Hello, I'm fairly new to RNA-Seq, and I'm working with a non-model organism. I've been working with some of the programs in Galaxy and trying to do my best with the information I have; however, I encountered three issues while using Tophat and Cufflinks in Galaxy.
1 - I'm using Tophat, Bowtie, or BWA, in Galaxy to map reads to a transcriptome assembled, via trinity, to the same reads I'm trying to map; therefore, I feel that all, or at least most, of the reads should map well. However, I'm only able to have about 10% of the reads actually map. A friend suggested that loosen the restrictions (change the default settings), which I've done, but whenever I do so, the job fails.
2 - For the reads that do map, when I run Cufflinks with the Bowtie or BWA file, the Cufflinks file comes up empty.
3 - I have an assembled transcriptome that has identified about 100,000 different genes (contigs). After I use cuffmerge to merge two cufflinks files, and the analyze the data with cuffdiff, the output only reports FPKM and differential expression testing data for around 1700 genes. I'm not sure if that is normal and some genes are just not reported, or if Cufflinks isn't looking at all of the genes.
Any help would be greatly appreciated. Thanks in advance.
1 - I'm using Tophat, Bowtie, or BWA, in Galaxy to map reads to a transcriptome assembled, via trinity, to the same reads I'm trying to map; therefore, I feel that all, or at least most, of the reads should map well. However, I'm only able to have about 10% of the reads actually map. A friend suggested that loosen the restrictions (change the default settings), which I've done, but whenever I do so, the job fails.
2 - For the reads that do map, when I run Cufflinks with the Bowtie or BWA file, the Cufflinks file comes up empty.
3 - I have an assembled transcriptome that has identified about 100,000 different genes (contigs). After I use cuffmerge to merge two cufflinks files, and the analyze the data with cuffdiff, the output only reports FPKM and differential expression testing data for around 1700 genes. I'm not sure if that is normal and some genes are just not reported, or if Cufflinks isn't looking at all of the genes.
Any help would be greatly appreciated. Thanks in advance.