Cufflinks assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples. It accepts aligned RNA-Seq reads and assembles the alignments into a parsimonious set of transcripts. Cufflinks then estimates the relative abundances of these transcripts based on how many reads support each one, taking into account biases in library preparation protocols.


cufflinks [options] <aligned_reads.(sam/bam)>

for example:

$ cufflinks -p 8 -G transcript.gtf --library-type fr-unstranded -o cufflinks_output tophat_out/accepted_hits.bam
  -o/--output-dir              write all output files to this directory              [ default:     ./ ]
  -p/--num-threads             number of threads used during analysis                [ default:      1 ]
  --seed                       value of random number generator seed                 [ default:      0 ]
  -G/--GTF                     quantitate against reference transcript annotations                      
  -g/--GTF-guide               use reference transcript annotation to guide assembly


Use to merge together several Cufflinks assemblies.

cuffmerge [options]* <assembly_GTF_list.txt>

Cuffmerge input files

cuffmerge takes several assembly GTF files from Cufflinks’ as input. Input GTF files must be specified in a “manifest” file listing full paths to the files.

Cuffmerge arguments


Text file “manifest” with a list (one per line) of GTF files that you’d like to merge together into a single GTF file.


Use to find significant changes in transcript expression, splicing, and promoter use.

cuffdiff [options]* <transcripts.gtf> \

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

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


Cuffdiff output Files

gene_exp.diff    Gene-level differential expression. Tests differences in the summed FPKM of transcripts sharing each gene_id


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