1. Align reads from each sample to the reference genome using tophat
For Illumina/basespace data:
For ABI SOLiD (colorspace) data:
The output file (accepted_hits.bam) is the alignment output which will be used in following steps.
-G transcripts.gtf can be used to align to transcriptome first and align only those that don't map to transcriptome to the genome. This is useful for speeding up tophat
2. Assemble transcripts using cufflinks on each alignment result
or to assemble transcripts using the assistance of a gfffile (this would assemble novel and known transcripts)
The output file, transcripts.gtf will be used in the following steps.
3. Merge the assembled transcripts to make a unified gff/gtf file.
Make a text file with locations of each sample's transcripts.gtf file, one line per sample.
The output file, merged.gtf will be used in the following steps (4a and 4b).
4a. Identify differentially expressed transcripts using cuffdiff
If you have more than one replicate for a sample, supply the SAM files for the sample as a single comma-separated list.
Several output files, consisting of raw and normalized counts for genes, isoforms and transcription start sites are generated. More about the output files at http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff_output
4b. Check for differences between the assembled transcripts and known transcripts.