TopHat Issue

[ashuchawla]

Hi,

I ran topHat with a transcriptome data file and then without the transcriptome file using all other parameters the same. I saw 2 million more reads that were aligned when I did not use the transcriptome GTF file. I used mm9 assembly.

As far as I understand, tophat first aligns reads to the genome and then to the transcriptome. Technically there should not be more aligned reads while mapping without the Transcriptome gtf file. Could anyone give me some pointers on this? Is it OK to see this different in alignment or is there something Wrong??

Following are the commands I used:

#Without genes.gtf
tophat -C -Q -p 24 --library-type fr-secondstrand -o <path/out_folder> --no-coverage-search /bowtie_indexes/mm9_c Libraries/Library15/F3/reads/RNAseq_L01_Library15_F3.csfasta Libraries/Library15/F3/reads/RNAseq_L01_Library15_F3.QV.qual

#With genes.gtf
tophat -C -Q -p 24 --library-type fr-secondstrand -G Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o <path/out_folder> --no-coverage-search /bowtie_indexes/mm9_c Libraries/Library15/F3/reads/RNAseq_L01_Library15_F3.csfasta Libraries/Library15/F3/reads/RNAseq_L01_Library15_F3.QV.qual

Thanks,

Ashu