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.
cufflinks [options] <aligned_reads.(sam/bam)>
-h/–help
Prints the help message and exits
-g/–GTF-guide
Tells Cufflinks to use the supplied reference annotation a GFF file to guide RABT assembly. Reference transcripts will be tiled with faux-reads to provide additional information in assembly. Output will include all reference transcripts as well as any novel genes and isoforms that are assembled.
-M/–mask-file
Tells Cufflinks to ignore all reads that could have come from transcripts in this GTF file. We recommend including any annotated rRNA, mitochondrial transcripts other abundant transcripts you wish to ignore in your analysis in this file. Due to variable efficiency of mRNA enrichment methods and rRNA depletion kits, masking these transcripts often improves the overall robustness of transcript abundance estimates.
-b/–frag-bias-correct
Providing Cufflinks with a multifasta file via this option instructs it to run our new bias detection and correction algorithm which can significantly improve accuracy of transcript abundance estimates. See How Cufflinks Works for more details.
-u/–multi-read-correct
Tells Cufflinks to do an initial estimation procedure to more accurately weight reads mapping to multiple locations in the genome. See How Cufflinks Works for more details.
–library-type
–library-norm-method
-m/–frag-len-mean
This is the expected (mean) fragment length. The default is 200bp.
Note: Cufflinks now learns the fragment length mean for each SAM file, so using this option is no longer recommended with paired-end reads.
-s/–frag-len-std-dev
The standard deviation for the distribution on fragment lengths. The default is 80bp.
Note: Cufflinks now learns the fragment length standard deviation for each SAM file, so using this option is no longer recommended with paired-end reads.
-N/–upper-quartile-norm
With this option, Cufflinks normalizes by the upper quartile of the number of fragments mapping to individual loci instead of the total number of sequenced fragments. This can improve robustness of differential expression calls for less abundant genes and transcripts.
–total-hits-norm
With this option, Cufflinks counts all fragments, including those not compatible with any reference transcript, towards the number of mapped hits used in the FPKM denominator. This option can be combined with -N/–upper-quartile-norm. It is active by default.
–compatible-hits-norm
With this option, Cufflinks counts only those fragments compatible with some reference transcript towards the number of mapped hits used in the FPKM denominator. This option can be combined with -N/–upper-quartile-norm. It is inactive by default, and can only be used in combination with
–GTF
Use with either RABT or ab initio assembly is not supported
–max-mle-iterations
Sets the number of iterations allowed during maximum likelihood estimation of abundances. Default: 5000
–max-bundle-frags
Sets the maximum number of fragments a locus may have before being skipped. Skipped loci are listed in skipped.gtf. Default: 1000000
–no-effective-length-correction
Cufflinks will not employ its “effective” length normalization to transcript FPKM.
–no-length-correction
Cufflinks will not normalize fragment counts by transcript length at all. Use this option when fragment count is independent of the size of the features being quantified (e.g. for small RNA libraries, where no fragmentation takes place, or 3 prime end sequencing, where sampled RNA fragments are all essentially the same length). Experimental option, use with caution.
-L/–label
Cufflinks will report transfrags in GTF format, with a prefix given by this option. The default prefix is “CUFF”.
-F/–min-isoform-fraction <0.0-1.0>
After calculating isoform abundance for a gene, Cufflinks filters out transcripts that it believes are very low abundance, because isoforms expressed at extremely low levels often cannot reliably be assembled, and may even be artifacts of incompletely spliced precursors of processed transcripts. This parameter is also used to filter out introns that have far fewer spliced alignments supporting them. The default is 0.1, or 10% of the most abundant isoform (the major isoform) of the gene.
-j/–pre-mrna-fraction <0.0-1.0>
Some RNA-Seq protocols produce a significant amount of reads that originate from incompletely spliced transcripts, and these reads can confound the assembly of fully spliced mRNAs. Cufflinks uses this parameter to filter out alignments that lie within the intronic intervals implied by the spliced alignments. The minimum depth of coverage in the intronic region covered by the alignment is divided by the number of spliced reads, and if the result is lower than this parameter value, the intronic alignments are ignored. The default is 15%.
-I/–max-intron-length
The maximum intron length. Cufflinks will not report transcripts with introns longer than this, and will ignore SAM alignments with REF_SKIP CIGAR operations longer than this. The default is 300,000.
-a/–junc-alpha <0.0-1.0>
The alpha value for the binomial test used during false positive spliced alignment filtration. Default: 0.001
-A/–small-anchor-fraction <0.0-1.0>
Spliced reads with less than this percent of their length on each side of the junction are considered suspicious and are candidates for filtering prior to assembly. Default: 0.09.
–min-frags-per-transfrag
Assembled transfrags supported by fewer than this many aligned RNA-Seq fragments are not reported. Default: 10.
–overhang-tolerance
The number of bp allowed to enter the intron of a transcript when determining if a read or another transcript is mappable to/compatible with it. The default is 8 bp based on the default bowtie/TopHat parameters.
–max-bundle-length
Maximum genomic length allowed for a given bundle. The default is 3,500,000 bp.
–min-intron-length
Minimum intron size allowed in genome. The default is 50 bp.
–trim-3-avgcov-thresh
Minimum average coverage required to attempt 3’ trimming. The default is 10.
–trim-3-dropoff-frac
The fraction of average coverage below which to trim the 3’ end of an assembled transcript. The default is 0.1.
–max-multiread-fraction <0.0-1.0>
The fraction a transfrag’s supporting reads that may be multiply mapped to the genome. A transcript composed of more than this fraction will not be reported by the assembler. Default: 0.75 (75% multireads or more is suppressed).
–overlap-radius
Transfrags that are separated by less than this distance get merged together, and the gap is filled. Default: 50 (in bp).
–3-overhang-tolerance
The number of bp allowed to overhang the 3’ end of a reference transcript when determining if an assembled transcript should be merged with it (ie, the assembled transcript is not novel). The default is 600 bp.
–intron-overhang-tolerance
The number of bp allowed to enter the intron of a reference transcript when determining if an assembled transcript should be merged with it (ie, the assembled transcript is not novel). The default is 50 bp.
–no-faux-reads
This option disables tiling of the reference transcripts with faux reads. Use this if you only want to use sequencing reads in assembly but do not want to output assembled transcripts that lay within reference transcripts. All reference transcripts in the input annotation will also be included in the output.
-v/–verbose
Print lots of status updates and other diagnostic information.
-q/–quiet
Suppress messages other than serious warnings and errors.
–no-update-check
Turns off the automatic routine that contacts the Cufflinks server to check for a more recent version.