[Isa0984]

So it is the amount of operations (substitution, insertion, delition) that I have to do, to change my 50bp read to my reference. But why do I have then reads mapped to my reference with partly more than 10 mismatches?

[dpryan]

You'd have to go through the exact command that the company used and compare that to how things are processed inside bwa (by looking at the code rather than the documentation).

[Isa0984]

That means other options in BWA can change the maximum of my 3 "mismatches" and allow more... wow that makes it even more complecated for me. They said they used default parameters with the only change in the options mentioned above.

[GenoMax]

You are not able to get the raw data and recreate the result yourself? You could be spinning your wheels for a long while trying to find an explanation for their results.

Note: Reading the beginning of the thread seems to indicate you have the raw data but are your results not matching the alignments the company did?

[Isa0984]

I have the raw data, but I am not working at the institut anymore... and at home with my old little labtop, not really. Iam writing my thesis and there you look sometime at things deeper, then you should have done it before. But maybe I should simply take it like it is. Its just the urge to understand it in detail. Thanks a lot for your patience!

[GenoMax]

I see the dilemma now. You could use the results provided by the company as is but then you may not be able to defend them if questioned during your defense.

Do you think the conclusion is going to differ if you were to do the analysis again? If you are sure the answer is no then you will have to make a decision.

[dpryan]

Neugier ist der Katze Tod, or perhaps "Curiosity killed the PhD thesis" would be a better variant

But anyway, I agree with GenoMax. If their results look problematic at all then it's worthwhile to repeat the alignment simply so you know exactly how things were processed.

[Isa0984]

Thats the point, but hopefully they will not ask... The good thing is, even Iam not so deep to this whole RNA-seq thing, Iam deeper then the others... so I just need a good explanation which they swollow.

Doing it again is no option. The only option I have is to terrorize the company, but customers service is not their strength and they explain me everytime that its too long ago, they are not able to look to the data... Which is incomprehensible for me.

[Isa0984]

Good statement, I still need something creative for my "plain site"... Until now my favourite is "Die Zeit promoviert jeden"

[dpryan]

This is RNAseq data that they aligned with bwa aln? I must have missed the RNAseq part earlier. I'm not sure how well bwa aln will deal with splicing...

BTW, I'm going to have to steal your saying

[Isa0984]

Yes it is, but it was aligned to a transcriptome (apple by the way, its not the best, but better then nothing).
Thats fine, do it... it did it too and I really love it.

[dpryan]

Ah, aligned to the transcriptome makes MUCH more sense! Your committee can't complain about that.

[Isa0984]

At least something positive