title: American Eel RBA15 RAD Sequencing
design: Whole genomic DNA was extracted from each individual using a                 standard phenol-chloroform procedure. Genomic DNA was digested with restriction                 enzyme EcoRI, ligated to a barcode-containing-adapter (to allow individual                 identification) and subsequently pooled and sheared to an average size of 500 bp.                 Sheared DNA was separated on a 2% agarose gel by electrophoresis and fragments in                 the 350-500 bp range were isolated. After dsDNA ends were treated with end blunting                 enzymes and 3’-adenine over-hangs were added, a second adapter was ligated. Finally,                 libraries were enriched by PCR amplification sand RADs for each individual were                 sequenced (10 individuals per sequencing lane). Processing Pipeline – First,                 sequence reads from the Illumina runs were sorted on the basis of the individual                 barcode tag. Quality filtering was carried out using FASTX-Toolkit                 (http://hannonlab.cshl.edu/fastx-toolkit), with all reads trimmed to a final length                 of 80 bp and using a minimum Phred score of 10 per nucleotide position. Sequence                 reads were aligned to the European eel genome draft (http://www. eelgenome.com)                 using Bowtie version 0.12.8 (Langmead et al. 2009). A maximum of two mismatches                 between the individual reads and the genome were allowed and alignments were                 suppressed for a particular read when more than one reportable alignment existed.                 Reference-aligned reads were processed using Stacks version 1.02 (Catchen et al.                 2011). First exactly-matching reads were aligned into stacks and subsequently into                 putative loci using a minimum stack depth of 10 reads. Second, SNPs were called                 using a maximum likelihood framework and a catalog of all loci and SNPs was built,                 against which all individuals were matched. Finally, all SNP data across individuals                 was processed using the program Populations in Stacks. Loci were only processed when                 present in at least 66.67% of the individuals at all sampling locations. As a                 further filtering step, we also excluded loci with higher-than-average number of                 reads (using as threshold twice the standard deviation from the mean number of                 reads), triallelic loci and those loci deviating from Hardy-Weinberg equilibrium. 