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Next-generation sequencing (NGS) is a powerful tool, and it’s use is growing in basic research and clinical laboratories. Illumina continues to raise the bar on genomic sequencing, democratizing NGS for virtually any sequencing application. The latest additions to its portfolio, the NextSeq 1000 and 2000, incorporate over seven breakthrough innovations, increasing capacity and throughput in efficient, cost-effective, and easy-to-use systems.
Decreasing sampling time and cost
Arizona State University (ASU) biodesign researcher and assistant professor Efrem Lim, PhD, began studying the human virome long before the arrival of SARS-CoV-2. In response to the global COVID-19 pandemic, the Lim lab has led research efforts to understand and prevent SARS-CoV-2 transmission. This includes NGS studies of SARS-CoV-2 to identify transmission patterns and functional studies to characterize virus mutations in clinical isolates.
“We have state-wide testing sites that send samples to ASU for COVID-19 testing. When positive samples come up in the lab, we sequence the genomes of these viruses to understand the mutations and variants that arise,” Lim said. “The NextSeq 1000 is much more cost effective for us. Compared to the MiSeq™, we are able to get about four times more savings per sample in terms of sequencing. With cost savings, that means we can sequence more cases of COVID-19 for the same budget, and that means the difference between 5,000 cases versus 10,000 cases. It’s a big difference.”
“Different applications require different read depths. The difference between the NextSeq 1000 and the NextSeq 2000 is the throughput and breadth of capabilities of the instruments,” said Sam Hester, senior product marketing manager, Illumina.
The cost and time required to run individual samples are no longer prohibitive restraints. Compared to previous sequencer models, the flow cells are not only smaller but also have greater output.
The NextSeq series has three flow cells for massively paralleled high throughput sequencing: P1 enables up to 100M reads, P2 equals 400M reads, and P3 equals 1.2B reads. The NextSeq 1000 can use both P1 and P2 flow cells while the NextSeq 2000 can run all three, resulting in 3x the output of the 1000.
As an example to illustrate the difference this makes, “on a P2 flow cell, if a project requires 50M reads per sample, eight samples can be analyzed in one run,” explained Hester. “On a P3, if a sample requires 50M reads, 24 samples can be analyzed at once, further decreasing the price per sample, run time, and the reagent footprint for storage and organization.”
Virtually any application
Virtually any sequencing application can be run on the NextSeq systems including shotgun metagenomic sequencing, sequencing-based spatial analyses, and single-cell RNA sequencing (scRNa-seq). In shotgun metagenomic sequencing, thousands of organisms are sequenced in parallel. With the ability to combine many samples in a single sequencing run and obtain high sequence coverage per sample, NGS-based metagenomic sequencing can detect low-abundance members of the microbial community that may be missed or are too expensive to identify using other methods.
scRNA-seq examines the transcriptomes of individual cells, providing a high-resolution view of cell-to-cell variation in complex tissues. These assays enhance the study of cellular function and heterogeneity in time-dependent processes, such as differentiation, proliferation, and tumorigenesis. scRNA-seq enables researchers to resolve transcriptional changes down to individual cell types to inform mechanistic and pathway models.
Magdy Alabady, PhD, is director of the Georgia Genomics and Bioinformatics Core Facility and an associate research scientist, plant biology, at the University of Georgia, Athens. His group uses the NextSeq 2000 for scRNA Seq, scATAC Seq, chromatin biology–related applications, metagenomics, exome sequencing, among other applications.
“I think there is a significant improvement in the sequencing efficiency in my lab and the amount of data, the cost, and efficiency of everything we do with the NextSeq 2000,” said Alabady. “We were all surprised when we saw how easy it is to operate the NextSeq 2000 and how easy it is to start the sequencing run. Having this small cartridge that has all things in it—the fluidics, the reagents, the waste—everything in the same cartridge and you just put the flow cell in it and put it in the machine. I can imagine people who are new to an Illumina sequencer will be pleased to start their experience with the NextSeq 2000.”
Upping the bar
A tremendous amount of technological innovation went into the development of the NextSeq systems. Five major advances include: blue/green two-color channel chemistry, super resolution optics, ultra-high density patterned flow cells, an integrated DRAGEN™ Bio-IT analysis platform, and a compact reagent cartridge to simplify the workflow.
The ultra-high density patterned flow cells contain organized nanowells. The DNA pulled into the nanowell amplifies quickly to fill the entire space, simplifying the imaging process. Instead of random clusters the evenly-spaced nanowells are easier to read, maximizing the capture rate. The improved blue/green two-color channel chemistry reduces the number of images needed, cutting the processing time in half compared to four-color systems.
“To improve tracking, we have super resolution optics that can better differentiate between the organized nanowells and the two colors, specifically resulting in clearer images,” Hester said. “This allows us to pack our flow cells more densely, and to sequence faster and less expensively than with older instruments. This is the strength of the Illumina technology.”
In past-generation sequencers, the fluidic system pulled the reagents out of the cartridge and pumped them through a series of internal tubes to automatically deliver them to the flow cell in the right order and quantity. This approach required wash cycles between runs to reduce cross-contamination risk. The NextSeq cartridges contain all of the reagents and deliver them to the flow cell which complexes to the cartridge. There are no wash cycles; no reagents enter the body of the instrument. “The NextSeq 1000/2000 are dry systems. You can immediately load your next cartridge,” Hester said.
After a run, typically a user would offload the sequencing output file and run it through a separate analysis pipeline. Now, analyses can be made directly on the instrument using the onboard server, computer screen, and DRAGEN Bio-IT platform that includes commonly used pipelines, such as gene expression and single-cell sequencing eliminating a step.
Compactness, ease, and, efficiency
Scribe Therapeutics is building new genetic modification platforms built on CRISPR technologies. The company rapidly generates hundreds of synthetic CRISPR molecules and novel technologies. Their first engineered molecule, X-editing (XE) technology, is built holistically on a novel CRISPR foundation to provide greater activity, specificity, and deliverability for therapeutic uses and for in vivo genetic modifications.
Research specialist Jasmine Amerasekera uses the NextSeq 1000 to perform amplicon sequencing to assess the directed evolution of Scribe’s next-generation CRISPR molecules. “If I could describe the NextSeq 1000 with just one word, I would have to say ‘efficient’. Having something that has that kind of data output and leading-edge technology that can also just sit on a benchtop and be used by anybody is really remarkable,” Amerasekera said.
For research use only. Not for use in diagnostic procedures.
Learn how NextSeq 1000/2000 can benefit your lab.