1. Align reads from each sample to the reference genome using tophat
For Illumina/basespace data:
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nohup tophat -p 4 -r <mate-inner-distance> -G <gfffile> <bowtie_index_prefix> <R1.fastq> <R2.fastq> &>tophat.log & |
For ABI SOLiD (colorspace) data:
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nohup tophat --bowtie1 -C -p 4 -r <mate-inner-distance> -G <gfffile> <bowtie_colorspace_index_prefix> <R1.fasta> <R2.fasta> <R1.qual> <R2.qual> &>tophat.log & |
The output file (accepted_hits.bam) is the alignment output which will be used in following steps.
2. 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.
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nohup cuffdiff -o <outputdirectory> <gfffile> <sample1_accepted_hits.bam> <sample2_accepted_hits.bam> <sample3_accepted_hits.bam> &cuffdiff.log & |
Several output files, consisting of raw and normalized counts for genes, isoforms and transcription start sites are generated. More about the output files at cufflinks manual