Once a luxury, single-cell sequencing is on its way to becoming routine, thanks to fast-paced technological developments that are giving scientists the tools they need to explore the implications of cellular heterogeneity. Cellular populations do not reflect the behavior of individual cells, and rare individual cells may play critical roles in disease or therapy resistance.
While initially focusing on interrogating DNA or RNA in isolation, sequencing has progressively become a component of multi-omics approaches, in which several layers of the biology are simultaneously captured and analyzed. One of the driving forces behind the emergence of multi-omics approaches is an increasing appreciation of the contribution of multiple regulatory pathways and paradigms and of their relevance to processes that govern development, health, and disease.
Helping single-cell genomics become mainstream
“In several years, single-cell genomics has gone from being a leading-edge technology, accessible to just a few sophisticated labs, to becoming a mainstream technology,” says Tarjei Mikkelsen, PhD, vice president of biology at 10x Genomics. In the few years that have passed since 10x Genomics developed its first gene expression technology, the company has introduced its Chromium system and an array of single-cell assay applications. These include a third-generation single-cell gene expression product and solutions for single-cell immune profiling, protein and antigen mapping, CRISPR screening, epigenomics with single-cell assay for transposase-accessible chromatin (scATAC-Seq), and copy number variation.
“We are continually gathering feedback on what users need beyond what we are providing now,” notes Mikkelsen. “This means we keep pushing the sensitivity of the technology and the ability to scale to larger and larger samples.”
As part of these endeavors, scientists at 10x Genomics are investing significant effort into understanding how to best perform sample preparation and help customers adapt the technology for different applications and sample types. “We see a lot of potential,” Mikkelsen emphasizes, “in continuing the development of pipelines and software tools that allow users to not just generate data but also extract highly valuable insight from it.”
Incorporating single-cell genomics in multi-omics approaches
“The technology is moving toward combining single-cell RNA sequencing data with protein analysis, a biological approach of combining ‘omes’ that researchers call multi-omics,” says Ranga Partha, PhD, global marketing director, single-cell genomics, BD Biosciences. In recent years, single-cell multi-omics has seen rapid adoption in the research community. To simultaneously measure RNA and protein levels in parallel, BD scientists developed BD AbSeq, a solution that uses antibodies coupled to unique oligonucleotides.
“After cells are labeled with the AbSeq antibodies, mRNA and protein information can be captured simultaneously in the single-cell workflow,” says Christina Chang, PhD, supervisor of genomics research at BD. To effectuate single-cell workflows, the company relies on its BD Rhapsody system, which uses microwell technology to partition individual cells with capture beads. The mRNA and antibody-conjugated oligonucleotides (Ab-oligos) in individual microwells are barcoded. After recovery of the oligonucleotide-coated beads, RNA and AbSeq sequencing libraries are generated in parallel using specific assays.
The direct measurement of mRNA and protein levels is critical, particularly for systems in which the two are not correlated to each other, or when mRNA expression is low and difficult to quantitate. BD Rhapsody supports targeted assays, which interrogate several hundreds of genes and are particularly helpful when the genomic target is known.
“We also have a whole transcriptome-based assay,” notes Chang. “It allows a more unbiased analysis at the single-cell level.”
Challenges in single-cell RNA sequencing include sample preparation, sample quality, and workflow synergy issues, which range from single-cell isolation to library preparation and sequencing. “The bioinformatics tools constitute an additional bottleneck for all of us to solve,” says Partha.
“On the more technical side, particularly in the clinical phase, having robust controls between experiments and controls, across platforms, and understanding how to better design experiments in terms of how many samples are enough to make meaningful conclusions, are all important considerations,” adds Chang.
Interrogating millions of cells and pushing the technology
“At this time, the single-cell space is primarily used in discovery, to get a deeper view of the underlying biology of several systems, but what we learn from those experiments will help guide where this will move in the future in terms of clinical utility,” says Jonathan Bell, director of product marketing, Celsee. Celsee’s Genesis System, which incorporates the company’s Celsingle™ Slides, can enable several types of applications to analyze molecular signatures from hundreds of millions of single cells. The Celsingle Slides use a gentle gravity-based approach to capture, isolate, and maintain the viability of single cells in discrete microwells. Then the cells are paired with a unique barcode and lysed to gain access to the mRNA, which is reverse
“This provides opportunities to look at individual cells in a population based on their mRNA expression and examine differences and similarities between subpopulations,” asserts Bell. The approach is scalable and provides a 10-fold increase in cell throughput compared to other technologies.
Another application of the Genesis System is the combination of protein quantification and gene expression analyses in the same single cell across thousands of cells. “That allows scientists to match up those two pieces of information to better characterize cell states,” Bell points out. The need to interrogate the biology of single cells has become an important part of research in several fields, including oncology, stem cell research, and immunology.
