1kb 33306654 0 0.00
TSS_up_5kb 148463534 0 0.00
TSS_up_10kb 265823549 0 0.00
TES_down_1kb 35215293 0 0.00
TES_down_5kb 152556214 0 0.00
TES_down_10kb 268614580 0 0.00
I don't really understand whats happening. It would be really kind of you guys if anyone could spare sometime and give useful inputs and help me solve the issue.
Thanks in advance..!