Bowtie woes

[nimrod337]

Hello all. First, let me just introduce myself, as I am very new here (first post)
My name is Austin, and I am a graduate student working on potato.
I have recently had my favorite potato clone sequenced using illumina's paired end sequencing. It took forever to get back, but I have it now, and I'd like to get some work done.
I am using bowtie to align the reads, but I am struggling to get anything to align.
The biotech center that did the sequencing included a fastQC check, and as far as I can tell, everything looks good. (again, I'm very new, so I could easily miss something.)
I've done some reading, searching, and pounding my head on my keyboard before posting this, but my searches were not infallible, and so I apologize if I missed the answer to this.
I couldn't get the paired end alignment to work, so I tried each set of data individually, to see if perhaps I was not using the paired end details correctly, but I cannot get the single sets to align at all (0.00%)

My first question is about indexes: I downloaded the .fasta file from potatogenome.net, and used the bowtie-build command to create an index from it. However, I have no idea if it was done correctly, as I have no control to align to it. It looks fine, but I am just covering my bases.

I'm basically not using any of the options available in bowtie for my first run, as I just want to have a basic understanding of the functions, but here is my code that I have tried thus far with zero sucess:

paired end try:

./bowtie -S -p 2 potatoindex -1 /run346.C287-Reprep_NoIndex_L001_R1.fastq -2 run346.C287-Reprep_NoIndex_L001_R2.fastq c287alignment

no luck there.

single end try:

./bowtie -S -u 10 potatoindex sequence/run346.C287-Reprep_NoIndex_L001_R2.fastq

(I added the -u 10 just so I didn't have to wait the whole time for it to try. I figured 10 reads would give me at least one that would pair... no such luck. Should I try more reads, just in case the first 10 are bunk?)

Any help you can give me would be awesome. I will answer any questions I failed to include.
Thank you so much!

[nimrod337]

Also, I forgot to ask, is there a potato index already available for bowtie that I could use as a control to see if I'm doing this right?

[dpryan]

This might have just been a copy-paste typo, but in the example commands you gave you are telling bowtie that your fastq files are in different directories. In the first one, you're saying that the first file is in the root ("/") directory, while the second file of reads is in the current directory. Perhaps that's just a copy-paste mistake.

A quick note regarding the single-end alignment attempt. Firstly, try the R1 file instead of the R2 file, it'll often give better results. Secondly, try a higher value for -u. Often, the first chunk of reads in a fastq file are really crappy (they come from the edge of the lane).

You might also try just blasting a few of the reads. I don't know if the potato genome is itself available in blast, but this might prove informative. I've also personally received the wrong sequencing results before, so don't write off the possibility that they just sent the wrong results.

I hope some of that ends up helping!

[nimrod337]

Yeah, that was a typo. I shortened the file paths to paste in here, but I didn't do a very good job. I will definitely blast some of the sequences just in case.
Thanks for the input!

[jimmybee]

Sample mix-ups happen frequently at service providers so its always good to check by BLASTing some reads (as previously mentioned).

Also, is there a reason that you are using bowtie? Maybe a gapped aligner like bowtie2 might help with the repetitive sequences in potato, or perhaps use BWA to see if you get the same results.

[amcloon]

You could see if anyone at your institute has a program like Geneious (sp?) or CLC Genomics workbench and test those (or test a demo version). The programs are expensive and perhaps not as powerful as some of the other analysis programs, but at least they are fairly idiot-proof and when mapping, they show you visually the reads that mapped... That seems a bit easier than guessing which reads to BLAST...

[nimrod337]

Okay, thank you all for your help. After looking at the general consensus, it seems that I should have checked to make sure the sequence I was returned was in fact, mine.

After BLASTing a few of the reads from the first run, it seems they all come from a particular "homo sapiens" organism... Last time I checked, I didn't know of any potato species named "homo sapiens"... Bummer. It seems I have the wrong sequence data.

Thank you all so much for your prompt and helpful responses.

Also, is there a list of bowtie comands listed somewhere other than the actual bowtie manual? It seems like there would be more than 3 commands within bowtie, but perhaps not. (like, wouldn't it be nice to have a command that just prints out the reads from your file?)

I'm sure I'll be back with more questions after receiving the correct data. Stay tuned! Thanks again,

-A

[dpryan]

Well, they do say that you are what you eat...

What's on the bowtie manual page is all there is. For printing the resulting alignments, just use samtools (the "view" command) or just "cat" or "less" if you haven't converted your SAM file to BAM yet. To view the fastq files, cat/less/zcat/etc. will work. Bowtie is an aligner, so I doubt the authors want to support a whole bunch of extraneous software

[GenoMax]

It is tempting to blame the sequence provider but the sample you had submitted may actually be the problem.

Until proved conclusively otherwise.

[amcloon]

To view some reads, you don't need Bowtie at all. If you want to look at your raw sequence reads (rather than the .bam file, which dpryan explained how to view), what you need is just a command like
>head <sequence_reads.fastq>

and that will show you the first few lines of your file of reads (if you are in/accessing the directory where your reads are).
>head -50 <reads.fastq>

will give you the first 50 lines, etc.

Sorry if this is patently obvious to you, I just figured I'd put it out there for anyone happening across this thread who is trying to use Bowtie without having first worked through a learn unix book/program...