Self-digitization chip for single-cell genotyping of cancer-related mutations

Abstract

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis.

Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation

“…Most array-based technologies suffer from sample loss in loading or digitization [38][39][40] . Previously, our device design comprised of parallelized straight channels with perpendicular inlet orientation 36 .…”

“…The HYPER-Melt platform enabled absolute quantification and methylation assessment of heterogeneously-methylated epialleles at fractions as low as 1 in 2 million 36 . While promising, this method, like most array-based techniques, requires considerably more sample volume than is ultimately analyzed, thereby limiting the number of analyses that can be performed on each sample [38][39][40] .…”