Mission Bio’s Tapestri device was originally developed for single-cell DNA sequencing and protein expression, allowing researchers to perform rapid, high-throughput analysis of genetic variations and surface proteins. But to establish clearer links between genotype and phenotype, scientists also need to explore the expression of different genes. With recent upgrades to Tapestri, researchers can now perform comprehensive single-cell sequencing, analysing the genome, transcriptome, and surface protein expression, all in one simple and powerful assay.
The Updated Tapestri Platform: Genotype, Gene Expression, and Beyond
The upgrades to Tapestri were implemented with our customers’ needs in mind. Our recent survey, conducted across the fields of oncology, hematology, and cell and gene therapy, revealed that more than 80% of participants felt that a targeted single-cell DNA + RNA assay would be valuable in their research. Let’s explore how we’ve adapted Tapestri for single-cell sequencing, why incorporating transcript analysis helps link genotype and phenotype, and compare targeted and whole transcriptome sequencing.
How does single-cell sequencing work on the Tapestri platform?
Until now, combined single-cell sequencing of the genome and transcriptome has been challenging; methods for doing so are generally low-throughput and labor-intensive, or lack the required sensitivity. Additionally, DNA and RNA sequencing methods require enzymatic reactions that are not compatible with each other. Tapestri’s multi-omics upgrade involves the incorporation of an in situ reverse transcription step, generating cDNA sequences for the mRNA transcripts present in each cell for downstream DNA and RNA sequencing. Molecular barcodes provide independent readouts of cDNA and genomic DNA sequences from each cell.
Single-cell sequencing analysis: Linking genotype and phenotype
Combining single-cell DNA sequencing and targeted single-cell RNA sequencing is key to understanding heterogeneous populations of cells, such as cell therapies, tumors, and blood samples. These layers of information help establish clear links between genotype and phenotype; while DNA provides the blueprint or outline of a cell’s potential, RNA adds color to the image, revealing which genes are expressed in which cells. With this targeted DNA + RNA analysis, researchers can understand cell populations with unprecedented depth and precision. DNA + protein analysis adds another critical layer, strengthening the link between genotype and phenotype.
Targeted vs. whole transcriptome sequencing
Sequencing the whole transcriptome and genome of individual cells is helpful for discovery research, but is typically low-throughput, comes at a high cost, and leaves researchers with a significant bioinformatics burden. This approach can also fail to detect low-frequency mutations that affect cell behaviour. With the new Tapestri workflow, researchers can perform single-cell sequencing of the genome and transcripts using targeted panels, allowing them to focus on their specific genes of interest. The high sensitivity and accuracy of Tapestri’s single-cell sequencing workflow means that even rare mutations and their effects on expression won’t go unnoticed.
Single-Cell Sequencing Applications with Tapestri
Single-cell sequencing of the genome and transcriptome with Tapestri has a wide range of applications across the fields of oncology, hematology, cell and gene therapy, and more. Let’s take a look at some key applications of the updated Tapestri platform, including how several Mission Bio customers adapted the workflow themselves.
Functional phenotyping of genomic variants with single-cell sequencing
Gene expression can be affected by both coding and non-coding variants in the genome. By incorporating single-cell RNA sequencing into the Tapestri workflow, researchers from the European Molecular Biology Laboratory (EMBL) and Stanford University School of Medicine were able to study the effects of non-coding variants on gene expression in both induced pluripotent stem cells (iPSCs) and primary B-cell lymphoma samples.
After validating the method in iPSCs using 28 DNA targets and 30 RNA targets, the authors demonstrated that this approach can be expanded to panels of hundreds of targets while retaining high sensitivity. Both coding and non-coding variants could be associated with gene expression changes in single cells with a high level of confidence. Further testing in primary B-cell lymphoma revealed the effects of genetic variants on gene expression and cell differentiation within tumors; cells with higher mutational burdens had enhanced B-cell receptor signaling and increased expression of tumorigenic genes.
This study highlights the power of Tapestri’s single-cell sequencing capabilities in the oncology space; using this multi-omics approach, researchers can better characterize and understand the complex tumor microenvironment and elucidate the effects of coding and non-coding variants on gene expression in tumor samples.
Single-cell sequencing in the study of clonal mosaicism
The combination of single-cell DNA and RNA sequencing can also be applied to the study of clonal mosaicism. As cells gain post-zygotic mutations over time, subpopulations of clones can expand and accumulate; these genetically distinct subpopulations of cells can be associated with normal aging, chronic diseases, and cancer. To better understand clonal mosaicism, scientists need to establish links between genetic mutations and cellular phenotypes by studying the genome and transcripts of individual cells within these heterogeneous subpopulations.
