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paired-end 150 bp sequencing on a MiSeq sequencer (Illumina, Inc, San Diego, CA). Sequence reads associated with each sample were identified by their respective indices. Te adapter sequences and sites with lower qualities were trimmed using the Cutadapt application (version 1.1; Contigs were assembled de novo for each species using ABySS (version 1.3.4; www.ſtware/abyss) (21). A custom Perl script (Supplementary Material) was used to retrieve the sequences for each gene for each species studied from the assembled contigs. Te aligned sequences for each gene were checked by eye to ensure accuracy using Geneious Pro v5.6.2 (Biomatters, Auckland, New Zealand; available at To ensure that only orthologous sequences were used in our final analyses, we only considered cases where the target returned a single hit. We were able to do this because our bait arrays were explicitly designed to target single copy genes in the 6 model organisms (based on a conservative 60% dissimilarity criterion used in the initial selection of target sequences). As a further test of orthology, we used a Hidden Markov Model (HMM), trained on a curated set of alignments of the 1449 single copy targets across the 6 model organisms (22) to assign captured sequences to their corre- sponding orthologous targets. In all cases, more than 90% of the captured sequences were assigned as orthologs by the HMM. We excluded all genes whose orthology was questionable from further analysis. We caution that paralogs resulting from gene duplication and subsequent asymmetric loss cannot always be ruled out with our approach. Te average identity between the captured sequences and the baits was calcu- lated using custom Perl scripts (Supple- mentary Material).

Results and discussion

Comparison across different classes of gnathostome vertebrates Te results we obtained varied depending on hybridization and washing conditions (Table 1). Under standard conditions, very few target sequences were successfully retrieved, except for the positive controls, where both bait and target library were derived from the same species. Te only exception to this was the result obtained for birds where 554 target sequences were captured for T. guttata (zebra finch) using G. gallus (chicken) baits. We speculate that this is a consequence of the unusually high level of sequence similarity seen among divergent species of birds relative to their

Vol. 54 | No. 6 | 2013

Table 2. The number of the 1449 target CDS captured for 13 chondrichthyans using baits based on the C. milii genome and the optimized capture protocol.

Target species

Callorhinchus milii Aetobatus narinari Leucoraja erinacea Neotrygon kuhlii

Rhinobatos schlegelii Torpedo formosa

Carcharhinus amblyrhynchos Chlamydoselachus anguineus Etmopterus joungi

Heterodontus portusjacksoni Isurus oxyrinchus Orectolobus halei Squatina nebulosa

No. of target CDS captured

1449 1309 1004 1284 1351 1036 1283 1217 1082 1294 1251 1206 1192

No. of CDS kept for final analysis

1242 1182

1232* 1163 1212 928

1150 1101 976

1167 1136 1087 1080

*Additional target sequences were retrieved from genome data available in GenBank (

non-avian vertebrate counterparts (23). By contrast, under the relaxed hybridization conditions, there was an eight-fold increase in the number of target sequences captured relative to experiments executed under standard conditions. Te difference in the effectiveness of gene capture was especially obvious where target species were highly divergent from bait species (Table 1). Performing 2 rounds of gene capture

further increased the number of targets captured relative to the initial round of capture by 68%, on average. Once again, the improvement was most conspicuous where the bait and target species were highly divergent (see Table 1). Te effec- tiveness of cross-species gene capture with the optimized protocol ranged from a minimum of 225 (for the amphibians) to a maximum of 1159 (between chicken and zebra finch) of a possible 1449 target CDS. Te lowest number of target genes successfully captured (225) resulted when baits designed from the X. tropicalis genome were used to capture corresponding genes in the axolotl, A. mexicanum. We hypoth- esize that this result may be due to compro- mising effects associated with repeats in the unusually large (~32 × 109

bp) (24) genome

of the axolotl. Te protocol we used for target enrichment used human cot-1 DNA in an effort to block and therefore mitigate the adverse effect of repetitive DNA during hybridization. We suspect that tailored cot-1 DNA derived from A. mexicanum may be necessary to effectively block repeat elements impeding capture for the axolotl. We investigated whether factors other

than bait-target divergence affected the efficacy of gene capture. We compared the GC content, target sequence length, and chromosomal position between the set of targets that were successfully captured, and


those that failed capture. Tis was carried out for the six positive controls (H. sapiens, G. gallus, X. tropicalis, A. carolinensis, D. rerio, and C. milii,) where the bait and target sequences are identical, allowing us to rule out any confounding effects that might be caused by sequence divergence. On average, the few targets that were not captured had higher GC contents (56%) and shorter lengths (235 bp) than those that were successfully captured (GC = 49%, length = 303 bp). However, there was overlap in both GC content and target length between the captured and non-captured targets suggesting that other influences may be involved when targets are not captured. Chromosomal position did not seem to have any effect on capture success. In summary, the efficacy of gene capture

was improved by incorporating both touchdown gene capture and conducting a second round of capture, but there was considerable variation in efficacy across the five classes of gnathostome vertebrates tested. We hypothesize that this was due to differences in rates of molecular evolution among the pairs of vertebrates, the presence of genomic anomalies such as repeats that are known to interfere with gene capture (25), or secondary structural features that inhibited hybridization to the baits (26).

Comparison within a class of vertebrates: chondrichthyan fishes A total of 13 hybridization reactions were carried out [elephant shark (C. milii), five skates and rays (Aetobatus narinari, Leucoraja erinacea, Neotrygon kuhlii, Rhinobatos schlegelii, Torpedo formosa), and seven sharks (Carcharhinus amblyrhynchos, Chlamydoselachus anguineus, Etmopterus joungi, Heterodontus portusjacksoni, Isurus

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