Next-generation sequencing (NGS) has arrived. “The expansion of users and applications shows no signs of abating,” notes Michael Rhodes, Ph.D., senior manager, SOLiD Sequencing at Applied Biosystems, a division of Life Technologies. “The uptick of transcriptomics applications has been faster than expected. Human genome resequencing projects will increase, and the existing eight published genomes will vastly increase.”
“NGS is an important area right now,” agrees Fred Ernani, Ph.D., product manager at Agilent Technologies. “Scientists look for solid tools and improved workflows. Making tools more efficient is crucial. Many lower throughput systems don’t lend themselves to automation so variability in results needs to be addressed.”
In addition to de novo and resequencing, instrument manufacturers see expansion into amplicon, transcriptome, and metagenomics. Response has been swift; product rollouts start at CHI’s upcoming “Exploring Next Generation Sequencing Conference” to be held later this month and continue through the fourth quarter and into next year.
“This conference is more of a pharmaceutical venue with an eye on clinical research,” says Timothy Harkins, Ph.D., director of marketing at Roche Applied Science.
“We were first to market with 454 sequencing, resulting in an undue amount of scrutiny, as we were first to challenge traditional Sanger technologies,” continues Dr. Harkins. “Ultimately, this was good for the industry, as we were held to a higher standard and thus set the pace. Downside, it slowed adaptation. Our platform is usually the first adopted for new applications.
“What we initially observed was a good snapshot of what happened on the biological level, as you need a lot of sequencing reads to identify transcription site binding events within the genome. As our competition increases the number of short reads for their respective platforms, we see that they are better suited for this specific application.”
By using Roche NimbleGen’s Sequence Capture technologies coupled with 454’s NGS, it is possible to readily sequence over 170,000 exons within the human genome in a single instrument run, adds Dr. Harkins. “We present a series of projects demonstrating performance of combining sequence capture arrays with the Genome Sequencer FLX. These projects include analysis of publicly available reference genomes to assess what coverage models are needed to identify genetic variants within the human exome.” [See the Assay tutorial on page 36 for more information on the combination of these products.]
Dr. Harkins notes that sequencing the whole human genome is neither practical nor feasible for labs outside of the genome centers.
“Sequencing the whole exome provides a practical approach to sequencing the complete protein coding portion of the human genome,” he says. “We have done several projects sequencing the whole exome and shown it to be effective in population studies and disease models in identifying all genetic variants within the coding regions.”
Abizar Lakdawalla, Ph.D., senior product manager, sequencing systems, Illumina, describes the Genome Analyzer as a massively parallel next-generation DNA sequencer. “It reveals information on the genome, epigenome, transcriptome, and the protein-nucleic acid interactome that had not even been imagined before,” he says. “In a few years, personal genomics will be relatively routine.”
Illumina’s sequencing technology relies on attaching randomly fragmented genomic DNA to a planar, optically transparent surface. Attached DNA fragments are extended and bridge amplified to create an ultrahigh density sequencing flow cell with hundreds of millions of clusters, each containing ~1,000 copies of the same template.
This platform uses the bridge-amplification process, eliminating the substantial challenges associated with emulsion PCR, explains Dr. Lakdawalla. These amplified templates are sequenced using a four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. This approach ensures high accuracy and true base-by-base sequencing, reducing sequence-context specific errors and enables sequencing through homopolymers and repetitive sequences, he claims.
“Most of the scientific excitement is derived from the application of the sequencing technology in gene expression, protein-nucleic acid interactions, and genomics. New data, new algorithms, novel insights, a change in our understanding of basic biology; a lot has changed in a short time,” says Dr. Lakdawalla. “True systems biology is now on the horizon. We already see exciting clinical applications—prenatal detection of chromosomal abnormalities at much higher effectiveness and lower cost than any other current technology.”