Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Halloween has come and gone, and Thanksgiving is on its way. The malls have had their Christmas decorations up for months. That can mean only one thing: It’s time to round up what’s new in the next-gen DNA sequencing space.
Sequencing instrumentation hasn’t changed since we last looked at the market in February 2014, but that’s not to say all is as it was. New sample-preparation instruments have arrived on the scene, in addition to new, ever-longer sequencing chemistries.
454 Life Sciences
As we noted in February, 454 Life Sciences announced in 2013 plans to discontinue sales of its sequencing hardware in 2015. The company will support its hardware through the end of 2016, says Susan Ulanowicz, sequencing marketing manager at Roche Diagnostics—not mid-2016, as previously reported.
Roche NimbleGen is still developing NGS sample-preparation tools, however. In January, the company launched the SeqCap Epi target-enrichment system, for capture of potentially methylated genomic DNA. More recently, NimbleGen launched its SeqCap RNA Enrichment System, which uses oligonucleotides to enrich for specific transcriptome subsets.
According to Ji Wu, international marketing director at Roche NimbleGen, the SeqCap RNA Enrichment System is available in three flavors: an lncRNA design “which captures 17.35 Mb representing 32,808 lncRNA and TUCP [transcripts of uncertain coding potential] transcript isoforms,” and two custom designs for capturing 7 Mb to 200 Mb material from as little as 10 ng total RNA.
“SeqCap RNA allows researchers to significantly improve the performance of RNA-Seq by focusing on the transcripts of interest with increased depth, sensitivity and accuracy,” says Wu. “This lowers the cost of sequencing and increases the power to discover and characterize novel exons, splicing variants, mutations and gene fusions.”
Illumina
Illumina has launched several new and updated sequencing chemistries in the past year, says Joel Fellis, senior manager, product marketing, sequencing systems and genomics services.
The HiSeq Rapid v2 Reagent Kit, shipping this month, will increase read lengths on the HiSeq 2500 to 2 x 250 bp (paired-end) in “rapid run mode,” providing some 300 Gb in 60 hours, according to a press release from the company.
The HiSeq X HD v2 Reagent Kit, also slated for November release, enables researchers to prepare sequencing libraries with a faster protocol and without PCR amplification, which can introduce biases, Fellis says. “For the best genome [assembly], we recommend the [TruSeq DNA] PCR-Free kit,” Fellis says, “because it offers superior coverage of challenging regions, such as those that are heavy in GC-rich content.”
In July, Illumina released its TruSeq Synthetic Long-Read DNA Library Prep Kit, a commercial implementation of the Moleculo technology the company acquired in 2012, which previously was available only as a service offering. As Fellis explains, the kit provides a way to computationally generate long-read data from Illumina’s relatively short-read technology. “It takes short reads and stitches them together to retain the long-read information to construct reads up to about 10 kb in length,” he says—information that can be useful for both chromosomal “phasing” questions and de novo assemblies.
Though it hasn't yet been upgraded, Illumina’s NextSeq 500 hasn't been overlooked; the instrument is slated to receive a chemistry upgrade sometime in the first half of 2015, Fellis says. Presented at the recent American Society for Human Genetics meeting, the new chemistry brings HiSeq-level accuracy to NextSeq 500’s more budget-conscious consumers. “You’ll be getting more accurate data than with the previous chemistry,” he says, “taking what we would argue is already best-in-class performance and making it more accurate.”
Ion Torrent
The Ion Torrent Ion PGM™ and Ion Proton™ instruments, now offered by Thermo Fisher Scientific, also have benefitted from recent chemistry upgrades.
Thermo released its new Hi-Q™ DNA polymerase for NGS in Q3, promising substantially reduced indel error rates, says Andy Felton, head of product management for instrumentation and reagents in the Ion Torrent Group at Thermo Fisher Scientific.
“In de novo assembly on small bacterial genomes, we see a 94% reduction in indel errors for S. aureus, whereas we see just under 80% for E. coli. For gene panels, it depends on the panel,” Felton says. In one case, involving a 2,300-target PCR amplicon-based resequencing panel, “we saw an approximately 43% reduction in indel error rate.”
Hi-Q has already launched for Ion PGM; an Ion Proton-ready formulation should launch in the first half of 2015.
