Motivated by our mission to prevent disease, advance treatment, and increase patient survival, The Bielas Laboratory pursues a broad-based methodological approach to elucidate the fundamental and clinical implications of nuclear and mitochondrial DNA mutations in the pathogenesis of cancer and age-related disease.
In our laboratory, the majority of projects set out to address long-standing intractable questions in mutation research that have remained unanswered largely due to technical limitations. Thus, our first step toward their resolution typically involves the development of new methods and technologies (featured examples highlighted below) to sensitively measure biological information. The application of these tools to the question at hand, more often than not, reveals new insights into biology that exceed the scope of the hypothesis being tested, driving us down new and exciting pathways of discovery in pursuit of our overarching mission.
As such, while mutagenesis remains at the core of our research program, the focus of the laboratory continues to diversify and expand. Current and ongoing areas of interest include nuclear and mitochondrial genomics, DNA repair, transcriptomics, metabolomics, single cell biology, tumor immunology, cancer therapeutics and diagnostics.
Single Cell Transcriptomics
Single cell RNA-sequencing (scRNA-seq) can be used to dissect transcriptomic heterogeneity that is masked in population-averaged measurements. We validated a fully-integrated and robust droplet-based system that enables 3’ mRNA digital profiling of thousands of single cells in a highly multiplex fashion. We demonstrate the clinical utility of our technology to characterize both immune cell subtypes and genotypes by integrating single cell digital RNA profiling with de novo single nucleotide variant (SNV) calling.
Random Mutation Detection
To permit the measurement of spontaneous and induced nuclear and mitochondrial mutations, we developed the digital Random Mutation Capture assay (dRMC). The dRMC permits the analysis of millions of nucleotides, and can identify one mutant base pair among 109 wild-type base pairs. In our approach, enrichment for mutant mtDNA with restriction endonucleases precedes single molecule amplification, effectively eliminating issues with polymerase fidelity.
Single Molecule Sequencing
Multiple independent studies have documented that the presence and quantity of tumor-infiltrating lymphocytes (TILs) are strongly correlated with increased survival. However, because of methodological factors, the exact effect of TILs on prognosis has remained enigmatic, and inclusion of TILs in standard prognostic panels has been limited. To address this limitation, we introduced a robust digital DNA-based assay, termed QuanTILfy, to count TILs and assess T cell clonality in tissue samples, including tumors.
Digital T-Lymphocyte Counting
Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. We developed a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection.