ddRADseq of the Giant Salamander

We performed double‐digest restriction site‐associated DNA sequencing (ddRADseq; Peterson et al., 2012 (link)), using the restriction enzymes EcoRI and PstI, both of which use six‐base‐pair recognition sites. We multiplexed individuals with 30 unique adapter barcodes. We size‐selected 500–600 bp fragments using a Pippin Prep Blue (Sage Science) and amplified final libraries using Phusion PCR master mix (Thermo Fisher Scientific). Due to the large genome size of A. barbouri (approximately 24 Gb; www.genomesize.com), we included only 30 individuals in each of four final libraries to attempt to increase sequencing depth across individuals; two individuals from each sampling location were duplicately sequenced in different libraries. A total of four libraries were sequenced on an Illumina HiSeq 2000 sequencing system at the University of Oregon Genomics Core Facility (gc3f.uoregon.edu) using single‐end, 100 bp reads.

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Beer M.A., Kane R.A., Micheletti S.J., Kozakiewicz C.P, & Storfer A. (2022). Landscape genomics of the streamside salamander: Implications for species management in the face of environmental change. Evolutionary Applications, 15(2), 220-236.

Publication 2022

Phusion PCR master mix

Base pair Restriction enzymes ecori

Corresponding Organization : Colorado State University

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Variable analysis

independent variables

Restriction enzymes used (EcoRI and PstI)

dependent variables

Sequencing depth across individuals

control variables

Genome size of A. barbouri (approximately 24 Gb)
Number of individuals included in each library (30)
Sequencing platform and read length (Illumina HiSeq 2000, single-end, 100 bp reads)

controls

Two individuals from each sampling location were duplicately sequenced in different libraries to serve as a form of positive control.

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