In the single-cell space, one of the rate-limiting factors involves finding the balance between scale, performance, and cost. While several droplet-based platforms are available commercially, scaling the number of cells influences the noise in the system in terms of the multiplet rate.
“This leads to the inability to get the data down to the single-cell resolution because droplets are overloaded with multiple cells,” explains Bell. From a practical standpoint, dealing with the scaling issue requires that many reactions be run, even though doing so can lower the cell throughput per reaction and increase overall
For a study to be sufficiently powered, the right number of cells is needed. But gaining the ability to obtain the right number of cells can mean accepting “a pretty high financial burden with the single-cell technologies that exist in the marketplace,” Bell suggests.
Another challenge is that many of the commercial droplet kits need to be used in a very prescribed manner, limiting the options to customize them for applications by end users. “But in science, people like to push technologies, invent new methods, optimize assays, and ask different kinds of questions,” observes Bell. “This is why flexibility, customizability, and openness are key focus areas for Celsee.”
Exploring transcript isoforms in the single-cell space
“Because of the long reads of full-length RNA sequences, the Pacific Biosciences SMRT® (Single Molecule, Real-Time) sequencing platform adds value to characterize and identify the transcriptome structure in single cells,” asserts Jonas Korlach, PhD, chief scientific officer, PacBio. The two main ways in which gene expression is regulated in cells involve gene product abundance and alternative splicing. “While the former has been quite extensively studied with short-read technologies, the latter has been inaccessible so far,” Korlach says. “This is something that has been appreciated by the scientific community in bulk studies, and taking that to the single-cell level will likely lead to important discoveries.”
The PacBio SMRT sequencing technology takes advantage of two developments: zero-mode waveguides and phospholinked nucleotides. Zero-mode waveguides enable a novel optical approach in which light illuminates only the bottom of a well that contains DNA polymerase-template complexes. This approach provides a simple and highly parallel method to study single-molecule dynamics at micromolar concentrations and at a microsecond resolution. Phospholinked nucleotides allow the immobilized complex to be visualized as DNA polymerase synthesizes a new DNA strand.
The PacBio SMRT sequencing technology, which generates up to 160 Gb DNA (with half of the data corresponding to read lengths of up to 50 kb), has >99% consensus accuracy and provides uniform coverage, promising a comprehensive view of genomes, transcriptomes, and epigenomes.
“With regard to the single-cell space, the inability to look at transcript isoforms” represents a gap that needs to be filled, Korlach maintains, adding that “PacBio provides an important solution for filling that gap.”
Another gap that PacBio fills is the interrogation of structural variants. SMRT sequencing can detect new structural variants at a single base-pair resolution at the breakpoints and provides a fivefold higher detection sensitivity compared to other technologies.
“Based on our record, I predict that we’ve seen just the beginning of what sequencing can provide for the community,” Korlach says. Compared to when PacBio first launched the system, the throughput has increased by over 10,000-fold. “This makes the technology suitable for larger projects, larger genomes, and many more samples,” Korlach concludes. “There will be many more solutions and improvements to develop the technology and applications further.”
Mapping chromatin accessibility genome-wide
“A couple of years ago, we entered the single-cell market with an RNA sequencing product,” says Carolyn Reifsnyder, director of global marketing, Digital Biology Group, Bio-Rad Laboratories. “This past September, we announced an early access program for scATAC-Seq. We are excited to begin shipping that new product this month.”
ATAC-Seq is an approach that maps chromatin accessibility genome-wide. This is achieved with a transposase that probes DNA accessibility by inserting sequencing adapters directly into open regions of chromatin, allowing those regions to be amplified and sequenced. The new solution from Bio-Rad combines this biochemistry with the company’s ddSEQ Single-Cell Isolator and Droplet Digital technology, to partition thousands of nuclei (or whole cells) into individual nanoliter-sized droplets to facilitate library preparation for ATAC sequencing.
“The droplet approach confers advantages in that it is cost-effective and scalable,” says Reifsnyder. It allows thousands of cells to be processed in parallel, in contrast to plate-based systems that are limited to hundreds of cells per plate. In addition to reagents and instrumentation for single-cell isolation, barcoding, and library generation, the solution also includes a robust bioinformatics pipeline to analyze the data.
“Our product offers users the best sensitivity by providing a high number of unique sequencing fragments that map to the nuclear genome, ATAC peaks, and transcription start sites,” Reifsnyder declares. “We are providing a tool to help researchers achieve better resolution and understand the mechanisms behind gene expression.”
This level of detail will be critical toward helping investigators understand the factors shaping cell differentiation and cell fate. By extrapolating these findings, investigators may identify markers for “response” versus “nonresponse” to therapies, allowing them to derive insights into many diseases.
Single-base resolution for precision medicine
“We provide the only platform that achieves single-base resolution,” asserts Charlie Silver, co-founder and CEO, Mission Bio. “It is particularly useful for studying therapy response and drug resistance in precision medicine.”
