Sea stars (Pisaster ochraceus) were collected from Santa Barbara, central California, Dabob Bay, Friday Harbor, Sokol Point, and Sitka. In all cases, tube feet were removed from Pisaster ochraceus with a razor blade and preserved using 95% undenatured ethanol.
The tube feet from Dabob Bay were preserved in dimethyl sulfoxide (DMSO) buffer per Wares (2000). DNA was subsequently isolated using a Puregene protocol as in Wares (2023) at our lab in Athens, Georgia, USA within the Department of Genetics at the University of Georgia. DNA concentration was quantified using Qubit fluorometry (Invitrogen, Waltham, MA, USA). DNA processing for Illumina sequencing was performed as follows: DNA samples were prepped for blunt-end ligation using End-ItTM DNA End-Repair Kit (Epicentre Biotechnologies) according to the manufacturer’s instructions. Next, DNA was cleaned using AMPure beads (Beckman Coulter) to select fragments of 100 bp or higher, and prepped for Illumina sequencing as in Ji et al (2018). Following elution into 43 μl of Tris–HCl, samples were combined with 50 μl A-tailing reaction in NEBNext dA-tailing buffer with Klenow fragment (3′–>5′ exo-) and incubated at 37 °C for 30 min. Following addition of A-tails, the DNA fragments were ligated to Illumina Truseq adaptors and again purified with AMPure beads. Amplification was achieved in a 50 μl reaction using PhusionTM High-Fidelity polymerase (Thermo Scientific) according to the manufacturer’s protocol and the following PCR thermocycler (Bio-RAD T100) specifications: an initial 95 °C for 2 min, then 98 °C for 30 s, followed by 15 cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 4 min and a final 10 min incubation at 72 °C. Primers were removed from the genomic DNA product with a third round of AMPure bead purification.
Samples collected from the remaining five sites (Santa Barbara, central California, Friday Harbor, Sokol Point, and Sitka) were processed as follows: DNA samples were prepped for blunt-end ligation using End-ItTM DNA End-Repair Kit (Epicentre Biotechnologies) according to the manufacturer’s instructions. Next, DNA was cleaned using AMPure beads (Beckman Coulter) to select fragments of 100 bp or higher, and prepped for Illumina sequencing as in Ji et al (2018). Following elution into 43 μl of Tris–HCl, samples were combined with 50 μl A-tailing reaction in NEBNext dA-tailing buffer with Klenow fragment (3′–>5′ exo-) and incubated at 37 °C for 30 min. Following addition of A-tails, the DNA fragments were ligated to Illumina Truseq adaptors and again purified with AMPure beads. Amplification was achieved in a 50 μl reaction using PhusionTM High-Fidelity polymerase (Thermo Scientific) according to the manufacturer’s protocol and the following PCR thermocycler specifications: an initial 95 °C for 2 min, then 98 °C for 30 s, followed by 15 cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 4 min and a final 10 min incubation at 72 °C. Primers were removed from the genomic DNA product with a third round of AMPure bead purification.
Sequencing of WGS data was performed on Illumina NovaSeq sequencing machines. Some samples were sequenced at the UC Davis Sequencing Core (those from central California), the rest were sequenced at Novogene.