Another new release from Thermo is the company’s Ion AmpliSeq™ Transcriptome Human Gene Expression Kit, a PCR-based sequence-enrichment tool that can capture and sequence amplicons representing more than 20,000 human transcripts per reaction, about one per gene. The design is intended mainly for transcript counting rather than isoform analysis, Felton notes. “We think that’s going to be a great way to measure gene-expression changes in cancer samples because of the ability to start with just 10 ng of FFPE DNA.”
In March, Thermo launched the Ion Chef™, a “fully automated” instrument that automates both clonal amplification and chip-loading steps for both PGM and Proton, Felton says. A new PII chip for Proton remains in development, he adds, with an “expected output of 200 to 300 million” 100-bp reads primarily for counting applications such as RNA-seq.
Oxford Nanopore Technologies
Users finally got their hands on Oxford Nanopore’s USB key-sized MinION™ sequencer this year, thanks to the MinION Access Program (MAP); some have already published their findings. Alexander Mikheyev and Mandy Tin, at the Okinawa Institute of Science and Technology, published in August an unflattering “first look at the Oxford Nanopore MinION sequencer,” claiming, among other things, low throughput and a high error rate.
Oxford Nanopore declined to speak with Biocompare. But writing at his blog Omics! Omics! computational biologist Keith Robison in September called out the analysis’ “spectacular flaws.”
“Because Mikheyev & Tin failed to explore a wide range of parameters, or to pay attention to the MAP community whilst they were rushing off their manuscript, and as a result they've generated a grotesquely flawed analysis. [sic] It would certainly be the honorable approach for the authors to retract this embarrassment before it is actually printed,” he wrote.
In the meantime, Oxford Nanopore's MAP continues. And Oxford Nanopore has expanded its instrumentation lineup. Joining the miniature MinION and scalable GridION is PromethION, “a tablet-sized benchtop instrument designed to run a small number of samples, possibly even one, on a very large number of nanopores or, instead, to run multiple samples in parallel with thousands of nanopore-sensing channels each,” according to the company’s website. (Neither GridION nor PromethION is yet available to customers.)
Pacific Biosciences
Last month Pacific Biosciences, which already led the NGS field in average read length (approximately 8,000 bases apiece), announced its newest chemistry. Called P6-C4 (for sixth-generation polymerase, fourth-generation chemistry), the new reagents promise single-molecule read lengths averaging 10,000 to 15,000 bases each, with some extending out to 40,000 bases.
“The throughput with the new chemistry is expected to be between 500 million to 1 billion bases per SMRT® Cell, depending on the sample being sequenced,” according to a company news release. (Pacific Biosciences declined to speak with Biocompare.) “By providing an increasing number of longer reads per instrument run, the new chemistry enables users to assemble genomes to a higher quality.”
PacBio’s long-read technology is proving popular for such applications as mapping transcript structure and infectious-disease monitoring. But at least one recent study has demonstrated the technology’s compatibility with de novo genome assembly, as well, mapping the genomes of five species from E. coli to man.
Third-party tools
Sequencing vendors are not the only ones making new tools for NGS.
Swiss automation firm Tecan, for instance, announced in September an NGS-focused automation system called the Freedom EVO® NGS workstation, including "qualified" protocols for Illumina and Ion Torrent libraries. "The Freedom EVO NGS workstation has been designed to offer robust and reliable automation—including library preparation, quantification, qPCR set-up, normalization, pooling and capture—even for inexperienced users," the company says.
Agilent Technologies, which markets sequence-enrichment kits under the SureSelect brand, announced in June its new SureSelectQXT kit, "a revolutionary next-generation sequencing target enrichment solution that produces sample-to-sequencing-ready-libraries in just seven hours with only 50 ng of input gDNA," according to a company press release.
Next-gen sequencing is reaching an ever-wider audience of life scientists, Illumina's Fellis notes. But the appetite for whole-genome human sequencing in particular, is "seemingly insatiable." In the company's last earning call, Illumina CEO Jay Flatley announced that, "We now have 15 customers [for the flagship HiSeq X Ten] who have ordered a total of 164 systems since the product launch in January." Clearly, the need for tools to drive those instruments is growing, as well.
Bottom line: Don't be surprised if there are yet more developments to announce next year, too.