The platform, called Tapestri, uses a proprietary droplet microfluidics solution for the fast detection of genomic variability within and across cell populations. “Several components of our technology differentiate us from everyone else in the market,” Silver emphasizes. One of these components is the platform’s underlying chemistry. After the platform partitions cells into individual droplets, it uses a two-protease mix to digest the proteins that would otherwise restrict access to DNA.
“We use a heat-sensitive protease so that, after lysis, we can turn it off to be able to get good downstream chemistry, even though the protease is still there,” explains Silver. After cell lysis and protease digestion, DNA is released for downstream applications. The cell lysates mix with barcoded beads, amplifying the genomic regions of interest. Each product is then tagged with an individual barcode to preserve its cell of origin identity. The two-step droplet workflow enables efficient chemistry on any single-cell analyte without the surface effects that plague plate-based approaches, and because the workflow retains all analytes in droplet it is flexible for all combinations of multi-omics.
“This is very much a translational and clinical research product,” Silver states. “We have been picked up across cancer centers and drug companies to support them through clinical discovery and pipelines.”
When interrogating and attempting to understand the emergence of resistance during therapy, investigators find that identifying the single cells is critical. The Tapestri platform helps explore cellular heterogeneity and clonal evolution in cancers.
“One of the early panels that we built was for acute myeloid leukemia, which is well-characterized at the genetic level,” Silver notes. “But the clonal evolution in leukemia becomes important for disease outcome and for anticipating treatment.”
In a recent study, scientists from Mission Bio and the University of Texas MD Anderson Cancer Center genotyped loci from >16,000 acute myeloid leukemia cancer cells from two patients, identified cells that harbor pathogenic mutations during complete remission, and uncovered clonal evolution events that could not be captured by bulk sequencing. These findings confirmed the previously hypothesized link between heterogeneity and cancer relapse—a discovery that holds the key to improving disease stratification and personalized treatment.
“We have applications and customers across the blood cancer space, but we also have customers in solid tissue,” Silver indicates. “Thanks to our universal protocol for nuclear separation, we can work with any tissue type.”
The sensitivity of Mission Bio’s Tapestri platform also makes it uniquely equipped to monitor measurable residual disease (MRD). The platform’s unique combination of specificity and sensitivity for identifying rare clones provides great confidence in using cells to monitor MRD through treatment.
In late March, Mission Bio announced that it is partnering with LabCorp to focus on clinical trials, drug development, and MRD detection. The precision offered by Mission Bio’s platform has great potential to streamline clinical trials and help providers manage their patients’ care more effectively.
With the power to identify mutation co-occurrence across distinct cell populations, the Tapestri platform also provides an unprecedented level of insight into the consequences of gene editing experiments. “When it comes to gene editing, the devil is in the details, and the stakes are so high that it’s important to characterize the details with great precision,” Silver maintains. “Through the application of single-cell DNA analysis, we can measure and better understand the impact of gene editing experiments across every cell of a sample. As this approach grows more prevalent across oncology, we’ll require such validation tools to bring new drugs to market.”
Human-on-Chip Model Simultaneously Measures Compound Efficacy and Toxicity
A reconfigurable “body-on-a-chip” model could transform drug development by simultaneously measuring compound efficacy and toxicity, for both target cells and other organs, such as the heart and liver, according to scientists. These findings, published in Science Translational Medicine, demonstrate the ability of a body-on-a-chip model to truly revolutionize biomedical research and personalized medicine through more accurate and efficient preclinical testing without the use of animal studies.
Hesperos, in collaboration with Roche and the University of Central Florida (UCF), has shown that one of its multi-organ in vitro model systems is able to realistically replicate in vivo responses to anticancer therapies for both the parent drugs and their metabolites to determine therapeutic index for both single drugs and drug-drug combinations, reports James J. Hickman, PhD, chief scientist at Hesperos and professor at UCF’s NanoScience Technology Center.
The therapeutic index measures relative safety of a drug and the range in which a drug dose is determined to have a therapeutic effect before significant toxicity begins to occur. The initial determination of efficacy at the same time can currently only be done at the preclinical stage in animals, and animal models are not always a good guide to how a drug will perform in humans.
“This is a game changer in the preclinical drug development process, which normally requires an animal model to measure therapeutic index, and in the case of many rare diseases requires testing in humans as there are no animal models available,” said Hickman. “In addition, our system will allow testing of different therapies on small samples of a specific cancer patient’s tissue to help inform doctors about which treatment works best for each individual.”
“With this system, medicinal chemists can test multiple variations of a drug candidate with milligram quantities of the compound, at the pre-animal stage. Normally to go into animals, scale-up needs to occur to manufacture grams to kilograms of a compound, which generally limits animal trials to one candidate because it is expensive,” added Michael Shuler, president and CEO at Hesperos and a professor emeritus at Cornell University.