Can I pool different species for RAD seq?

[sw8362]

Hi,

is it possible to pool different species (after ligating individual barcodes off course) for RAD seq on Illumina?

[SNPsaurus]

I would say probably (my lab developed RAD-Seq). We almost always ran multiple projects in a lane, with different species in each. I don't think we ever, however, mixed different species in the amplification. Usually we would pool 16-24 samples before shearing, so there was little reason to pool across species if carrying out a project of 48 flies and another of 72 mimulus. We would make 2 pools of 24 flies and 4 pools of 18 plants.

If one project was cut with SbfI and another with PstI, you would get different shearing efficiencies (better shearing for the SbfI project) and would have difficulties getting the reads balanced as you would like, but otherwise there is little reason to suspect pooling would cause problems. Just little reason to do so.

[sw8362]

Thanks! Ok, so if I understand you correctly: it is lttle risk to mess up completely if we pooled two different species (to a total of 48 samples, all cut with the same enzyme) prior to shearing and PCR but after ligating barcodes? But what could occur is that one species get a higher number of reads than the other?

[SNPsaurus]

Right. We would normally pool the two species after the shearing and second ligation, but you could do it before.

[sw8362]

Great, thanks again. Our initial data shows reads on all 48 barcodes so I hope we will get some data from most. You've been really helpful!