Reports Materials and methods


Proof of concept To demonstrate the efficacy of gene capture across highly divergent taxa we selected a set of target genes that would be (i) present in all taxa tested and (ii) unambiguously identifiable (unique) within each genome. We targeted coding DNA sequences (CDS) from single-copy protein-coding genes that are shared across gnathostome vertebrates. We compared the published and annotated genomes of human (Homo sapiens), chicken (Gallus gallus), western clawed toad (Xenopus tropicalis), green anole (Anolis carolinensis), zebrafish (Danio rerio), and elephant shark (Callorhinchus milii), and identified the putatively orthologous genes within each respective genome. A within-species BLAST search (http://blast.ncbi.nlm.nih.gov/Blast. cgi) (17) was used to identify gene regions determined to be single-copy according to a 60% dissimilarity criterion (i.e., there were no genomic regions present that were more than 40% similar to the potential target according to the within-species BLAST search criterion). We then used among- species BLAST searches to verify that the identified targets occurred in all six verte- brates. The EvolMarkers software for selecting such sequence sets for any combi- nation of species is freely available at http:// bioinformatics.unl.edu/cli/evolmarkers/ (18). We identified a total of 1449 candidate single-copy CDS markers that were shared across the 6 vertebrate species using this approach. Te size of the target CDS varied from 151 bp to 2390 bp.


Bait development We developed separate custom bioti- nylated RNA bait libraries based on the identified CDS for the H. sapiens, G. gallus, X. tropicalis, A. carolinensis, D. rerio, and C. milii genomes using the MYBaits target enrichment system (MYcroarray, Ann Arbor, Michigan).


D. rerio Target sp. Genome size (pg)


Time of divergence from bait species (MYA)


1Standard capture 2Relaxed 1st 3Relaxed 2nd capture capture


Each resulting bait library comprised a pooled series of 120 bp baits designed for each target gene. We included a 60 bp overlap between baits, allowing for 2× complete coverage of each target gene (2 × tiling). If the length of the target gene was less than 120 bp, the sequence was extended in length to 120 bp by adding thymine (T) nucleotides.


Experimental design We carried out two experiments. First, in an effort to evaluate the effectiveness of the method across broadly different classes of gnathostome vertebrates, we used baits to capture the selected set of 1449 target single-copy genes from (i) genomic libraries of the species from which the baits were derived (as a positive (+ve) control) and (ii) distantly related species belonging to the same verte- brate class as the species from which the bait sequence was derived. Tus, for fish we used baits designed from the D. rerio genome to capture target DNA sequences from D. rerio (+ve control) and the stickleback (Gasterosteus aculeatus); for amphibians we used baits from the X. tropicalis genome to capture targets in X. tropicalis (+ve control) and the axolotl (Ambystoma mexicanum); for reptiles we used baits from the A. carolinensis genome to capture targets in A. carolinensis (+ve control) and the painted turtle (Chrysemys picta); for birds we used baits from the G. gallus genome to capture targets in G. gallus (+ve control) and the zebra finch (Taeniopygia guttata); and for mammals we used baits from the H. sapiens genome to capture target genes in H. sapiens (+ve control) and the gray short- tailed opossum (Monodelphis domestica). Te similarities of the target CDS ranged from 89% to 61% among the 6 vertebrates (ultraconserved element approaches target highly conserved sequences with similarities typically greater than 90% (14,15)). We compared the effects of two different


hybridization and washing schemes on the capture results. Standard conditions


Table 1. The number of the 1449 target CDS captured using different protocols, for 10 vertebrates. Bait sp.


X. tropicalis 1.78 ~ 0


1349 (93%)


1387 (96%)


(4.6%) 329


(23%) 643


(44%)


1125 (78%)


1412 (97%)


1428 (99%)


21.85–48 ~ 298.6 0 53


(4%) 225


(16%) A. carolinensis


comprised 65°C for both hybridization and washing temperature, following the manual for the MYBaits Target Enrichment System (Mycroarray, Ann Arbor, Michigan). Te relaxed conditions comprised hybridization under a touchdown gene capture scheme similar to Mason et al. (10) but with a much lower final temperature: 65°C for 11 h, followed by 60°C for 11 h, 55°C for 11 h, and 50°C for 11 h, using 45°C instead of 65°C for the last three washing steps (see detailed protocol). We also tested whether capturing twice (i.e., using the captured products aſter re-amplification as templates to perform a second round of capture) increases the number of captured target genes. Secondly, in an effort to explore the


effectiveness of the method within a class of gnathostomes, we applied the optimized protocol to a diverse suite of chondrichthyan fishes (sharks, skates, rays, and chimaeras), the group that represents the most basal gnathostome class and whose origins date back to the Devonian period, more than 400 million years ago (19). We used baits based on the elephant shark (C. milii) genome to capture the 1449 target genes from C. milii (+ve control), five skates and rays (Aetobatos narinari, Leucoraja erinacea, Neotrygon kuhlii, Rhinobatos schlegelii, Torpedo formosa), and seven sharks (Carcharhinus amblyrhynchos, Chlamydoselachus anguineus, Etmopterus joungi, Heterodontus portusjacksoni, Isurus oxyrinchus, Orectolobus halei, Squatina nebulosa). For detailed experimental design infor-


mation, please refer to the protocols for library preparation using the with-bead method and gene capture that are provided as Supplementary Material.


Sequencing and analysis We pooled 20 indexed samples in equimolar ratios aſter gene capture. We then measured the pooled product using the CFX Connect Real-Time PCR system (Bio-Rad, Hercules, CA) and used 600 µL of 8 pM sample for


G. gallus 1.61–3.06 2.57–3.22 1.25 ~ 0 958 (66%)


1365 (94%)


1374 (95%)


1Standard capture: the standard protocol using 65°C for the hybridization and the second washing step. 2 1.25 3.5 H. sapiens


D. rerio G. aculeatus X. tropicalis A. mexicanum A. carolinensis C. picta G. gallus T. guttata H. sapiens M. domestica 1.68–2.28 0.58–0.70


3.5 ~ 0 ~ 254.4


1216 (84%)


67 ~ 244.2 ~ 0 ~ 106.4 ~ 0 14


(1%) 377


(26%) 777


(54%)


1259 (87%)


1420 (98%)


1413 (98%)


554 (38%)


1057 (73%)


1159 (80%)


1124 (78%)


1297 (90%)


1355 (94%)


~ 163.9


132 (9%)


438 (30%)


1027 (71%)


Relaxed first capture: relaxed hybridization conditions, i.e., touchdown


hybridization scheme of 65°C for 11 h, followed by 60°C for 11 h, followed by 55°C for 11 h,completed by 50°C for 11 h, and using 45°C instead of 65°C for the final 3 washing steps (see detailed protocol). 3


were calculated from “The TimeTree of Life” (29). The genome size data were retrieved from the Animal Genome Size Database (www.genomesize.com). Vol. 54 | No. 6 | 2013 322 www.BioTechniques.com Relaxed second capture: products from the first capture were used as templates to perform another round of capture. Divergence times


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