To get the most precise genomic information on a cell, scientists need to sequence its—and only its—nucleic acids. With single-cell sequencing, a researcher can study a cell’s genome or transcriptome. It doesn’t sound easy, and it’s not, but the payoff in focused molecular information can be unrivaled.
“The most complex part is capture, and preservation of the single cell to minimize damage to the DNA before cell lysis and processing of the DNA for microarray or next-generation sequencing,” says Samuel Rulli, global product manager genomics at QIAGEN. “Depending on the technique being used, damage to the DNA can result in drop-outs and corrupt copy number–variation measurements.” To address this challenge, Rulli explains, QIAGEN “incorporated a cell preservation reagent into its REPLI-g Advanced DNA Single Cell Kit.”
As this article will show, scientists already use single-cell genotyping in many ways. Still, this technology creates some challenges.
Less expensive and more effective
One challenge is cost. “The most costly part of single-cell genotyping is missed data,” according to Rulli. “There is no way to go back and re-analyze a failed NGS library or bad microarray experiment.”
Consequently, scientists want to use top-quality reagents from cell capture through analysis. As an example, Rulli points out: “The customers that we work with appreciate that the QIAGEN REPLI-g Advanced DNA Single Cell Kit has a simple one-tube protocol requiring only a few steps to go from a single cell to enough material for microarrays or a NGS library, saving time and costs.”
At some point, most processes run into a bottleneck, but it’s not just one thing with single-cell sequencing. “Bottlenecks really depend on the application that is being done and how the cells are being obtained,” Rulli explains. “Tissue-disruption techniques can be very labor intensive and take a long time, while collecting a rare cell under a microscope can be a slow process.” He adds, “Most instruments being designed to address single-cell genotyping are now being specially designed to preserve cell viability and structure.”
Adding editing
From the Max Planck Florida Institute for Neuroscience, Hiroki Taniguchi—research group leader for the development and function of inhibitory neural circuits—explains that his research team is using single-cell genotyping to study “molecular mechanisms underlying cortical interneuron wiring using CRISPR/Cas9.” He adds, “We use single-cell genotyping to definitely correlate phenotypes and genotypes.”
Combining this gene-editing technology with single-cell genotyping, Taniguchi’s team “found genes that are necessary for axonal branching and synapse formation of cortical interneurons,” he explains. “Thanks to single-cell genotyping, we were able to find that heterozygous knockout of a certain gene causes a significant phenotype.”1
At this point, Taniguchi believes that his approach can be scaled up considerably. “We don’t think that there is a limit of cell numbers with our genotyping method,” he explains. “We established this method to be able to analyze biochemical and morphological features of the cells and afterwards genotype them.”
Image: Single-cell sequencing can be combined with gene editing, such as CRISPR/Cas9, to correlate changes in genes with resulting phenotypes. Image courtesy of Hiroki Taniguchi.
This approach also works with limited cells to sample. As Taniguchi points out, his method works well even with “a small number of sparse cells in the cortex being analyzed,” and these cells “would not be picked up by methods like [fluorescence-activated cell sorting].” In the article noted, Taniguchi and his colleagues analyzed gene loci one by one. “The number of loci analyzed with our method could be a point of improvement in the future,” he notes.
Delving deeper
The exciting findings from Taniguchi’s lab indicate the new things that can be revealed with single-cell sequencing. Some other groups are also exploring brain-related questions with this sequencing approach.
As an example, one international team of scientists applied single-cell sequencing to the pineal gland.2 This structure, deep inside the brain, makes melatonin, which plays a fundamental role in sleep and other cycles. These scientists reported: “Identification of at least nine distinct cell types in the rat pineal gland has been made possible, allowing identification of the precise cells of origin and expression of transcripts for the first time.”
Beyond looking at structures, scientists can also use single-cell genotyping to learn about behaviors. For instance, Hermona Soreq, professor at The Edmond and Lily Safra Center for Brain Science and the Life Sciences Institute at The Hebrew University of Jerusalem, and her colleagues applied single-cell sequencing to neurons in the central nervous system. Specifically, this team sequenced RNA and applied various analytical techniques. They reported: “We used this pipeline to analyze cortical transcripts of schizophrenia and bipolar disorder patients.”3
Soreq and her colleagues pointed out that these different diseases have similar transcriptional features, but the known sexual dimorphisms have not been explained. “Our method reveals the differences between afflicted men and women and identifies disease-affected pathways of cholinergic transmission and gp130-family neurokine controllers of immune function interlinked by microRNAs,” the scientists noted. “This approach may open additional perspectives for seeking biomarkers and therapeutic targets in other transmitter systems and diseases.”
Single-cell RNA sequencing (scRNA-seq) can actually be applied to all sorts of neuron-related questions. Scientists from Australia, as an example, used it to study sensory neurons that participate in breathing. These scientists wrote: “Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs.”4 These cells—composed of various developmental lineages and phenotypes—must work together for healthy breathing and to fight off respiratory contaminants. These scientists applied scRNA-seq to these sensory neurons to “provide a deeper insight into their molecular profiles.” These results even revealed differences in the ion channels, which are fundamental to the signaling process of neurons.
From breathing to brain-related disorders, single-cell sequencing helps scientists understand and apply the information in the nucleic acids that produce different phenotypes. Plus, gene-editing techniques can be used to modify the DNA and then study the phenotypic impact. To make the most of this technology, though, scientists need to start with healthy, isolated cells, and that’s still not easy to do. But when scientists use single-cell sequencing to tease apart fine differences in neurological disorders and more, a little challenge—even a big one—won’t stop the work.
References
1. Steinecke, A., Kurabayashi, N., Hayano, Y. et al. In vivo single-cell genotyping of mouse cortical neurons transfected with CRISPR/Cas9. Cell Rep. 2019. 28(2):325–331.e4. [PMID: 31291570]
2. Coon, S.L., Fu, C., Hartley, S.W., et al. Single cell aequencing of the pineal gland: the next chapter. Front. Endocrinol. 2019. 10:590. [PMID: 31616371]
3. Lobentanzer, S., Hanin, G., Klein, J., Soreq, H. Integrative transcriptomics reveals sexually dimorphic control of the cholinergic/neurokine interface in schizophrenia and bipolar disorder. Cell Rep. 2019. 29(3):764–777. [PMID: 31618642]
4. Mazzone, S.B., Tian, L. Moe, A.A.K. et al. Transcriptional profiling of individual airway projecting vagal sensory neurons. Mol. Neurobiol. 2019. [Epub ahead of print]. [PMID:31630330]