Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double-digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single-end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11 000 polymorphic loci per library of 6–30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost-effective generation of variable and reproducible genetic markers.
Table_1
Table_1 contains the locus sequences and coded genotypes of each individual used to recreate genomic composition of the Alpine x Baltic cross and their offspring. SNPs that were fixed between parents (following standard genetic mapping procedure) were used in the Structure analysis. Each individual contains two columns per locus with their genotype. Numbers refer to the four different DNA bases: A = 1, C = 2, G = 3, T = 4. 0 = missing data.
Table_2
Table_2 contains all the locus sequences (sheet 1: ‘Locus sequences’) and SNP information (sheet 2: ‘Genotypes’) that were used for the inference of phylogenetic relationships of Arctic charr and European whitefish.
Library1_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library2_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library3_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library4_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library5_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library6_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library7_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library8_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library9_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library10_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library11_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library12_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
Library13_SNPs
Sequences of all loci containing SNPs within the genomic library are listed in the first sheet ('Sequence'). Each SNP, its position on the respective locus and the genotype of each individual for that SNP is stored in the second sheet ('SNPs').
RAxML_treefile
The tree file contains three Arctic charr and three European whitefish individuals from different localities in Europe
Replicate_1_genotypes_8x
Input file for estimation of the SNP error rate. The R script to analyse this file is provided by Mastretta-Yanes et al. (2014).
Replicate_1_genotypes_16x
Input file for estimation of the SNP error rate. The R script to analyse this file is provided by Mastretta-Yanes et al. (2014).
Replicate_2_genotypes_8x
Input file for estimation of the SNP error rate. The R script to analyse this file is provided by Mastretta-Yanes et al. (2014).
Replicate_2_genotypes_16x
Input file for estimation of the SNP error rate. The R script to analyse this file is provided by Mastretta-Yanes et al. (2014).