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Capturing protein-coding genes across highly divergent species
 
Chenhong Li1,3, Michael Hofreiter2, Nicolas Straube3, Shannon Corrigan3, and Gavin J.P. Naylor3
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Supplementary Material


Figure 1.  Average identity of the 1242 successfully captured gene sequences when compared with the bait sequences of C. milii. (Click to enlarge)







Figure 2.  Length distribution of the captured genes for chondrichthyan fishes. (Click to enlarge)




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 information from the associated flanking regions that are generally more variable (14, 15, 27, 28). The 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. This has been achieved by maximizing retention of target material by significantly lowering the hybridization temperature (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 ultraconserved 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. The rate of evolutionary 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. The targets of the method described here are well characterized, protein-coding orthologs that are easy to align and predisposed to evolutionary analysis with little bioinformatic preprocessing.

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. The modifications presented here extend the power of gene capture to comparisons among highly divergent sequences and taxa. This has obvious implications for molecular systematics 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

This 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.

Correspondence
Address correspondence to Gavin J P Naylor, Hollings Marine Laboratory, College of Charleston, Charleston, SC, USA. E-mail: [email protected]

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