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Fig. 2. A general description of the workflow common to next-generation sequencing methods (with the exception of single molecule sequencing platforms). These methods transform (a) genomic DNA to short pieces through (b) fragmentation, amplification, or DNA capture. (c) Platform-specific adaptors are then ligated in vitro to template sequences (barcodes for multiplexing may also be ligated at this time). (d) Using the adaptors as priming sites for hybridization, templates or fragments are immobilized to a bead (Roche 454 pyrosequencing, SOLiD and Polonator G.007) or a solid surface (Illumina GAIIx). (e) Clonal amplification is the final step in sequencing feature generation (see text section amplification-based sequencing for expla- nation) and is followed by (f) sequence interrogation using chemoluminescence or fluorescence. (g) Sequences are assembled to a scaffold for large genomic regions or to a reference sequence for short amplicons.
a variant of traditional PCR using the adapter sequence as priming sites or hybridization targets (Fig. D–E). Emulsion- or bead-based PCR (Nakano et al. ) is used in pyrosequencing (Roche), SOLiD sequencing, and Polonator G. sequencing, whereas PCR employing primers covalently bonded to a flow cell (i.e., bridge PCR; Adams and Kron ) is used for the Illumina Genome Analyzer IIx. Following amplification, separate but parallel sequencing of each of the millions of single clonally amplified targets is performed on a substrate (Fig. F), for example within a micrometer-sized well on a microtiter plate ( pyrosequencing) or directly on the tile of a flow cell (Illumina Genome Analyzer IIx).
light emission (Ronaghi et al. , Margulies et al. ). e strength of the luciferase signal is proportional to the number of nucleotides incorporated in a template so that regions of sin- gle nucleotide repeats (homopolymers) may be read with a single light pulse. e drawback to the pyrosequencing method is that the light signal reaches an asymptote with increasing length of the single nucleotide repeat region. us, the ability to determine the length of a homopolymer drops off the longer the chain gets, such that homopolymers of bp or longer are not reliably deter- mined and homopolymers of as few as bp can be questionable in our experience.
At this point, the chemistries diverge dramatically, result- ing in either large numbers of short reads (< bp) from Illumina, SOLiD, and the Polonator or smaller amounts of longer reads (– bp) from pyrosequencing. Both Illumina and He- licos use reversible dye-terminators in which a single nucleotide bound to a terminator is added by DNA polymerase and detected in real time by fluorescence, followed by removal of the termi- nator group (Ju et al. , Mitchelson ). is method of “sequencing by synthesis” is continued by the addition of a differ- ent nucleotide (with terminator), and so on for a predetermined number of cycles. Pyrosequencing also uses a sequencing-by- synthesis method, but instead of reversible dye-terminators, pyrophosphate is released during nucleotide incorporation, fu- eling a downstream series of reactions that results in luciferase
Alternatively, sequencing by ligation is used in SOLiD se- quencing (Brenner et al. , Shendure et al. ), where oligos of all possible di-mers (bound as degenerate -mers) are ligated to the single-stranded template DNA and read by fluorescence cor- responding to their unique di-mers. Terminal nucleotides and the fluorescent group are cleaved, which is followed by succes- sive rounds of ligation, detection, and cleavage up to either bp or bp of template DNA (depending on run specifications). e unique dual-base interrogation method of SOLiD sequencing pro- vides very high sequencing accuracy at lower cost than many of the other methods, making it a particularly good platform for SNP discovery. e higher output ( Gbp) from SOLiD at a lower cost per mega base pair (Mbp) also makes this an attractive sequencing method for projects that can suffice with short reads.