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URL: https://dx.doi.org/DOI:10.17504/protocols.io.khuct6w

Authors: Adam Vivian-Smith, Jørn Henrik Sønstebø

Summary: Double digested Restriction-site Associated DNA (ddRAD) sequencing is a powerful approach for identifying and analyzing genome-wide SNP variation. Many studies have now used ddRAD protocols for population genetic studies. Here we have adapted the protocol from Peterson (2012) for Ion Torrent sequencing to produce a significantly streamlined workflow capable of having fully sequenced ddRAD libraries in two days. A reduced number of steps for producing a ddRAD library is achieved through the use of a unidirectional double digestion-ligation reaction with adaptors having fusion barcodes in the 5' and 3' ends. This also allows for the immediate pooling of sets of compatible barcoded samples for downstream processing. The A-adaptor contains the standard Ion Torrent barcodes and a key sequence, while the P1-adaptor contains a divergent Y-adaptor with a paired-end code for increased multiplexing. The described system and adaptors are compatible with the SbfI, PstI and NsiI restriction endonucleases in the A adaptor, while the P1-adaptor has compatible overhangs with NdeI or MseI, and, MspI or HpaII. The later two restriction enzymes were chosen as isoschizomers for their differentially sensitivity to CpG methylation, thus allowing the use of this protocol for epigenetic ddRAD profiling at genomic loci with 5-methylcytosine and 5-hydroxymethylcytosine modifications. This protocol takes an advantage in that the Ion Torrent platform has scalability with different sequencing chip sizes, and that the protocol has a range of compatible restriction endonucleases with different motif lengths. This ensures a versatile, cost-effective and flexible method to which you can tune the number of ddRAD loci being profiled, for both small and large genomes, with relative speed and with the ability to observe the performance from small pilot scale reactions.

Associated Publications: Daemi-Saeidabad M, Shojaeiyan A, Vivian-Smith A, Stenøien HK, Falahati-Anbaran M (2020) The taxonomic significance of ddRADseq based microsatellite markers in the closely related species of Heracleum (Apiaceae). PLoS ONE 15(5): e0232471. doi: 10.1371/journal.pone.0232471

Affiliations: Norwegian Institute of Bioeconomy Research (NIBIO), Norwegian Institute of Bioeconomy Research (NIBIO)

External URL: https://doi.org/10.1371/journal.pone.0232471

Version: 1

Publication Date: 2017

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