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“Next-generation” sequencing is a broad term for high-throughput technologies for nucleic acid sequencing. The two platforms employed by the genomics core are Illumina sequencing by synthesis and Pacific Biosciences single molecule real-time sequencing.
The Illumina sequencing platform is based on reversible dye terminators and can generate a massive amount of sequence information per run. It is the method of choice for most major sequencing centers. At full capacity, the HiSeq 2500 can produce >4 billion individual sequences (“reads”) from DNA samples in less than two weeks. It is suited to sequencing whole human genomes or exomes, counting abundance of transcripts in RNA samples and identifying genome-wide epigenetic signatures. Illumina has released a new “personal” sequencer for low-throughput needs. The MiSeq will have a capacity of up to 15M reads per run, and will complete within a day; this machine will be used in the facility for pilot projects and clinical genetic testing.
The Pacific Biosciences platform is exquisitely sensitive; it is able to continuously sequence a single molecule for up to 6 kilobases. The technology is based on a recombinant polymerase and a cutting edge imaging system for visualization of individual base incorporation events during DNA synthesis. The platform is well suited for de-novo sequencing of small genomes and polishing of genomic assemblies for large genomes. Because the platform performs at the speed of the polymerase (2 bases per second), DNA samples are sequenced within hours instead of days. Rapid sequencing is of use when time is of the essence, as in a clinical testing or pathogen outbreak situation. Additionally, this platform allows unprecedented insight into epigenetic organization of genomes beyond CpG methylation. Using the kinetic information gleaned during sequencing, modified bases such 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 8-oxoguanine can be characterized.