RAD Seq Data Analysis without barcode

Question

I received a ddRAD Seq data from my supervisor without barcodes and restriction enzymes. I asked him for both and he said I don't need it since the data has been cleaned by the company. Now, I am kinda confused and don't know where to start.

Answer 1

Wow Biobash, that's kinda harsh. It is cool that sometimes you unexpectedly learned something new. What Michael mentioned is pretty interested, no one has ever mentioned that or I have never heard that.

So, my approach would be that you now are doing a Poolseq study, since you can't identify individuals. The other way to try to catch the paralogues is to map your reads to a sister species. My question is what are you trying to estimate? Genetic differentation? Genetic diversity? Pop Gen structure? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116966/ https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.5240 https://www.g3journal.org/content/9/12/4159

One semi-robust approach to assess usefulness is thinking about your system and defining what you expect to find, let's say in terms of genetic differentiation or genetic structure, and estimating that from your data. If that hypothesis is confirmed, your dataset might be useful. You can test the same by first estimating the loci by mapping your reads to a sister reference genome, to try to detect paralogues. Since you don't have barcodes, then each "locus" is independent, and you lose power, but there is nothing else you can do.

Good luck;

Answer 2

The fundamental problem with RAD is that if you hit paralogue - you're done.

Identifying a paraologue isn't necessarily easy depends on the species. Essentially if you're doing pop gen you need homologues and RAD has too many situations where it can fall over without you knowing.

What happens if you've a paralogue is that you start confusing concerted evolution (pop gen of a genome) with population structure between individuals. I do agree that without barcodes identifying individuals - thats a bit tricky ;-) , however RAD an approach loaded with pitfalls.

Pop gen means being able to identify populations and on face value that will not be trivial without barcodes (I suspect they are still there), but RAD is far from an ideal tool and can give weird and wonderful metrics which have little to do with the population structure.

Its not the only problem with RAD for the purpose you are using it.