A team of researchers from Weill Cornell Medicine recently used Tapestri to explore clonal mosaicism in phenotypically normal esophageal (PNE) biopsies across a range of ages that had high exposure to tobacco and alcohol. By focusing on a targeted panel of 118 genomic amplicons across six known driver genes, the authors of the study identified that most cells carried at least one genetic mutation, with two particular mutations dominating the clonal landscape.
The team then used 86 RNA targets that act as markers of phenotype to categorize cells and associated these categories with the identified driver mutations. This genotype-to-phenotype mapping approach revealed that clones with specific mutations were biased towards increased cell cycling phenotypes, while others were biased only towards early differentiation. Rare, double-mutant clones were biased towards increased proliferation, supporting their likely role in tumor development.
This study demonstrates how comprehensive single-cell sequencing with Tapestri can help establish strong links between genotype and phenotype in heterogeneous populations of cells with high accuracy, allowing researchers to elucidate mechanisms of disease and tumorigenesis.
Tracking stem cell lineages using single-cell sequencing
During the aging process, we also experience clonal expansions in our blood stem cells, and tracking these different lineages of cells throughout the process of hematopoiesis can help us understand development, aging, and disease. However, there are several obstacles to successfully tracing these lineages in human subjects, and the methods for doing so do not always provide an accurate picture of clonal phenotypes.
To tackle this problem, researchers from the Barcelona Institute of Science and Technology recently used Tapestri to analyze the DNA, RNA, DNA methylation, and protein expression profiles of stem cell clones in blood samples. Incorporating a DNA methylation-sensitive restriction enzyme into the Tapestri workflow to digest unmethylated DNA, the authors were able to keep only methylated CpG sites or ‘epi-mutations’ for sequencing.
They demonstrated that epi-mutations can serve as a clonal barcode, providing information about clonal identity and cell state, using 120 RNA targets to confirm the accuracy of these cell-state annotations. Using hundreds of methylated DNA amplicons, the team created an accurate map of hematopoiesis. The authors also applied this method to other samples, such as mature immune cells, lung tissue, and bone marrow, indicating that Tapestri’s comprehensive single-cell sequencing approach can be used to track cell differentiation across many different sample types.
Cell therapy characterization with single-cell sequencing
Cell therapies, particularly gene-edited cell therapies, are complex biologics that are inherently variable; a batch of genetically engineered cells can contain many subpopulations harbouring different edits, expressing different genes, and secreting different proteins. To maximize therapeutic efficacy, avoid unnecessary risks to patients, and minimize regulatory delays, cell therapy developers need robust assays to characterize their drug product.
Comprehensive single-cell sequencing with Tapestri is now a powerful method for the characterization of cell therapy products, revealing single-cell differences in genotype, immunophenotype, and gene expression, all in one simple assay. Developers of cell therapies can assess editing efficiencies, explore the range of clonotypes in batches of edited and expanded cells, monitor the expression of therapeutically relevant genes, and track changes in drug product fitness over time.
Importantly, the Tapestri platform can link vector copy number with the expression of transgenes or edited genes and therapeutically relevant proteins, and reveal molecular signatures associated with the potency, persistence/engraftment, dysfunction, and exhaustion of cell therapy products. This is particularly relevant in oncology, where T cell exhaustion is a major obstacle.
Using Tapestri for Single-Cell Sequencing of DNA and RNA in Your Own Research
Using the Tapestri single-cell sequencing platform, DNA and RNA sequencing can be combined with protein expression in a single, powerful, high-throughput assay. This allows researchers to correlate genetic mutations with gene expression with high levels of precision and accuracy, and the applications of this multi-omics approach go far beyond the few examples we’ve discussed above.
Using Tapestri, researchers can now analyze mechanisms of therapeutic resistance in oncology applications, trace clonal evolution patterns, refine our understanding of disease mechanisms and classifications, speed up biomarker and drug discovery processes, predict and monitor therapeutic responses, validate gene editing, and identify low-frequency mutations in rare populations of cells, among other applications.
Importantly, Mission Bio offers customizable panels of DNA and RNA targets for single-cell sequencing, allowing researchers to focus on their targets of interest and reduce costs. With high-throughput capability and user-friendly tools for data analysis, single-cell sequencing with Tapestri is ideal for researchers looking to incorporate multi-omics into their own research. To get started, chat with a single-cell sequencing expert today.
