cufflinks/cuffdiff command line

Question

[dimitrischat]

i ve been trying to figure out the command line for cufflinks/cuffdiff but i have been getting error again and again. Trying the command line with 2 bam files from tophat :

cuffdiff -p 2 --labels esred1,esred2 - b /Users/../mm9genome.fa  /Users/.../accepted_hits-red1.bam,/Users/.../accepted_hits-red2.bam
error(s) : number of labels must match number of conditions

cuffdiff -p 2 - b /Users/.../mm9genome.fa  /Users/.../accepted_hits-red1.bam,/Users/.../accepted_hits-red2.bam
error : cannot open reference GTF file - for reading

cuffdiff -p 2 - L esred1,esred2 - b /Users/.../mm9.fa  /Users/.../accepted_hits-red1.bam,/Users/.../accepted_hits-red2.bam
error : cannot open reference GTF file - for reading

any help ?

Answer 1

[Santosh Anand]

Your command line is wrong. -L specifes labels for different samples, and different samples must be separated by whitespace (not comma; comma seperates the replicates). Also you have not provided any transcripts (GTF/GFF) file.

See manual: http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/

cuffdiff [options]* <transcripts.gtf> \

<sample1_replicate1.sam[,…,sample1_replicateM.sam]> \

<sample2_replicate1.sam[,…,sample2_replicateM.sam]> … \

[sampleN.sam_replicate1.sam[,…,sample2_replicateM.sam]]

transcripts.(gtf/gff)

A transcript annotation file produced by cufflinks, cuffcompare, or other source.

sample1.(sam/bam/cxb)

A SAM file of aligned RNA-Seq reads. If more than two are provided, Cuffdiff tests for differential expression and regulation between all pairs of samples.

[dimitrischat]

whitespace is ...\ .. right ?

also if i cant run cufflinks how will i get the trascripts .gtf file? "produced by cufflinks" ..

sorry i guess its a "stupid" question . Thanks for your reply

[Santosh Anand]

whitespaces normally means SPACEs or TABs.

you may like to follow this protocol http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

[WouterDeCoster]

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