Mike May earned an M.S. in biological engineering from the University of Connecticut and a Ph.D. in neurobiology and behavior from Cornell University. He worked as an associate editor at American Scientist, and he is the author of hundreds of articles for clients that include Nature, Science, Scientific American and many others.
Next-generation sequencing (NGS) covers a grab bag of technologies, all of which collect the order of nucleic acids faster than ever. Still, the concept of “fast” in sequencing evolves quickly. In brief, scientists keep expecting more from sequencing, and tool providers keep providing new and improved platforms and related technologies.
In sequencing, the reach of a read is one key consideration. “The use of technology to achieve long-range reads is advancing rapidly, with companies like 10x Genomics making a major difference by enabling scientists to use their standard sequencers while still expanding their ability to see data not possible with standard short-read technology,” says Curtis Knox, global strategic marketing manager at Promega. Knox also points out the value of getting phased reads, which reveal which parent strand provided a specific part of the sequence: “Being able to get phased reads of the DNA in question is critical to truly understanding what’s going on in a genome.”
Other experts in NGS agree on the benefit of longer reads. “The trend in NGS has been moving towards information that spans longer distances/range,” says Jonas Korlach, chief scientific officer at Pacific Biosciences. “Whether it’s long-read SMRT Sequencing, which captures every base in reads averaging 10 to 15 kilobases and longer, or scaffolding technologies like Hi-C or non-sequence-based optical mapping, which give a high-level view of genome structure without base-level resolution, scientists are asking for technologies that more accurately reconstruct complex genomes.”
Longer reads provide more opportunities.
“With information from long reads, scientists are able to assemble highly repetitive, heterozygous and even polyploidy genomes, allowing them to study areas of low recombination, large structural variations and other features previously undetectable,” Korlach explains.
Suggested specs
At the HudsonAlpha Genome Sequencing Center, Jeremy Schmutz, faculty investigator and leader of the informatics group, and colleagues have used various NGS platforms, including ones from 454 Life Sciences (acquired by Roche and later discontinued in 2013), Illumina and Pacific Biosciences. When asked about the key benefits of NGS technology, Schmutz says, “From Illumina platforms, the ability to collect significant overrepresentation of a genome or transcript sequence allows us to apply it to a broad range of genomics applications, and longer read lengths from PacBio are a key benefit for sequencing new organisms.”
Anyone interested in using NGS should consider several features. “Streamlined library preparation, robust data collection and accuracy of collected sequence data are important,” Schmutz says.
Other features to consider depend on how the platform will be used. “For research and RNA sequencing,” says Schmutz, “we are mostly driven by the amount of sequence that can be collected for an assay at a specific price point.” He adds, “For de novo genome sequencing, we are interested in long reads that can be used to assemble genomes.” The sample to be sequenced also matters. “For plants, particularly,” Schmutz explains, “we need reads that span through complex repetitive regions that allow us to construct the reference sequence of species.”
Arenas of application
As NGS platforms provide broader reads more easily, scientists and technologists will put this technology to work in more places. One of those places is medicine.
“The application of NGS in oncology—specifically, liquid-biopsy applications—is moving so fast it’s hard to keep up,” says Knox. “We are just scratching the surface of how we can truly use circulating cell-free DNA and circulating tumor cells to increase our depth of knowledge regarding individual cancers and tailor potential therapies.”
For clinical applications in particular, ease of operation will determine how far and how fast NGS penetrates the industry. In all labs, though, making NGS easier—while retaining its ability to decipher information—will expand its use. That will require new platforms that can do more.
“We are going to see more use of DNA and RNA simultaneously in a single workflow for a comprehensive look at the genome,” says Knox. He points out that you can already use total nucleic acid as the sample with some technologies, including the FusionPlex Kits from ArcherDx and the Oncomine Comprehensive Assay from Thermo Fisher Scientific. “As these strategies gain momentum,” Knox says, “I think you will see more applications like this in the near future.”
Advances in NGS also enable important research applications
“The availability of sequencing technologies, such as SMRT Sequencing that can produce long-read data with uniform coverage across targeted regions or whole genomes, are providing scientists with more complete information for their research,” Korlach explains. “For scientists focused on human population sequencing, this capability has provided structural variation information that can be coupled with existing single nucleotide variation information from short-read technologies.”
Korlach also describes the value of deeper genome dives: “This same capability can also be used to produce full-length genes phased and at high accuracy to provide scientists more insight into their research area, as opposed to only exons or single nucleotide polymorphisms.”
Open advantages
At the University of California, Los Angeles, collaboratory fellow Michael Weinstein says, “I use and assist in NGS analysis all the time.” His group helps labs that have NGS data, but lack the experience in sequence analysis. The ongoing advances in this technology make it difficult for scientists to keep up, even on the terminology. “I’m starting to move towards calling it high-throughput sequencing,” Weinstein says. “It’s been around long enough that it doesn’t feel quite so next-generation anymore.”
When asked about the key benefits of “high-throughput sequencing” for his purposes, Weinstein says that one is “the ability to ask questions of the sequence in a manner we could not before.” He adds, “The old paradigm was very focused sequencing with relatively low sensitivity for detection of sequence variants, which worked well for larger batches of homogenous DNA samples, like plasmid preps or PCR products of genomic DNA. It tended to be more difficult when looking for rare, novel—for example, viral—sequences or underrepresented sequences, such as mosaicism in the genomic DNA.” Higher throughput also provides other benefits, Weinstein says, including “the advantage of looking at more ‘quantum’ sequence data, where we can try to estimate the frequency of a given contig or variant in the sample, sometimes with very high sensitivity.”
As for the features that matter most in a sequencing platform, Weinstein looks for openness. “The more open-sourced that this technology becomes, the more outside developers can write their own analysis tools, the more value and utility this technology gains,” he explains. “I shudder to think what the technology would be like now if the companies had adopted a vertically integrated model where they kept control over the entire process from sample prep to platform to alignment and analysis, and took steps to exclude outside development of methods.”
Spread ahead
New systems and platforms promise even more use of NGS.
“With the launch of the Sequel System last year, the up-front capital and running costs of long-read SMRT Sequencing were significantly reduced,” Korlach says. “We have both chemistry and software releases planned in the next year, which will further increase throughput and reduce cost.” As he explains, “These improvements will open up new applications that were previously cost-challenged, such as routine, high-throughput sequence-based detection of genetic variants in the biomedical, industrial and agricultural research fields, where the additional information provided by PacBio long-read sequencing can provide new biological insights.”
Tomorrow’s targeting may be even more specific. As an example, scientists at Pacific Biosciences are exploring “methods that use the Cas9 endonuclease to perform targeted sequencing of regions of interest without a polymerase chain reaction amplification step,” says Korlach. “This will provide scientists with highly accurate sequence data, with uniform coverage across their region of interest, that is fully phased and includes epigenetic information, such as methylation information.”
NGS arose from advancing technology, and new platforms and methods will continue to drive this approach to analyzing nucleic acids. Scientists already talk about next-NGS, and much more is sure to come.
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