Questions about using Bowtie2

Question

[fk566938]

Hello I am a Research Technician doing some bioinformatics which isn't something I've had prior experience in. I am using Bowtie 2 and I am aligning RNAseq files downloaded from the illumina bodymap website (16 tissues) to hg19 index files that were created in bowtie2. This is the message I get from the alignments:

-bash-4.1$ bowtie2 -p 22 -x software/bowtie2-2.2.6/scripts/hg19 -U ERR030858.fastq -S 030858.sam
77229855 reads; of these:
  77229855 (100.00%) were unpaired; of these:
    29907119 (38.72%) aligned 0 times
    30792402 (39.87%) aligned exactly 1 time
    16530334 (21.40%) aligned >1 times
61.28% overall alignment rate

I am not sure if this is good or bad, if I'm getting the right thing or not. Is there any way of knowing?

Answer 1

[Antonio R. Franco]

Bowtie2 is not splicing aware. If you used the whole genome as reference, it contains introns, and all of the reads that are spliced, will be unmapped

Use a splice aware program such as TopHat, STAR, BBMap, etc to do the mapping

[fk566938]

do you know what the command would be for tophat? I have it loaded, however I've never used linux before and so all of this has been quite a challenge for me.

[Antonio R. Franco]

Tophat has many choices, but after installing the program you can run the following

tophat >& help.tophat

less help.tophat

And you will have the chance to read the instructions