Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification

Authors: MAGBANUA, ZENAIDA - Mississippi State University; HSU, CHAN-YU - Mississippi State University; PECHANOVA, OLGA - Mississippi State University; ARICK II, MARK - Mississippi State University; GROVER, CORRINE - Iowa State University; PETERSON, DANIEL - Mississippi State University

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2022
Publication Date: 2/1/2023
Citation: Magbanua, Z.V., Hsu, C., Pechanova, O., Arick Ii, M., Grover, C.E., Peterson, D.G. 2022. Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification. Analytical Biochemistry. https://doi.org/10.1016/j.ab.2022.115001.
DOI: https://doi.org/10.1016/j.ab.2022.115001

Interpretive Summary: While whole genome comparison of individuals is extremely powerful, but it is not always financially or technically practical. Comparison of gene-enriched subsets of DNA (i.e., DNA markers) provides many of the advantages of whole genome comparisons at a fraction of the cost. Here we describe a DNA-based marker technique that is an improvement of the single-nucleotide polymorphism (SNP) discovery technique know as ddRAD-Seq. The modified ddRAD-Seq method employs restriction enzyme digestion of DNA samples while eliminating buffer exchange steps. Fragment amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the amplified genomic regions. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared DNA sequence differences detected using our protocol with those from whole genome resequencing data from two cotton species. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

Technical Abstract: We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.