Attachment 01 Molecular methods_Maier.docx

Dr. Paul Maier’s original molecular methods used to develop the Yosemite toad ddRADseq libraries and sequencing data.

Double-digest RADseq libraries were constructed following the protocol of Peterson et al. (2012; S1 Protocol).

1. A total of 200–500 ng DNA was digested with 5U SbfI-HF and MspI (New England Biolabs), for 3 hr at 37°C.

2. Digested DNA was cleaned with 1.5x Agencourt Ampure XP beads (Beckman Coulter) and quantified by Qubit 2.0 Fluorometer (Life Technologies).

3. Digested DNA was ligated to oligo-nucleotide adapters with one of 8 unique 5 bp MID barcode sequences at 25°C for 30 min, followed by a 10 min heat kill at 65°C.

4. Ligated DNA was cleaned with the AMPure XP and pooled by adapter.

5. Ligated and pooled DNA was then size selected between 424 and 525 bp using a 1.5% gel cassette (Pippin Prep; Sage Science). This optimal fragment size range was chosen to balance number of loci with projected coverage per locus.

6. Size-selected DNA was amplified with Illumina primers containing one of 12 unique indices using a Phusion PCR kit (New England Biolabs). The following cycle profile was used: 98°C for 30 s, [98°C for 10 s, 72°C for 20 s @ 16% ramp], 72°C for 10 min, 4°C hold.

7. Finally, amplicons were bead-cleaned, quantified by BioAnalyzer (Agilent Technologies), and pooled in equimolar amounts for sequencing. This combinatorial approach allowed 8×12 unique samples to be sequenced in parallel on a single Illumina flowcell, and use of double-restricted fragments dramatically increased locus recovery across samples.

8. All ddRAD libraries were 2×100 bp sequenced on seven lanes of an Illumina HiSeq 2500 (96 samples per lane) at the Institute for Integrative Genome Biology, UC Riverside, CA.