MedGenome Inc. Broadens Single Cell Transcriptome and Epigenome Profiling: From Tissues to single nuclei RNA (snRNA) Sequencing and Data Analysis

Neha Verma, Kushal Suryamohan, Derek Vargas, Ekkirala Chaitanya Ramesh

Single-cell transcriptomics has revolutionized genomics and is now an integral part of therapeutics and diagnostics research. Single cell RNA sequencing (scRNA-seq) enables the analysis of gene expression at single cell resolution via droplet-based cell capture methods that rely on microfluidic instruments such as those developed by 10x genomics. While this technology is now routinely used to study a variety of research areas including the study of cellular development, the identification of cell types and states, the exploration of human disease and the development of stem cell technologies, there are several instances where it is much harder to obtain intact cells (e.g., interconnected neurons, flash frozen or cryopreserved tissues). Furthermore, the logistics and budgetary challenges of obtaining fresh tissues can often lead to significant delays for obtaining research projects. Approaches to isolate cells from such difficult sources such as flow cytometry places the cells under stress, which substantially alters gene expression. In such cases, the use of nuclei for RNA-seq avoids the difficulties involved in obtaining undamaged whole cells. To overcome this limitation, MedGenome has developed a streamlined protocol to isolate nuclei from different sources of cells (fresh and cryopreserved cells). We demonstrate that these isolated nuclei can be used for downstream applications including gene expression profiling and epigenome profiling.

Multiple Platforms for Single Cell Genomics to Enable Biomarker Discovery in Immunotherapy :: SLAS 2020

Ankita Das, Jing Wang, Kayla Lee, Derek Vargas, Gavin Washburn, Niyati Thosani, Vasumathi Kode, Nitin Mandloi, Angelica Lavallee, Amit Chaudhuri and Papia Chakraborty

Single cell genomic approaches can provide valuable insights into the complexity and heterogeneity of the cell types in the context of a tissue or tumor. However, challenges with the preparation of single cell suspensions, good cell viability and efficiently capturing diverse cell types in a mix via appropriate cell capture methods can override the utility of the approaches. With the aim to accommodate a wide range of cellular throughput and single cell genomics assays, at MedGenome, and we have validated and integrated multiple options for single cell library preparation workflows downstream of different cell capture platforms. We have generated proof-of-concept data to show the utility of the different platforms for successfully generating libraries from different workflows. We will present data on our platform agnostic approach to generating single cell gene expression libraries, starting with a range of single cell input types using the Chromium (10X Genomics), the plate-based SMART-Seq and the iCELL8 workflows (Takara Bio). Our results from the validation studies can help to determine the number of cells needed for an approach, and the number of cells and genes per cell recovered from the different approaches, and help researchers determine the single cell approach that will work for their research needs.

Recent studies have suggested that capturing additional information on cellular phenotypes or features can also provide valuable information on cell identities otherwise missed and additional heterogeneity, which in turn facilitate discovery of meaningful biomarkers and understanding of molecular mechanisms of development and disease. To enable for such discovery, we have validated several different feature barcoding solutions, and epigenomic analyses namely the CUT&TAG, and CITE-Seq approaches. We will present data demonstrating the utility of CUT&TAG in studying chromatin accessibility and as well as understanding epigenomic regulation and mechanisms of disease.

Multiple Platforms for Single Cell Genomics to Enable Biomarker Discovery in Immunotherapy :: IO Summit 2019

Ankita Das, Kayla Lee, Derek Vargas, Gavin Washburn, Niyati Thosani, Vasumathi Kode, Nitin Mandloi, Jing Wang, Amit Chaudhuri, and Papia Chakraborty

Single cell genomic approaches can provide valuable insights into the complexity and heterogeneity of the cell types in the context of a tissue or tumor. However, challenges with the preparation of single cell suspensions, good cell viability and efficiently capturing diverse cell types in a mix via appropriate cell capture methods can override the utility of the approaches. We provide validation and application data generated from a range of single cell input types (number of starting cells, viability, and research question) and diverse commercially available platforms (the 10x Genomics Chromium and the FACS based SMART-Seq (Takara Bio). We demonstrate the utility of using the appropriate single cell genomics approach to get relevant information. Recent studies have shown that capturing additional information on cellular phenotypes or features can provide valuable information on cell identities otherwise missed and additional heterogeneity, which in turn facilitates discovery of meaningful biomarkers and understanding of molecular mechanisms of development and disease. To enable for such discovery, we have validated the sC-ATAC seq and CITE-Seq approaches and present data on the utility of those approaches and will present data to highlight the capabilities at MedGenome.

• We present a platform agnostic and flexible approach to utilize single cell profiling of gene expression depending on the research question and the number of cells available.
• We present single cell epigenomic profiling data on immune cells using single cell ATAC seq and show that examining chromatin accessibility can identify cellular identities and mechanisms of response to stimuli.
• We conclude the multi-omic approaches have been validated at MedGenome and can offer it as a service.

Applications of TCR repertoire analysis for biomarker discovery and beyond :: World Pharma Week 2019

Ankita Das, Vasumathi Kode, Kayla Lee, Priyanka Shah, Xiaoshan “Shirley” Shi, Nitin Mandloi, Ravi Gupta, Amit Chaudhuri and Papia Chakraborty

T cell immunity provides significant therapeutic benefit to cancer patients treated with checkpoint inhibitors, however a very small fraction of patients typically respond to checkpoint inhibitors, and a smaller fraction of them have any long-term benefit. This can be attributed to the lack of prognostic and predictive biomarkers. The infiltration levels of CD8 T cell in tumors is often used as a characteristic biomarker and can be correlated with response, but recent studies have shown that examining the functional state of the T cells and other immune cell types in the tumor, and the immunogenic neoantigen burden and the TCR repertoire clonality, might give a more appropriate representation of what might be going on in the tumors and hence can be used as predictive biomarkers for response. At MedGenome we have built a suite of tools that can be utilized to: a) Study the tumor microenvironment using the OncoPeptTUME analysis pipeline, b) Predict and validate the immunogenic neoantigens using OncoPeptVAC & OncoPeptSCRN, and c) A suite of workflows to analyze the TCR repertoire from a wide range of sample types using bulk and single-cell approaches. Here we present the data highlighting the applications of TCR repertoire as a biomarker for immunotherapy and also present our capabilities of providing these solutions as a service.