Reports
Figure 1. Average identity of the 1242 successfully captured gene sequences when compared with the bait sequences of C. milii.
oxyrinchus, Orectolobus halei, Squatina nebulosa)]. Touchdown gene capture and the double capture protocol were deployed as in the first set of experiments across classes of gnathostome vertebrates. For the cross-species captures, we were able to obtain full sequences for 1004 of the 1449 target sequences in the worst case, and 1351 of 1449 target sequences in the best case (Table 2). We obtained 1449 of 1449 sequences for the positive control (C. milii baits tested against a C. milii library). Tese results show greater homogeneity in the efficacy of capture than was evident in the survey across vertebrate classes, confirming the expectation that the methods would be more consistent when applied to a denser taxonomic sample of more closely related lineages. A small subset of genes that were either not captured for the majority of taxa, or showed some evidence of potential paralogy due to local gene duplication were excluded from our final data set. Overall, more than 90% of the captured
sequences were assigned as orthologs by HaMSTR (22). Te average identity between baits and successfully captured target sequences ranged from 61% to 98% (Figure 1). In total, we obtained 338,822 orthologous base pairs (of 418,475 initially targeted) across the 13 chondrichthyan orders (i.e., 81% of the original target set) with a maximum sequence divergence between bait and target of 39%. Te final chondrichthyan data set included 1242 successfully captured putatively orthol- ogous CDS that ranged in length from 112 to 5091 bp, with 51 CDS being more than 600 bp in length (Figure 2). Capturing homologous genes across
species is not a new idea. However, previous approaches have either (i) focused on bait- target divergences that are much shallower than those used in the current study (12,13), or (ii) targeted highly conserved regions in an effort to extract useful sequence
Vol. 54 | No. 6 | 2013
Figure 2. Length distribution of the captured genes for chondrichthyan fishes.
information from the associated flanking regions that are generally more variable (14,15,27,28). Te method described here is distinct from both of these approaches. It is designed to explicitly target known orthologous protein-coding genes across a range of divergences (up to 39% bait-target dissimilarity) using a single set of probes. Tis has been achieved by maximizing retention of target material by signifi- cantly lowering the hybridization temper- ature (e.g., our touchdown hybridization temperature ended at 50°C in comparison to 60°C used by Mason et al.) (10); by lowering the temperature of the second wash to 45°C compared to 65°C under the standard protocol; and by deploying 2 rounds of enrichment. By way of comparison, the ultracon-
served element (14,27,28) and anchored enrichment methods (15) are strategies to determine sequences in the variable regions that flank known conserved genomic elements. A significant proportion of the sequences that occur in these flanking regions have been found to be noncoding, or of unassigned function. Te rate of evolu- tionary change per site generally increases exponentially across these flanking regions with increasing distance from the conserved ultraconserved element core. Such patterns of rate variation have been reported as an asset by advocates of ultraconserved element methods (15) because they allow for the collection of sequences exhibiting a range of evolutionary rates, which may be helpful for identifying markers relevant to particular phylogenetic questions. However, these same patterns also contribute to uncertainties in orthology assignment, alignment, and data analysis. Te targets of the method described here are well charac- terized, protein-coding orthologs that are easy to align and predisposed to evolu- tionary analysis with little bioinformatic preprocessing.
325 Gene capture technology allows
researchers to target, isolate, and sequence hundreds or thousands of genes of interest from genomic libraries. As powerful as it is, the technique is currently restricted in scope to comparisons within species, among species with limited divergence or by targeting highly conserved fragments across otherwise divergent taxa. Te modifi- cations presented here extend the power of gene capture to comparisons among highly divergent sequences and taxa. This has obvious implications for molecular system- atics and evolutionary genetics. Perhaps more importantly, our approach holds promise for comparative biochemistry, physiology and medicine as it will expand the range of evolutionary comparisons that can be efficiently explored for pre-specified genes associated with particular biochemical pathways, physiological adaptations, and disease conditions.
Acknowledgments
Tis research was funded by the National Science Foundation (DEB award -1132229) to GJPN. CL is also supported by the Leading Academic Discipline Project of the Shanghai Municipal Education Commission, project number: S30701. We thank Johanna L. A. Paijmans for technical assistance in the lab.
Competing interests
The authors declare no competing interests.
References 1. Sulem, P., D.F. Gudbjartsson, S.N. Stacey, A. Helgason, T. Rafnar, M. Jakobsdottir, S. Steinberg, S.A. Gudjonsson, et al. 2008. Two newly identified genetic determinants of pigmentation in Europeans. Nat. Genet. 40:835-837.
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