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January 2010

  • speCiaL reviews in ornitHoLogy

5

Fig. 1. Comparison of traditional capillary sequencing with next-generation sequencing methods. Capillary methods produce a single consensus read from input template in which low peaks on a chromatogram representing sequence variants cannot be distinguished from background noise. Next- generation sequencing methods produce a single sequence read for each input template, allowing the detection of low-copy variants.

are good targets for de novo sequence assembly, given their smaller genome sizes and lower quantity of repetitive DNA in comparison to other tetrapod taxa (Hughes and Piontkivska ). e benefit of generating massive amounts of data may itself pose a problem for some researchers, because bioinformatics support may be nec- essary to parse the data. Finally, a single run or lane may produce vastly more data than is actually necessary for a project, thus raising the cost per required base. erefore, for projects that either require small amounts of sequence (in the range of a few kilobases) or have limited samples or individuals, traditional Sanger sequencing may remain the method of choice.

Clearly, the advantages of NGSMs will outweigh the few drawbacks for many avian studies and study systems. In fact, certain types of questions and large data sets may only be truly approachable using NGSMs. Given the relative simplicity of the laboratory techniques needed to generate next-generation se- quencing libraries, we envision broad application of such methods in ornithology. Here, we provide a gateway for the avian biologist by comparing next-generation sequencing platforms, describing preparatory techniques to make such methods more broadly ap- plicable, and discuss previous and potential applications in several subfields of avian biology.

expensive to buy or maintain for a single-researcher laboratory, with the exception of the Polonator G. and the recently announced

  • 

    Junior, a next-generation bench-top pyrosequencer to be mar-

keted in  for small to medium labs. Sequencing facilities will perform much of the benchwork of sample preparation, which can be tricky depending on the platform, as part of the sequencing ser- vice for a modest fee. To choose the most appropriate platform for a project, some knowledge of the differences among methods is useful. Here, we briefly describe the methods and the unique benefits and drawbacks of each platform (see Table ). ere are several reviews that describe the technologies in greater detail, as well as animated demonstrations on the company websites (Hudson , Mardis

  • 

    , Shendure and Ji ; see Table  for company websites).

Commercial next-generation sequencing methods can be distinguished by the role of PCR in library preparation. Four main platforms are amplification-based: Roche  GS FLX, Illumina Genome Analyzer IIx, ABI SOLiD  Plus System, and Polonator G. (Table ). Two single-molecule sequencing methods (i.e., not PCR-based) are either very recently available or nearly avail- able: Helicos Genetic Analysis System and Pacific Biosciences SMRT technology, respectively (Table ).

amplification-based sequencing

Comparison of next-Generation platforms

Currently, the specifics of the chemistries and sample preparation for NGSMs are largely irrelevant because the machines are too

Next-generation sequencing libraries for amplification-based meth- ods comprise short (– bp) DNA templates called “sequencing features.” Preparing sequencing features requires ligation of plat- form-specific adapters to template DNA (Fig. A–C) followed by

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