Using molecular markers in ecology and evolution is moving massively parallel. Not only can the new era of genotyping using next-generation sequencing give high resolution through generating thousands of markers but, with sufficient genomic knowledge, can be used to localize polymorphisms across the genome. Restriction site associated DNA sequencing (RADseq) of various different forms (see a very nice recent review of approaches here) is currently a major method of choice for researchers studying complex genomes and without requiring prior genomic resources.
We have recently developed a new genotyping-by-sequencing RAD sequencing approach for the Ion Torrent platform. The method can robustly generate thousands of loci using a modification of the ddRADseq protocol developed by Hopi Hoekstra's lab based on RADseq and applied on Ion Torrent/ Ion Proton/ Ion PGM semi-conductor sequencing.
The method is modular, has a low error rate, is economical, and importantly can be very fast. It is generally a low to medium through-put approach and so is likely especially suitable for researchers who have an Ion platform on hand. We have tested this method on a number of different non-model species that we are current interested in, such as common lizards, Arctic charr, European whitefish, and fire salamanders. The method can of course just as easily and successfully be applied to the so-called ‘model organisms’.
As recently presented at Population Genetics Group Sheffield, we offer a step-by-step bench protocol (in the manuscript Supporting Information) to anyone interested in trying the approach. Please be in touch if you have any questions.
Inherent to semiconductor sequencing, the reads generated by Ion Torrent differ in length around a median. We also provide a short Unix command in combination with an R script to maximize the number of base-pairs that will be retained for further analysis. Just like illumina-based RAD sequencing data, the reads can then be analyzed using customized RADSeq scripts or programs such as STACKS.
We ran the method with PI chips -- if Life Technologies releases the promised PII and PIII chips in the future, which should have the same chemistry and well technology so no need for new adapters, then the efficiency of this ddRADseq-ion method will increase many-fold.
The paper is in press with Molecular Ecology Resources. (IF = 5.626)
The work was supported by collaboration with Glasgow Polyomics, a University of Glasgow John Robertson Bequest grant, University of Glasgow start-up, and Wellcome Trust Institutional Strategic Support Fund Pilot funding from Glasgow Polyomics to KRE.
Recknagel Hans, Jacobs Arne, Herzyk Pawel, Elmer Kathryn R (2015) Double-digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq-ion) with non-model organisms. Molecular Ecology Resources. in press.