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.
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.
The authors declare no competing interests.
Address correspondence to Gavin J P Naylor, Hollings Marine Laboratory, College of Charleston, Charleston, SC, USA. E-mail: [email protected]
1.) Sulem, P., D.F. Gudbjartsson, S.N. Stacey, A. Helgason, T. Rafnar, M. Jakobsdottir, S. Steinberg, S.A. Gudjonsson. 2008. Two newly identified genetic determinants of pigmentation in Europeans. Nat. Genet. 40:835-837. 2.) Ng, S.B., E.H. Turner, P.D. Robertson, S.D. Flygare, A.W. Bigham, C. Lee, T. Shaffer, M. Wong. 2009. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272-276. 3.) Jones, F.C., M.G. Grabherr, Y.F. Chan, P. Russell, E. Mauceli, J. Johnson, R. Swofford, M. Pirun. 2012. The genomic basis of adaptive evolution in threespine sticklebacks. Nature 484:55-61. 4.) Vasta, V., S.B. Ng, E.H. Turner, J. Shendure, and S.H. Hahn. 2009. Next generation sequence analysis for mitochondrial disorders. Genome Med. 1:100. 5.) Hofreiter, M., and T. Schoneberg. 2010. The genetic and evolutionary basis of colour variation in vertebrates. Cell. Mol. Life Sci. 67:2591-2603. 6.) Kalujnaia, S., I.S. McWilliam, V.A. Zaguinaiko, A.L. Feilen, J. Nicholson, N. Hazon, C.P. Cutler, R.J. Balment. 2007. Salinity adaptation and gene profiling analysis in the European eel (Anguilla anguilla) using microarray technology. Gen. Comp. Endocrinol. 152:274-280. 7.) Hickey, A.J., G.M. Renshaw, B. Speers-Roesch, J.G. Richards, Y. Wang, A.P. Farrell, and C.J. Brauner. 2012. A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (Hemiscyllum ocellatum). J. Comp. Physiol. B 182:91-100. 8.) Hodges, E., Z. Xuan, V. Balija, M. Kramer, M.N. Molla, S.W. Smith, C.M. Middle, M.J. Rodesch. 2007. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 39:1522-1527. 9.) Gnirke, A., A. Melnikov, J. Maguire, P. Rogov, E.M. LeProust, W. Brockman, T. Fennell, G. Giannoukos. 2009. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27:182-189. 10.) Mason, V.C., G. Li, K.M. Helgen, and W.J. Murphy. 2011. Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens. Genome Res. 21:1695-1704. 11.) Cosart, T., A. Beja-Pereira, S. Chen, S.B. Ng, J. Shendure, and G. Luikart. 2011. Exome-wide DNA capture and next generation sequencing in domestic and wild species. BMC Genomics 12:347. 12.) Bi, K., D. Vanderpool, S. Singhal, T. Linderoth, C. Moritz, and J.M. Good. 2012. Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales. BMC Genomics 13:403. 13.) Hancock-Hanser, B.L., A. Frey, M.S. Leslie, P.H. Dutton, F.I. Archer, and P.A. Morin. 2013. Targeted multiplex next-generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics. Mol Ecol Resour 13:254-268. 14.) McCormack, J.E., B.C. Faircloth, N.G. Crawford, P.A. Gowaty, R.T. Brumfield, and T.C. Glenn. 2012. Ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis. Genome Res. 22:746-754. 15.) Lemmon, A.R., S.A. Emme, and E.M. Lemmon. 2012. Anchored hybrid enrichment for massively high-throughput phylogenomics. Syst. Biol. 61:727-744. 16.) Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 17.) Altschul, S.F., W. Gish, W. Miller, E. Myers, and D. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. 18.) Li, C., J.J. Riethoven, and G.J.P. Naylor. 2012. EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies. Mol Ecol Resour. 12:967-971. 19.) Inoue, J.G., M. Miya, K. Lam, B.H. Tay, J.A. Danks, J. Bell, T.I. Walker, and B. Venkatesh. 2010. Evolutionary Origin and Phylogeny of the Modern Holocephalans (Chondrichthyes: Chimaeriformes): A Mitogenomic Perspective. Mol Biol Evol.. 20.) Martin, M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17:10-12. 21.) Simpson, J.T., K. Wong, S.D. Jackman, J.E. Schein, S.J. Jones, and I. Birol. 2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19:1117-1123. 22.) Ebersberger, I., S. Strauss, and A. von Haeseler. 2009. HaMStR: profile hidden markov model based search for orthologs in ESTs. BMC Evol. Biol. 9:157. 23.) Avise, J.C., J.C. Patton, and C.F. Aquadro. 1980. Evolutionary genetics of birds. Comparative molecular evolution in New World warblers and rodents. J. Hered. 71:303-310. 24.) Straus, N.A. 1971. Comparative DNA renaturation kinetics in amphibians. Proc. Natl. Acad. Sci. USA 68:799-802. 25.) Fu, Y., N.M. Springer, D.J. Gerhardt, K. Ying, C.T. Yeh, W. Wu, R. Swanson-Wagner, M. D'Ascenzo. 2010. Repeat subtraction-mediated sequence capture from a complex genome. Plant J. 62:898-909. 26.) Lane, S., J. Evermann, F. Loge, and D.R. Call. 2004. Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays. Biosens. Bioelectron. 20:728-735. 27.) Crawford, N.G., B.C. Faircloth, J.E. McCormack, R.T. Brumfield, K. Winker, and T.C. Glenn. 2012. More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs. Biol. Lett. 8:783-786. 28.) Faircloth, B.C., J.E. McCormack, N.G. Crawford, M.G. Harvey, R.T. Brumfield, and T.C. Glenn. 2012. Ultraconserved Elements Anchor Thousands of Genetic Markers Spanning Multiple Evolutionary Timescales. Systematic biology.. 29.) Hedges, S.B., and S. Kumar. 2009. The TimeTree of Life. Oxford University Press, New York.