Quantification of RNA-seq with Cufflinks (no de-novo assembly) for FASTQ files (workflow)

Workflow title

Quantification of RNA-seq with Cufflinks (no de-novo assembly) for FASTQ files

Provider

geneXplain GmbH

Workflow overview

Description

This workflow offers a possibility to discover new genes and transcripts (splice variants) and measure transcript expression in a single assay from RNA-seq data. This workflow is described in “Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks”, Nat. Protoc. 7:562-578, 2012.

The first step of the workflow is alignment of sequence reads with TopHat (http://tophat.cbcb.umd.edu/). TopHat aligns reads to the genome and discovers transcript splice sites. TopHat uses Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) as an alignment ‘engine’ and breaks up reads that Bowtie cannot align on its own into smaller pieces called segments.

Output files from TopHat are tables and tracks with insertions, deletions, splice junctions and the alignments. The second step of the workflow is performed by Cuffdiff, part of the Cufflinks package, which calculates expression in two or more samples and tests the statistical significance of each observed change in expression between them. Cuffdiff allows for supplying multiple technical or biological replicate sequencing libraries per condition. With multiple replicates, Cuffdiff learns how read counts vary for each gene across the replicates and uses these variance estimates to calculate the significance of observed changes in expression.

The last step of the workflow comprises several conversions and filtering steps of some Cuffdiff output files. The output is a folder with different tracks and tables including Differentially expressed genes, Differentially expressed TSS promoters and regulated promoters.

 

Parameters

Experiment fastq files

Control fastq files

Output folder