RAD-seq pool or not newbie question

[alaminos]

Dear all,

So my question is regarding pooling individuals or not for RAD-seq.
First of all we will use 3 lanes of illumina hi seq 2500 and my project involves sequencing of bees across 18 sites. Sites that differ in terms of landscape.
So we will use RADseq for both identifying demographic parameters from neutral markers and candidate loci undergoing selection for complex habitats.
Since we have one plate and 3 lanes of illumina we could either genotype 5 individuals per population (site) or we pool about 25 individuals (thats how much I have) per population.
Which of the two ways do you think is preferred for the purpose of our study.

Thanks in advance

P

[SNPsaurus]

I think it is difficult to get accurate SNP frequency estimates from pooled RAD data. You could identify SNPs present/absent in each pool though.

I'm not sure why your project is limited to 1 plate? Could you sequence 1 plate of individuals in each lane for 285 total? Unless you have many loci that should be possible.

[alaminos]

Thanks for your answer.
If we use 2 plates that means 95 additional RAD libraries and our budjet unfortunately is limited.

[SNPsaurus]

I think if there is flexibility, then I would genotype 10 individuals from each population in 2 lanes of sequencing, 95 individuals per lane. You can re-use the same adapters in each of the two lanes, of course, so the extra cost of making the libraries should be easily offset by the cost of the third lane that is no longer needed.

You'd end up with solid genotypes of 20 chromosomes per population (are you sequencing diploids?), giving a pretty accurate estimate of allele frequency at 5% resolution from each site.

[alaminos]

Thanks a lot for your suggestion. I will ask regarding how flexible we are with the setup.