Tophat

TopHat is a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to mammalian-sized genomes using the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons.

Tophat 2.0.3 is installed on Fourierseq as of 06/11/12.

Very basic Tophat command:

nohup tophat -p 4 -r <mate-inner-distance> -G <gfffile> <bowtie_index_prefix> <R1.fasta> <R2.fasta> &>tophat.log &

• – bowtie1 can be added to ask tophat to use bowtie1 instead of bowtie2 (bowtie2 does not support colorspace data).
• - C for colorspace (provide csfasta and quality files when using the flag)

Example 1: For mouse:

nohup tophat -p 4 -r 130 -G /usr/local/genome/references/mmu_ncbi37/mm9_ucsc-known.gff3 /usr/local/genome/references/mmu_ncbi37/mmu_masked_ncbi37.fasta sim.test1.forward.fa sim.test1.reverse.fa &>tophat.log &

Example 2: For human:

tophat --transcriptome-index=/usr/local/genome/references/hg19/bowtie_gtf_index/hg19.gtf /usr/local/genome/references/hg19/bowtie_index/hg19.bs.bowtie <reads.fq>

Cufflinks

Cufflinks 2.0.0 is installed on Fourierseq as of 05/24/12

Very basic cufflinks command:

nohup cufflinks accepted_hits.bam -o cufflinks_outputdir &>cufflinks.log &

• -G reference.gtf can be added to use reference annotation to assemble transcripts. This will not assemble novel transcripts.
• -g reference.gtf can be added to use the reference annotation as a guide to assemble transcripts. This will include reference transcripts and novel transcripts.

• accepted_hits.bam : bam file created by tophat

Errors encountered:

An error during the segment mapping step with tophat 2.0.2 (Error: segment-based junction search failed with err =1) has been encountered that is solved by not using multiple threads (-p option).