Cuffdiff - Error in running cuffdiff by using the merged transcriptome assembly along with the BAM files from TopHat

Question

[evashan96]

Hi I'm new to RNA-Seq analysis. I ran the following command with cuffdiff: cuffdiff -o /home/silicon/mouse/diff_out -b /home/silicon/mouse/GRCm38/Sequence/Bowtie2Index/genome.fa -p 7 -L g1,g2 -u /home/silicon/mouse/assembly/merged_asm/merged.gtf, \ .//home/silicon/mouse/alignment/Tophat_S2/accepted_hits.bam, /home/silicon/mouse/alignment/samp12_alignment/accepted_hits.bam

and I got the following error: Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu). Error: cannot open reference GTF file /home/silicon/mouse/assembly/merged_asm/merged.gtf, for reading

Can anyone help me out?

[Prakash]

See below link regarding deprecation of Tophat2-cufflink-cuffdiff

Diverging read alignment and counts in TopHat vs. HISAT2

RNA-Seq Cufflinks normalization methods

RNA Seq Pipeline Question (CuffMerge)

[bioinfo_ga]

Try this if you have one control and one treated sample cufflinks-2.2.1.Linux_x86_64/cuffdiff -u -p 25 --library-type fr-firststrand -o /Cuffdiff/Control_VS_Treated /Cuffmerge/merged.gtf Control/A_Sorted.bam Treated/C_Sorted.bam, -L Control,Treated

In case of replicates try the following command (replicates should be comma separated) cufflinks-2.2.1.Linux_x86_64/cuffdiff -u -p 25 --library-type fr-firststrand -o /Cuffdiff/Control_VS_Treated /Cuffmerge/merged.gtf Control/A_Sorted.bam,Control_2/A1_Sorted.bam Treated/C_Sorted.bam,Treated_2/C1_Sorted.bam -L Control,Treated Also check the location of your merged gtf file (cuffmerge output)