Next Generation Sequencing
This Review focuses on commercially available technologies from Roche/454, Illumina/Solexa, Life/APG and Helicos BioSciences, the Polonator instrument and the near-term technology of Pacific Biosciences,
Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly, and current NGS platform performance to provide guidance on how these technologies work and how they may be applied to important biological questions.
Sequencing technologies include a number of methods
that are grouped broadly as template preparation,
sequencing and imaging, and data analysis.
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There are two methods used in preparing templates for NGS reactions: clonally amplified templates originating from single DNA molecules, and single DNA molecule templates
Clonally amplified templates. most imaging systems have
not been designed to detect single fluorescent events, so
amplified templates are required. The two most common
methods are emulsion PCR and solid-phase
amplification
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emulsion PCR (emPCR)
solid-phase amplification
Single-molecule templates. Although clonally amplified
methods offer certain advantages over bacterial cloning,
some of the protocols are cumbersome to implement
and require a large amount of genomic DNA material
(3–20 μg).
The preparation of single-molecule templates is more straightforward and requires less starting material (<1 μg). more importantly, these methods do not require PCR, which creates mutations in clonally amplified templates that masquerade as sequence variants. AT-rich and GC-rich target sequences may also show amplification bias in product yield, which results in their underrepresentation in genome alignments and assemblies.
Two different methods are used to attach the template to a solid support :
- Sequencing methods are classified as cyclic reversible termination (CRT), single-nucleotide addition (SNA) and real-time sequencing.