e Auk 127(1):4−15, 2010
e American Ornithologists’ Union, 2010.
Printed in USA.
Special ReviewS in ORnithOlOgy
ProsPects for the Use of Next-GeNeratioN seqUeNciNG
Methods iN orNitholoGy HeatHer r. L. Lerner1 and robert C. FLeisCHer
Center for Conservation and Evolutionary Genetics, National Zoological Park, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012 MRC 5503, Washington, D.C. 20013, USA
N of avian biology, from behavior to con- servation to systematics, have benefited from the application of molecular methods over the past two decades. New technolo- gies of high-throughput DNA se- quencing are sparking a revolution in the life sciences that is sure to fur- ther transform our understanding of avian systems. Next-generation (also called “massively parallel”) sequenc- ing methods were first introduced commercially just years ago (Roche GS FLX; Margulies et al. ), yet their capacity to process millions of sequences in parallel, in contrast to the conventional -capillary capac- ity of Sanger sequencing, has rapidly placed them at the forefront of ge- netic research (Fig. ). Although in- dividual read lengths are currently limited (< base pairs [bp] for most platforms), the depth of coverage per base pair and advanced sequence- assembly software allow sequencing of .– giga base pairs (Gbp). us, in a single run, depending on the plat- form (Table ), it is possible to sequence anywhere from one half to times the size of the chicken genome, or a minimum of , mitochondrial genomes. ese methods extend far beyond simply genome sequencing and have already greatly benefited the field of biology, leading to advances in evolution (Shendure et al. , Toth et al. ), epidemiology (Cox-Foster et al. , Palacios et al. ), phylogenetics (Moore et al. , ), comparative genomics (Romanov et al. ), microbial diversity (Edwards et al. , Sogin et al. , Roesch et al. ), DNA marker dis- covery, and studies of gene function and expression (Morozova and Marra , Varshney et al. ). ese new tools make this an exciting time for ornithol- ogy because vast quantities of genetic data can be obtained affordably and with relative ease. With a little creativity, next- generation sequencing methods (NGSMs) can be used to address new and long-standing questions previ- ously inhibited by technological and financial limitations (e.g., obtaining enough sequence data to resolve phy- logenies for adaptive radiations, study of avian major histocompatibility complex [MHC] while eliminating cloning bias, and whole-genome se- quencing). At the very least, the huge increase in available DNA sequence will enable development of impor- tant markers for population genetic, systematic, behavioral, “evo-devo,” and gene mapping studies. e benefits of NGSMs in com- parison to traditional capillary- based sequencing include () massive amounts of sequence data for a single or multiple individuals in a single run; () low cost per base; () reduction of the role of cloning and polymerase chain reaction (PCR) and, thus, reduced bias in resulting sequences; and () the ability to identify rare variant sequences rather than a single consensus sequence (see Fig. ). ere are three main drawbacks to NGSMs. e main obsta- cle at present, although this is beginning to change, is the relatively short read length for most platforms in comparison to traditional methods. For highly repetitive or complex genomes or genome re- gions, short reads (even at high coverage) may not suffice to gener- ate an alignment. us, de novo sequence assembly is not always possible unless a reference or scaffold sequence of the same species or a very closely related species is available. Avian species, however, “these new tools make this an exciting time for ornithology because vast quantities of genetic data can be obtained affordably and with relative ease. With a little creativity, next-generation sequencing methods can be used to address new and long- standing questions previously inhibited by technological and financial limitations.”
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