Cuffdiff is part of Cufflinks. Cuffdiff find significant changes in transcript expression, splicing, and promoter use. Please cite: Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren MJ, Salzberg SL, Wold B, Pachter L. Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms. Nature Biotechnology doi:10.1038/nbt.1621
Know what you are doing
There is no such thing (yet) as an automated gearshift in expression analysis. It is all like stick-shift driving in San Francisco. In other words, running this tool with default parameters will probably not give you meaningful results. A way to deal with this is to understand the parameters by carefully reading the documentation and experimenting. Fortunately, Galaxy makes experimenting easy.
Cuffdiff takes Cufflinks or Cuffcompare GTF files as input along with two SAM files containing the fragment alignments for two or more samples.
Cuffdiff produces many output files:
All of the options have a default value. You can change any of them. Most of the options in Cuffdiff have been implemented here.
Cuffdiff parameter list
This is a list of implemented Cuffdiff options:
-m INT Average fragment length (SE reads); default 200 -s INT Fragment legnth standard deviation (SE reads); default 80 -c INT The minimum number of alignments in a locus for needed to conduct significance testing on changes in that locus observed between samples. If no testing is performed, changes in the locus are deemed not significant, and the locus' observed changes don't contribute to correction for multiple testing. The default is 1,000 fragment alignments (up to 2,000 paired reads). --FDR FLOAT The allowed false discovery rate. The default is 0.05. --max-mle-iterations INT Sets the number of iterations allowed during maximum likelihood estimation of abundances. Default: 5000 --library-norm-method Library Normalization method : Geometric (default), classic-fpkm, quartile --dispersion-method Dispersion estimation method : Pooled (default), per-condition, blind, poisson -u Multi read correction tells Cufflinks to do an initial estimation procedure to more accurately weight reads mapping to multiple locations in the genome. -b ref.fasta bias correction. Bias detection and correction can significantly improve accuracy of transcript abundance estimates. --no-effective-length-correction Use standard length correction --no-length-correction Disable all length correction. --library-type ff-firststrand,ff-secondstrand,ff-unstranded,fr-firstrand,fr-secondstrand,fr-unstranded,transfrags --mask-file (gff3/gtf) Ignore all alignment within transcripts in this file --time-series Treat provided sam files as time series --compatible-hits-norm With this option, Cufflinks counts only those fragments compatible with some reference transcript towards the number of mapped fragments used in the FPKM denominator. Using this mode is generally recommended in Cuffdiff to reduce certain types of bias caused by differential amounts of ribosomal reads which can create the impression of falsely differentially expressed genes. --total-hits-norm With this option, Cufflinks counts all fragments, including those not compatible with any reference transcript, towards the number of mapped fragments used in the FPKM denominator --max-bundle-frags Sets the maximum number of fragments a locus may have before being skipped. Skipped loci are listed in skipped.gtf. --num-frag-count-draws Cuffdiff will make this many draws from each transcript's predicted negative binomial random numbder generator. Each draw is a number of fragments that will be probabilistically assigned to the transcripts in the transcriptome. Used to estimate the variance-covariance matrix on assigned fragment counts. --num-frag-assign-draws For each fragment drawn from a transcript, Cuffdiff will assign it this many times (probabilistically), thus estimating the assignment uncertainty for each transcript. Used to estimate the variance-covariance matrix on assigned fragment counts. --min-reps-for-js-test Cuffdiff won't test genes for differential regulation unless the conditions in question have at least this many replicates.