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was able to resolve two divergent clades of wooly mammoth, .–
. million years apart, much like the pattern found for Asian
elephants (Fleischer et al. ). Interestingly, PCR and Sanger se- quencing of mtDNA control regions of mammoths from Eur- asia and North America suggest that this mixed pattern resulted from late Pleistocene recolonization of Eurasia by mammoth lin- eages that originated in North America (Debruyne et al. ). Two of the main obstacles in ancient DNA studies are separating the desired DNA from a high level of background DNA and re- moving PCR inhibitors. As described above, DNA capture meth- ods will be useful for the former, and single-molecule sequencing could obviate the need for PCR entirely.
other creative applications
Several interesting applications of NGSMs have been developed, in addition to those discussed above. One useful method involves assessment of prey items from (noninvasive) fecal material. An ex- citing recent paper by Deagle et al. () used PCR with redun- dant primers to amplify fish and cephalopod S (mitochondrial) and S (nuclear) rDNA sequences from genomic DNA isolated from Australian Fur Seal (Arctocephalus pusillus) feces but used blocking primers to keep the Fur Seal DNA from being amplified.
e products were pyrosequenced using a GS-FLX platform,
and the aligned contigs were compared with databases of fish and cephalopod sequences to identify species of bony fish, spe- cies of cartilaginous fish, and cephalopod species from fecal samples. e coverage, assuming that no bias in amplification oc- curred, should indicate the relative frequency of the prey items in the Australian Fur Seal’s diet. is study identified more diet spe- cies of Australian Fur Seals in a single analysis than were found over multiple years using traditional, extensive hard-parts analysis of samples. Comparing individuals from three distant study sites showed site-specific variation in both type and frequency of prey.
Another recent study (Soininen et al. ) compared the traditional DNA barcoding method and direct pyrosequencing of plant remains from stomach samples of two rodent species but found that the barcoding via PCR outperformed the pyrosequenc- ing in identifying a range of plant taxa. We feel that a tagged, tar- geted approach using PCR or array capture would have greatly increased the resolution in that study. Pyrosequencing methods hold much promise for use in prey assessments for other organ- isms, including birds (e.g., Marrero et al. ).
Next-generation sequencing generates volumes of data that are both a blessing and a curse. e process of sorting quality reads and aligning and analyzing hundreds of thousands or millions of base pairs is both time intensive and computationally intensive. Technology for data acquisition is proceeding faster than infor- mation technology in many cases, and data-processing time may well exceed sample-handling time until new methods of analysis are developed. New programs and updated versions of traditional applications are being released daily to fill this gap. Given the rap- idly changing nature of software programs, we have not attempted to detail them here. Several online resources feature up-to-date lists and descriptions of software packages for analysis of next- generation sequencing data. Two of the currently most useful sites are provided in the Acknowledgments.
Next-generation sequencing methods have been applied at a blinding pace to a wide range of fields in biology and medicine. In addition, NGSMs have changed the scope and speed of standard sequencing methods by several orders of magnitude. Evolutionary biologists, ecologists, and ornithologists have been particularly slow to adopt and adapt these methods to their research programs (except perhaps a few of those involved in ancient DNA stud- ies), but creative uses—such as targeted, tagged pyrosequencing; gene capture; and assessments of gene expression through cDNA coverage—are currently being developed. And the first whole ge- nomes are being produced by NGSMs, with many more proposed for a huge suite of non-model organisms, including a large number and wide sampling of birds (Genome K Community of Scien- tists ). We predict that there will be a major acceleration in the near future in the application of NGSMs to ornithology and that many important findings in avian biology will arise from such studies. It is indeed an exciting time for ornithology.
We thank M. Meyer, M. Hofreiter, F. Hailer, D. Locke, S. Schus- ter, and K. Helgen for elucidation and discussion of NGSMs and the National Science Foundation (DEB-) for funding. O. Ryder and A. R. Hoelzel provided valuable comments on an earlier version of the manuscript. Online resources for analysis of next- generation sequencing data are available at the following websites: seqanswers.com/forums/showthread.php?t=, and http://www. sanger.ac.uk/resources/software/.
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