TopHat protocol error at cuffdiff

[dherndon23]

Hi all - I've been trying to work through the TopHat protocol from the March issue of Nature Protocols but ran into an issue at step #5 (Running Cuffdiff). The protocol describes only two labels but lists six merged files. Any ideas of how to run the protocol with only two labels on all six datasets?

[dpryan]

The 6 bam files are broken up into two lists of three (each list is comma separated). So the label is for the list of files. The command they list in the paper looks correct. If running it, or something like it, produces different results for you then it'd be helpful for you to provide the command and an example of the output.

[dherndon23]

Thanks for the quick response, here is the command and output:
[herndon@usda02 rnaseqexp]$ cuffdiff -o diff_out -b genome.fa -p 8 -L C1,C2 -u merged_asm/merged.gtf ./C1_R1_thout/accepted_hits.bam, ./C1_R2_thout/accepted_hits.bam, ./C1_R1_thout/accepted_hits.bam, ./C1_R3_thout/accepted_hits.bam, C2_R1_thout/accepted_hits.bam, ./C2_R2_thout/accepted_hits.bam, ./C2_R3_thout/accepted_hits.bam
cuffdiff: /lib64/libz.so.1: no version information available (required by cuffdiff)
You are using Cufflinks v1.3.0, which is the most recent release.
Error: number of labels must match number of conditions

[dpryan]

You have C1_R1_thout twice and spaces after the commas, as well as only one list once you fix all of that.

What you want is probably something like this:

cuffdiff -o diff_out -b genome.fa -p 8 -L C1,C2 -u merged_asm/merged.gtf C1_R1_thout/accepted_hits.bam,C1_R2_thout/accepted_hits.bam,C1_R3_thout/accepted_hits.bam C2_R1_thout/accepted_hits.bam,C2_R2_thout/accepted_hits.bam,C2_R3_thout/accepted_hits.bam

[dherndon23]

Yep - that worked. Aside from the misplaced commas, I was running the program from the directory where the tophout ouptut directories were located.

Thanks for the help!

[dpryan]

Cool, glad I could help. Best of luck with the analyses.

[jfofly]

not reading BAM file

I'm using the same Nature Protocol on my samples and am running into a different problem at the cuffdiff step. The program is not reading my accepted_hits.bam files. I've tried converted to .sam and still no luck. .sam file looks ok to my eyes. And .bam file worked for cufflinks step. Any ideas?

$ cuffdiff -o ./results-NCBIref082011_cuffdiff/cuffdiff/ -b /Users/Fontana/Genomes/Drosophila_melanogaster/NCBI/build5.3/Sequence/Bowtie2Index/genome.fa -p 2 -L Mid,nonMid -u ./results-NCBIref082011_cuffdiff/merged_asm/merged.gtf \ ./results-NCBIref082011_JF001g1/accepted_hits.bam,./results-NCBIref082011_JF003g1/accepted_hits.bam \ ./results-NCBIref082011_JF002g1/accepted_hits.bam
You are using Cufflinks v2.0.0, which is the most recent release.
open: No such file or directory
File ./results-NCBIref082011_JF001g1/accepted_hits.bam doesn't appear to be a valid BAM file, trying SAM...
Error: cannot open alignment file ./results-NCBIref082011_JF001g1/accepted_hits.bam for reading

-Note : also tried running with some of my older BAM files that I know are good. Still spitting out same error

[dpryan]

Perhaps it's because I'm seeing this on my phone, but you appear to have a number of "\ " instances in your command. I imagine that you don't want to escape those spaces.

[jfofly]

Perhaps it's because I'm seeing this on my phone, but you appear to have a number of "\ " instances in your command. I imagine that you don't want to escape those spaces.

Thanks for the reply. Those "\ " characters are written in the protocol before the samples for each condition used. Am I interpreting it wrongly? I'm unfamiliar with what that character would be doing. Should I be leaving it out?

[dpryan]

Yeah, maybe they should have mentioned this detail in the paper since I lot of the readers following it won't be familiar with the command line. The backslashes are used to allow you to split a command over multiple lines. So, you need to hit return/enter after each one. Alternatively, you can leave them out and just put everything on one line. The version with the backslashes increases readability, but will lead to problems like the one you ran into for those unaccustomed. Try either of the following and see if that fixes your problems:

All on one line:

cuffdiff -o ./results-NCBIref082011_cuffdiff/cuffdiff/ -b /Users/Fontana/Genomes/Drosophila_melanogaster/NCBI/build5.3/Sequence/Bowtie2Index/genome.fa -p 2 -L Mid,nonMid -u ./results-NCBIref082011_cuffdiff/merged_asm/merged.gtf ./results-NCBIref082011_JF001g1/accepted_hits.bam,./results-NCBIref082011_JF003g1/accepted_hits.bam ./results-NCBIref082011_JF002g1/accepted_hits.bam

Split across lines:

cuffdiff -o ./results-NCBIref082011_cuffdiff/cuffdiff/ -b /Users/Fontana/Genomes/Drosophila_melanogaster/NCBI/build5.3/Sequence/Bowtie2Index/genome.fa -p 2 -L Mid,nonMid -u ./results-NCBIref082011_cuffdiff/merged_asm/merged.gtf \
./results-NCBIref082011_JF001g1/accepted_hits.bam,./results-NCBIref082011_JF003g1/accepted_hits.bam \
./results-NCBIref082011_JF002g1/accepted_hits.bam

[jfofly]

That did it. I appreciate the help!

[mbk0asis]

why are there '.' in front of '/' in bam files?

[dpryan]

You don't need the "./" at all. If you just used "/file.bam", though, then "file.bam" would need to be in the root directory, which should never occur. "./" just mean "the current working directory".

[dpryan]

You don't need the "./" at all. If you just used "/file.bam", though, then "file.bam" would need to be in the root directory, which should never occur. "./" just mean "the current working directory".

This has to be the most inept attempt at a spam bot I've ever seen.