Single-Cell Sequencing in Modern Research

Single-cell DNA sequencing (scDNA-seq) has marked a significant evolution in genetic research. 20 years ago, scientists were limited to sequencing small DNA fragments, but now they can sequence and assemble entire genomes from individual cells. These advances in technology have led to an explosion of information, providing unique insights into cellular diversity and disease pathology.

Single-cell sequencing vs bulk sequencing

Ellie Juarez, Ph.D., Business Unit, Manager of NGS Commercial Product Management at Integrated DNA Technologies (IDT), emphasized the transformative nature of this technology. “The advent of multi-omic and single-cell DNA sequencing has unlocked a level of biological resolution that previously was not possible. Unlike traditional sequencing methods—which only arrive at the average of many cells—single-cell sequencing reveals cell population differences, cellular evolutionary relationships, and highlights the heterogeneity of cells in tissues.”

Echoing this sentiment, Renyu Li, Field Application Scientist at Innomics, noted that scDNA-seq, “offers several advantages for studying cellular diversity, identifying rare cell types, and detecting somatic genetic variations or mutations that may be masked in bulk sequencing.” While traditional (i.e., bulk) sequencing maintains its utility, the higher resolution offered by scDNA-seq has allowed for a more detailed analysis of cell-to-cell variability. 

Applications in cancer research

Both cancer research and drug discovery heavily rely on addressing cellular heterogeneity, explained Li. “Single-cell DNA sequencing plays a crucial role in resolving genomic differences between these cells, providing valuable insights into cancer cell evolution and aiding in drug discovery efforts.”

Juarez elaborated on this by stressing that, “human disease often relies on dynamic and complex interactions between a variety of cellular types.” She detailed that this is most exemplified in a tumor ecosystem where crucial transitions occur, including tumor initiation, local expansion, metastasis, and therapeutic resistance.

One of the most noteworthy contributions of scDNA-seq is its ability to reconstruct the evolutionary paths of these tumor cells. This development allows researchers to trace back the mutations that occurred and the paths that different cells took during the progression of cancer. Most importantly, this information deepens our understanding of how tumors begin, evolve, and eventually form metastases. Researchers have applied this methodology to investigate various cancer types, including acute myeloid leukemia¹, breast cancer², and hepatocellular carcinoma³, among many others.

The profound impact of scDNA-seq in cancer research is exemplified by collaborative efforts such as the Human Tumor Atlas Network, an integral component of the National Cancer Institute (NCI) Cancer Moonshot Initiative. Juarez explained that this interdisciplinary group is employing the power of multi-omic longitudinal single-cell sequencing to “identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions” in cancer, with the hope of improving patient outcomes.⁴

Developmental biology

The insights brought about by scDNA-seq in cancer are some of the most well-known, but this technology has also accelerated research in developmental biology. “Single-cell DNA sequencing has proven valuable in the field of reproductive research for studying the development of eggs and sperm, providing crucial insights into infertility,” explained Li. “By analyzing the genomic profiles of individual cells, scDNA-seq has shed light on the genetic changes and regulatory mechanisms that influence reproductive cell development.” This deeper comprehension of reproductive disorders could be instrumental in the creation of more effective treatments.

The application of scDNA-seq in developmental biology has also emerged as a pivotal tool in examining mammalian embryos with chromosomal irregularities, such as aneuploidy.^5,6 In this context, scDNA-seq allows researchers to closely monitor the development of embryos in a similar way to how it tracks the development of tumor cells. This line of research is essential to deepening our understanding of embryogenesis and preimplantation development as embryonic aneuploidy is a major contributor to early embryo loss.

Innovations and the journey ahead

Since its inception, scDNA-seq technology has undergone a continuous series of refinements and enhancements. One of the most apparent improvements is the growing number of cell isolation methods allowing labs of different sizes and budgets to perform their own single-cell research. Other notable changes have been in the improvements to key processes of the library preparations. For example, Juarez noted that IDT's Adaptase™ technology allows researchers to create sequencing libraries from low-input, post-conversion ssDNA, maximizing the capture of DNA molecules in single-cell experiments. This was particularly highlighted in a recent pre-print that showed comprehensive DNA methylation diversity across the entire mouse brain.⁷

Li also described important updates during library preparation steps like Innomics’ implementation of Multiple Displacement Amplification to perform unbiased whole genome amplification. Due to the complexities of the data, scDNA-seq has also resulted in enhancements to many analysis workflows. Li noted that their own bioinformatics pipeline was outfitted to allow for the detection of variations such as SNPs, indels, and CNVs from single-cell samples.

Another significant development in the field of single-cell sequencing is its synergistic integration with other emerging technologies. “Advances in single-cell sequencing from the past decade have paved the way for recent advances in spatial profiling,” noted Juarez. “Coupled together, these technologies allow researchers to investigate multi-omic endpoints down to the cellular level in morphologically intact tissues.” With this technology, researchers are able to explore the cellular interplays between tumor, stromal, and immune cells in the tumor microenvironment. Juarez stated that this type of work will be paramount to advancements in the development of checkpoint inhibitors and other tumor therapies.

In addition to identifying drug targets, Li explained that healthcare providers can also utilize scDNA-seq to monitor the treatment process. “This enables more precise and personalized treatment planning, leading to improved patient outcomes,” explained Li. “Furthermore, single-cell DNA sequencing has immense potential in studying organism development and DNA modifications. Research outcomes in these areas can advance our understanding of tissue regeneration and aging processes, paving the way for future breakthroughs.”

References

1. Morita K, Wang F, Jahn K, et al. Clonal evolution of acute myeloid leukemia revealed by high-throughput single-cell genomics. Nature Communications. 2020;11(1):5327. doi:https://doi.org/10.1038/s41467-020-19119-8

2. Demeulemeester J, Kumar P, Møller, Elen K, et al. Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing. Genome Biology. 2016;17(1):250. doi:https://doi.org/10.1186/s13059-016-1109-7

3. Duan M, Hao J, Cui S, et al. Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing. Cell Research. 2018;28(3):359-373. doi:https://doi.org/10.1038/cr.2018.11

4. Rozenblatt-Rosen O, Regev A, Oberdoerffer P, et al. The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution. Cell. 2020;181(2):236-249. doi:https://doi.org/10.1016/j.cell.2020.03.053

5. Daughtry BL, Rosenkrantz JL, Lazar NH, et al. Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion. Genome Research. 2019;29:367-382. doi:https://doi.org/10.1101/gr.239830.118

6. Brooks KE, Daughtry BL, Davis B, et al. Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development. Development. 2022;149:dev198341. doi:https://doi.org/10.1242/dev.198341

7.Liu H, Zeng Q, Zhou J, et al. Single-cell DNA Methylome and 3D Multiomic Atlas of the Adult Mouse Brain. bioRxiv. Published online January 1, 2023:2023.04.16.536509. doi:https://doi.org/10.1101/2023.04.16.536509