RADseq library preparation restriction enzymes

[LoeraI]

Hey there,

I’m preparing some libraries for RADseq sequencing of a plant species using double enzyme digestion sequencing. I already digested the genomic DNA of some samples with two enzymes (EcoRV and MSE1). I’m going to use double pair end Illumina sequencing so I’m interested in fragments around 200-500 bp.

I attached a photo of the digestion. As you can see for most of the samples (sample 1 (S1) has 1ug of genomic DNA whereas for the digestion of the other samples (S2-S4) I used 400ng) I can see smear around 100 and 600 bp. So I think these digestions could work for further ligation of barcoded adaptors, PCR enrichment and gel purification.

You think it’s OK to continue (I have to decide if these enzymes are OK before ordering all the primers with the barcodes for 26 populations) with the construction of libraries? Or should I try with other combination of enzymes? I’m working with a non-model organism so there’s no reference genome.
I have never prepared libraries before so any advice would be very useful.
Hope this is clear and thanks in advance.

Cheers.

[NRP]

Hi Loera1 - I just saw your post and I am curious what the outcome of your library prep was. I am currently trying to generate my first RAD libraries and some of my genomic DNA samples are pretty degraded. Thanks.