speCiaL reviews in ornitHoLogy
e Polonator G. also uses ligation for sequencing, but
a single base rather than two bases is interrogated per ligation attempt (Dressman et al. , Shendure et al. ). e main benefit of Polony sequencing by ligation is that the cost of the ma- chine and recurrent reagent costs are low. Furthermore, all asso- ciated protocols and software are open-source, which maximizes flexibility.
One single-molecule sequencing system is currently available commercially: the Helicos Genetic Analysis System, which uses technology developed by Braslavsky et al. (). Sequencing features are prepared for the Helicos library by the simple ad- dition of poly-A tails to DNA templates, with no amplification of the template necessary. e template DNA is then hybridized to poly-T oligos anchored to a slide. Using a sequence-by-syn- thesis method (as described above) with reversible fluorescent dNTP terminators, the sequence of single-molecule templates is determined. By ligation of both a poly-A tail at the ′ end and an adaptor to the ′ end of the template, a template can be read twice, in this manner: after sequencing from the poly-A tail is completed, one strand can be removed by denaturation and the DNA can subsequently be sequenced from the ′ end using the adaptor sequence as a priming site for initiation of synthesis. is bidirectional sequencing greatly improves ac- curacy (from –% deletion error rate with one pass to .–% with two passes, and a raw substitution rate of ∼.% for two passes). e simplicity of library preparation and complete in- dependence from PCR or cloning (and related errors and bi- ases; Kanagawa , Acinas et al. ) make this a highly attractive option (see section on ancient DNA below). Single- molecule sequencing and faster methods of massively parallel sequencing are an active area of research and development (e.g., Oxford Nanopore and Pacific Biosciences).
e speed of the five previously described NGSMs is limited
because each addition of a different nucleotide species (G, T, A, or C) is necessarily separated in time by several steps, particu- larly nucleotide detection, washing away the previous nucleotide species, and introducing a new species for incorporation. For in- stance, a single cycle (i.e., steps from one nucleotide incorpora- tion to the next) requires – h, depending on the platform (with the exception of Roche at s cycle–). A single molecule- sequencing method under development by Pacific Biosciences (Eid et al. ), SMRT observes the real-time sequencing of a single template by using a chip of thousands of nanometer-scale cham- bers (zero-mode waveguides; Levene et al. ), each with a sin- gle anchored polymerase molecule. When the DNA polymerase incorporates nucleotides with phosphate-linked fluorescent la- bels, a window at the bottom of the zero-mode waveguide is used to detect the fluorescence signal. As polymerase incorporates the nucleotide, it naturally cleaves the phosphate group (including the phosphate-linked fluorescent label), and through random diffu- sion the next nucleotide in the sequence becomes available to the polymerase. Because all four nucleotide species are present at any given time, this method is limited only by the polymerase’s rate of incorporation and the machine’s speed of detection. By using circularized templates and a strand-displacing enzyme, multiple independent reads can be obtained from each DNA template to
improve the sequencing accuracy to the desired level. Read lengths using the SMRT technology will also not be as limited as in other methods, currently averaging bp and reaching , bp in some cases. Such long read lengths make this a particularly prom- ising method for de novo sequencing projects. Test platforms that employ the SMRT technology will be available for preselected test laboratories in January , with commercial availability depen- dent on their success.
Multispecies, Multilocus studies (Multiplexing)
Next-generation sequencing methods were, for the most part, originally designed with funding from genome-sequencing initia- tives. us, runs on most platforms can be subdivided only to a limited degree (– divisions; Table ), which allows only a few individuals (or otherwise unique samples) to be sequenced in a single run. Most ecological and evolutionary studies require ho- mologous sequence data, sometimes from multiple genomic re- gions and often for many individuals. High levels of sequence divergence among samples could make it possible to separate sam- ples simply on the basis of the resulting sequence alignments (Pol- lock et al. ). For individuals of the same or a closely related species, however, samples are better distinguished by attaching a unique sequence-based tag to each sample’s template before pre- paring the sequencing library. Both PCR-based and ligation-based techniques of tag attachment have been developed by indepen- dent researchers (Binladen et al. ; Meyer et al. , ), and some additional platform-specific barcodes exist (e.g., bar- codes for the SOLiD Plus System; Table ). Tags can be attached either to short pieces of genomic DNA or to PCR amplicons and then pooled in equimolar ratios for preparation of the sequenc- ing library. e pooling of tagged templates, called “multiplexing,” can be done at several levels in a single library. For instance, multi- ple PCR amplicons of different genomic regions from the same in- dividual can be labeled with the same tag, pooled in an equimolar ratio, and then pooled again with similar libraries that bear other unique tags. Combining tags with subdivided runs can vastly in- crease the number of unique samples processed in a single run to hundreds or thousands.
Targeted resequencing, also called “genome partitioning” or “DNA capture,” is a revolutionary way to isolate large amounts of homologous sequence data from genomic DNA for downstream sequencing applications (Hodges et al. , Summerer et al.
). Two primary methods exist, both based on hybridization
to sequence-specific probes either in solution (Agilent and Invit- rogen) or on a microarray. Such methods are particularly appro- priate for studies using ancient DNA and fecal material, in which background or contaminating DNA would compete with target DNA in next-generation sequencing libraries, greatly reducing the number of useful sequences obtained (e.g., Briggs et al. ). At present, these methods are applicable only to single-species stud- ies and have been applied predominantly to human studies, but development of multispecies probe applications is underway in several laboratories. Combining DNA capture with multiplexing followed by next-generation sequencing is likely to transform ge- netic research in the future.