Anyone doing GBS/ RAD? A question about using selective primers

[Pauline dlp]

So I saw several papers referencing a method used by Sonah (2013) where they used a selective primer that has one or two additional bases to further reduce their GBS library. This step will further reduce complexity but increases the read depth.

My question is, how necessary/useful is this step for non-plant species? I know Sonah et al used that to improve depth coverage in soybean GBS libraries since plant genomes have lots of highly repetitive fractions. The thing is, I'm working with an insect genome and other studies on insects I've read also used selective primers though I haven't seen a justification for it other than citing Sonah's method.

[nucacidhunter]

This is to reduce the number of tags and hence sequencing requirement. Sequencing is much cheaper than 5 years ago and may not be a factor in experiment design.

One need to estimate tag number if possible and expect on average 5% polymorphism and required SNP number for application to decide if further reduction is required.

This process has risks as well if the oligo synthesis quality is low or 3’ end is not exonuclease resistant which will result in truncated primers so the reduction will be incomplete and many tags may not be common among samples requiring deeper sequencing.

[Pauline dlp]

Thank you for your reply. So if one were to do this method, the primer should have the phosphorothioate modification to make it exonuclease resistant, is this right?

[nucacidhunter]

Yes, specially if you are using proof reading polymerase. The primers also will be too long and would need gel or HPLC purification which will increase costs.

It will be more cost effective if you can choose the RE that gives required number of RAD-tags.