Gene Expression Analysis


cuffdiff [options]* <transcripts.gtf> <sample1_replicate1.sam[,…,sample1_replicateM.sam]> <sample2_replicate1.sam[,…,sample2_replicateM.sam]> … [sampleN.sam_replicate1.sam[,…,sample2_replicateM.sam]]


-h/–help Prints the help message and exits -o/–output-dir Sets the name of the directory in which Cuffdiff will write all of its output. The default is “./”. -L/–labels Specify a label for each sample, which will be included in various output files produced by Cuffdiff. -p/–num-threads Use this many threads to align reads. The default is 1. -T/–time-series Instructs Cuffdiff to analyze the provided samples as a time series, rather than testing for differences between all pairs of samples. Samples should be provided in increasing time order at the command line (e.g first time point SAM, second timepoint SAM, etc.) –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. It is inactive by default. –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. It is active by default. -b/–frag-bias-correct Providing Cufflinks with the multifasta file your reads were mapped to via this option instructs it to run our bias detection and correction algorithm which can significantly improve accuracy of transcript abundance estimates. See How Cufflinks Workshow_it_works/index.html#) 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. -c/–min-alignment-count 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 signficant, and the locus’ observed changes don’t contribute to correction for multiple testing. The default is 10 fragment alignments. -M/–mask-file Tells Cuffdiff 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. –FDR The allowed false discovery rate. The default is 0.05. –library-type –library-norm-method –dispersion-method -m/–frag-len-mean This is the expected (mean) fragment length. The default is 200bp. Note: Cuffdiff 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: Cuffdiff now learns the fragment length standard deviation for each SAM file, so using this option is no longer recommended with paired-end reads. –max-mle-iterations Sets the number of iterations allowed during maximum likelihood estimation of abundances. Default: 5000 -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. –poisson-dispersion Use the Poisson fragment dispersion model instead of learning one in each condition. –emit-count-tables Cuffdiff will output a file for each condition (called _counts.txt) containing the fragment counts, fragment count variances, and fitted variance model. For internal debugging only. This option will be removed in a future version of Cuffdiff. -F/–min-isoform-fraction <0.0-1.0> Cuffdiff will round down to zero the abundance of alternative isoforms quantified at below the specified fraction of the major isoforms. This is done after MLE estimation but before MAP estimation to improve robustness of confidence interval generation and differential expression analysis. The default is 1e-5, and we recommend you not alter this parameter. –max-bundle-frags Sets the maximum number of fragments a locus may have before being skipped. Skipped loci are marked with status HIDATA. Default: 1000000 –max-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. Default: 100. –max-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. Default: 50. –min-reps-for-js-test Cuffdiff won’t test genes for differential regulation unless the conditions in question have at least this many replicates. Default: 3. **–no-effective-length-correction Cuffdiff will not employ its “effective” length normalization to transcript FPKM. –no-length-correction Cuffdiff 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.

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