Genomic Library Prep
We sampled 157 individuals from 103 localities throughout the geographic distribution of the Antrodiaetus unicolor species complex (Supplementary Table S1; Figure 1). We extracted genomic DNA from leg tissue for each individual using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. DNA was quantified using a Qubit 3.0 Fluorometer (Life Technologies, Inc., Grand Island) and checked for quality using an agarose gel.
RADseq libraries were prepared following the Adapterama III protocol (Bayona-Vásquez et al., 2019), which is a modified version of ddRAD (Peterson et al., 2012) that uses three enzymes for digesting genomic DNA (3RAD). This protocol alleviates the need for large quantities of DNA since the third enzyme prevents adapter dimers and DNA chimaeras from forming during the reaction (Bayona-Vásquez et al., 2019; Graham et al., 2015; Hoffberg et al., 2016). Based on previous studies that tested different enzyme combinations (Bayona-Vásquez et al., 2019; Burns et al., 2017), we chose EcoRI-HF, MspI, and ClaI as our cohort of enzymes for genomic DNA digestion.
For each sample, 100 ng of genomic DNA were digested for 2 hr at 37 °C in an 18 µl solution consisting of 1X Cutsmart buffer, 20U EcoRI-HF, 20U MspI, 10U ClaI, 0.28 µM each of forward and reverse adapters. Immediately following digestion, the reaction was brought to 24 µl total consisting 0.25x Ligase buffer, 100U T4 DNA Ligase, 0.75 µM rATP, and incubated at 22 °C for 20 min and 37 °C for 10 min for 3 cycles and a final 20 min enzyme kill step at 80 °C. Libraries were pooled and cleaned with 1.5X volume Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh, PA) and quantified using the Qubit.
PCR was used to attach the iTru5 primer. Six replicate 50 µl reactions consisting of 10 µl of pooled DNA, 1X Kapa HiFi Fidelity Buffer (Kapa Biosystems, Massachusetts, EUA), 0.3 µM dNTP mix, 0.5 µM iTru5_8N primer, and 1U KAPA HiFi Hotstart DNA polymerase with conditions at 95 °C for 2 min of initial denaturation, followed by 98 °C for 20 sec, 61 °C for 30 sec, and a 5.5 min 72 °C extension. The pools were combined and then 2X volume bead-cleaned. Twelve iTru7 primers were added using PCR with three reactions consisting of 10 µl of pooled DNA, 1X Kapa HiFi Fidelity Buffer, 0.3 µM dNTP mix, 0.5 µM P5 primer, 0.125 µM of four different iTru7 primers, and 1U KAPA HiFi Hotstart DNA polymerase with conditions at 95 °C for 2 min; 6 cycles at 98 °C for 20 sec, 61 °C for 15 sec, and 72 °C for 30 sec; and a final extension at 72 °C for 5 min. The products were pooled and 2X volume bead-cleaned and quantified.
Size selection was performed using Pippin Prep (Sage Science, Beverly, MA) to capture 570 bp +/- ~18% (470-670 bp range) fragments using a 1.5% agarose cassette. The last enrichment PCR for the final size-selected pool was run with 10 µl of pooled DNA, 1X Kapa HiFi Fidelity Buffer, 0.3 µM dNTP mix, 0.5 µM each of P5 and P7 flowcell binding primers, and 1U KAPA HiFi Hotstart DNA polymerase with conditions at 95 °C for 2 min; 9 cycles at 98 °C for 20 sec, 61 °C for 15 sec, 72 °C for 45 sec; and lastly an extension at 72 °C for 5 min. Final libraries were sent to the Georgia Genome and Bioinformatics Core or UC Davis Genome Center for paired-end 150 bp mid-output sequencing with the Illumina NextSeq 550 platform.