RAD-seq SNPs: pooled population library prep?

[a.cardilini]

G'day Everyone,

I am new to genomics and hope that I don't annoy you by asking a basic question.

I am thinking of using RAD-seq SNP discovery to identify SNP markers for a population genetics project in a non-model species. At the moment, I am trying to figure out if it is realistic for me to go down this path and have been reading the protocol by Etter et al 2011. I have a question about pooling populations that I hope someone can help me with:

Can I pool the DNA of individuals before I digest the DNA with the appropriate restriction enzyme? Meaning I would only need to do 1 digest per library rather than ~20.
If not, what is the reason behind not doing this?

Thank you in advance for your help and time.

Kind regards,
Adam

[uranotaenia]

It depends on if you want to have individual-level data at the end. If you pool before you ligate your barcodes (P1 adapter ligation comes after restriction digest) then you will not be able to sort out your individuals in the final reads.

So if you want to maintain individual-level data then you will want to do the individual restriction digests, ligate your barcodes (P1 adaptors) on and then pool for shearing.

[jboone123]

Uranotaenia hit it on the head.

Also keep in mind- when doing pools together with local de novo assembly of RAD pe reads, assembly of pe reads can get messy if the level of variation in the pool is high.

Good luck and tell us how it goes.