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:
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
Cuffmerge:
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
<assembly_GTF_list.txt>
Text file “manifest” with a list (one per line) of GTF files that you’d like to merge together into a single GTF file.
Cuffdiff
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]> … \
[sampleN.sam_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
cuffdiff过程中同一处理的多个样本间用逗号分隔，不同处理间空格分隔.
