Heterogeneity is defined as the quality or state of being diverse in character or content. Heterogeneity drives our differences as humans in a series of minute variations in our DNA, the basic building block of our cells, resulting in the differences we see in our attributes, appearance, senses and ability to live a long, healthy life.

Throughout our lives, small and seemingly inconsequential changes to our basic genetic blueprint can and do occur. Some of these changes or variants include single nucleotide level mutations while other larger structural changes of chromosomes swapping, duplicating, or losing large sections of genetic material also occur. These modifications then affect the way our cells behave. BioSkryb is enabling the ability to study this process of cellular evolution. In some cases, our cells' evolution is a positive selection to better adapt to our environment. But changes  can have very negative effects, such as the development of diseases such as cancer.

Malignant tumors are highly genetically heterogeneous at the cellular level.  In fact, we actually don’t know how heterogeneous the genomes of tumors are on a cell-to-cell basis. Further, the development of treatment resistance is driven in a large part by this heterogeneity.  Each time these cells are exposed to a new drug, they accumulate additional mutations, resulting in even greater heterogeneity. The net effect of this process is the creation of cells that are capable of growing uncontrollably despite using our most effective treatments available. The solution to this challenge again returns to understanding the genetic changes that allow these lethal cells to survive and continue growing. One of our goals at BioSkryb is to enable highly accurate analyses of these single cells.

Unfortunately, the barrier that has existed to the accurate analysis of genomes from individual cells is the ability to amplify the single molecule of DNA from a single cell. The pioneering efforts from Professor Roger Lasken’s lab at Yale University led the development of Multiple Displacement Amplification or MDA. The method relies on the ability to create long copies of the DNA from our genome with an isothermal amplification reaction. The MDA method opened a completely new era of scientific discovery and remains the market leading method for the amplification of whole genomes to this day. However, MDA is not without drawbacks. In the process of amplification, errors are introduced. Due to the MDA reaction copying long fragments, these errors are then re-copied during the process of amplification.  In addition, the exponential process results in non-uniform amplification of the initial DNA template. These errors and lack of uniformity result in a reduced ability to accurately identify variants in MDA-amplified cells or samples.

To overcome the challenges with current methods, BioSkryb’s co-founder Dr. Charles Gawad, a pediatric oncologist, invented a new method of DNA amplification, termed Primary Template-directed Amplification or PTA. The method makes use of a proprietary set of nucleotides to prevent recopying of the amplification products. The reaction conditions were then modified to allow robust amplification, that drives the primers back to the original template, preventing re-copying of daughter amplicons. By creating shorter fragments, which are not favorably re-copied during the amplification reaction, errors are reduced and genome coverage is dramatically increased as the amplification reaction becomes significantly more uniform.  This novel mechanism of DNA amplification is the core technology of BioSkryb. (See technology video here)

BioSkryb was founded to solve the challenge of accurately calling variants in genomes, including cancer genomes, one cell at a time. This degree of resolution is required to properly delineate many aspects of basic biology. Our early collaborators are trying to answer a range of questions, such as: to what degree does the genome variation effect development in the brain and can we identify the key mutations in rare circulating tumor cells. Others have interest in using the ResolveDNA (formerly SkrybAmp) PTA method to analyze small amounts of input DNA for ancestry and forensics applications.

A particularly important application of the ResolveDNA PTA technology is the ability to differentiate the genomic heterogeneity of hematologic diseases, including malignancies. Leukemias and lymphomas constitute approximately 10% of cancers worldwide and affect both pediatric and adult patients. While great strides have been made in the fight against the leukemias and lymphomas, the fight is far from over. The treatment regimen is complex due to the complexity of the cancers themselves. These therapeutic regimens are prolonged, and designed to eliminate the cancer in stages as the pool of cancer cells contain a vast array of genomic alterations that make them resistant to different drugs.

In many cases of leukemia, the initial treatment offered is effective with only a small number of cancer cells persisting as minimal residual disease or MRD. MRD describes these cells that survive the initial therapeutic cycle. However these resistant cells begin to repopulate the bone marrow where they acquire even more genetic alterations.  By combining Fluorescent Activated Cell Sorting (FACS), we are able to isolate these rare but deadly MRD cells down to a sensitivity of 1:10,000. Although droplet based single-cell platforms have high cell throughput, they cannot enrich for these rare, but important cell types. Furthermore, the ResolveDNA Ultra-low DNA input system is able to detect the minute but critical modifications across the entire genome of each MRD cell. This is also largely not possible today with standard bulk sequencing, as the use of populations of thousands of cells mixed together cannot detect these rare variants or determine if any two variants reside within the same cell.

New Applications/Development Pipeline: 

Our team is passionate about finding innovative solutions to complex biological scientific challenges. The research and development team at BioSkryb is developing a new suite of applications that build on the core technology. Our goal is to provide insights that matter to researchers, clinicians, and patients.

Multi-Omic Analysis 

An exciting derivation of the core PTA technology is the integration of multi-omic approaches to elucidate phenotypic changes driven by the DNA blueprint. Using the novel PTA genome amplification technology, we are able to combine the analysis of additional classes of cellular macromolecules.  One of the most important modalities in single cell and cancer research today is the analysis of the gene expressed within cells. We have recently realized the ability to perform a multi-omic, combining the ability to analyze the entire transcriptome and genome of each cell. This multimodal analysis allows us to create linkages between each cell’s phenotype and genotype. For example, we will have the ability to see the cell type, differentiation state, and expression profile, meaning the single-cell phenotype with the blueprint DNA sequence.

Contact us for more information about the method, to ask about collaborating with our R&D team, or to request a copy of our AGBT 2020 posters on Single-Cell Multi-Omic or Bacterial Analyses.

Bacterial Analysis

We have extended the use of ResolveDNA PTA technology to the more accurate assembly and identification of single cell bacterial genomes. In this application, single bacterial cells are enriched through FACS and then amplified using an optimized version of the ResolveDNA protocol for ultra-low DNA input and single cells. The application allows the assembly of stretches of the DNA genome, or  contigs, that are hundreds of thousands of base pairs long and span up to 97% of the genome. Using this method, we are able to unmask significant diversity of human microbiome samples, enabling the detection of single bacterial species within the sample, as well as differentiating when a sample has two bacteria present. We have also identified a previously unannotated bacterial genus or species. This application provide significant advantages over metagenomic studies where the generation of contigs from a highly mixed population creates significant challenges in assembling intact novel and complete genomes of known and novel bacterial species.

Contact us for more information about the method, to ask about collaborating with our R&D team, or to request a copy of our AGBT 2020 posters on Single-Cell Multi-Omic or Bacterial Analyses.

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