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Hi all,
I am new to NGS RNA analysis and am trying to analyze differential expression of genes using cuffdiff between 3 groups of cells (Heterozygous, Knock-out and wildtype).
Completed steps by our NGS core:
1. prepare samples from FQ files to BAM files.
2. prepared transcripts.gtf for every sample
Then, what I did:
2. create a assemblies.txt that lists the location and assembly file (transcripts.gtf) for every sample.
3. Ran cuffmerge which created a merged.gtf file
Now, at step 4 of running CuffDiff I am getting an error called
"Error: cuffdiff requires at least 2 SAM files"
This was my command:
cuffdiff -o diff_out -b genome.fa -p 8 -L HZ,KO,WT -u merged_asm/merged.gtf \
./HZ_1_unp/accepted_hits.bam,./HZ_2_unp/accepted_hits.bam,./HZ_3_unp/accepted_hits.bam\
./KO_1_unp/accepted_hits.bam,./KO_4_unp/accepted_hits.bam,./KO_5_unp/accepted_hits.bam\
./WT_1_unp/accepted_hits.bam,./WT_2_unp/accepted_hits.bam,./WT_3_unp/accepted_hits.bam,./WT_4_unp/accepted_hits.bam
Then, I receive the error. I get the feeling that it is not able to find the BAM files? It seems incorrect that cuffdiff would like to have SAM files, since according to the Article of Trapnell at all. it states " 5) Run Cuffdiff by using the merged transcriptome assembly along with the BAM files from TopHat for each replicate"
How can i make cuffdiff do it's thing after all?! Thanks!
I am new to NGS RNA analysis and am trying to analyze differential expression of genes using cuffdiff between 3 groups of cells (Heterozygous, Knock-out and wildtype).
Completed steps by our NGS core:
1. prepare samples from FQ files to BAM files.
2. prepared transcripts.gtf for every sample
Then, what I did:
2. create a assemblies.txt that lists the location and assembly file (transcripts.gtf) for every sample.
3. Ran cuffmerge which created a merged.gtf file
Now, at step 4 of running CuffDiff I am getting an error called
"Error: cuffdiff requires at least 2 SAM files"
This was my command:
cuffdiff -o diff_out -b genome.fa -p 8 -L HZ,KO,WT -u merged_asm/merged.gtf \
./HZ_1_unp/accepted_hits.bam,./HZ_2_unp/accepted_hits.bam,./HZ_3_unp/accepted_hits.bam\
./KO_1_unp/accepted_hits.bam,./KO_4_unp/accepted_hits.bam,./KO_5_unp/accepted_hits.bam\
./WT_1_unp/accepted_hits.bam,./WT_2_unp/accepted_hits.bam,./WT_3_unp/accepted_hits.bam,./WT_4_unp/accepted_hits.bam
Then, I receive the error. I get the feeling that it is not able to find the BAM files? It seems incorrect that cuffdiff would like to have SAM files, since according to the Article of Trapnell at all. it states " 5) Run Cuffdiff by using the merged transcriptome assembly along with the BAM files from TopHat for each replicate"
How can i make cuffdiff do it's thing after all?! Thanks!
Cuffdiff is expecting at least two groups of files to compare against each other (here, a group can consist of one or more members). The groups should be separated from each other by spaces, e.g., "group1_1.bam,group1_2.bam group2_1.bam,group2_2.bam". What the "\" does is tell the shell that you're splitting a single command over more than one line, generally to make things easier to read (if you're basing everything off of the nature methods paper, their usage of "\" there ends up confusing a LOT of people new to the command line). So, your command is identical to:
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