Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

Abstract

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach

“…The capability of DEPArray TM technology to deliver pure cells from a mixed population has been widely described in oncology research [11,15,[17][18][19][20][21] but it clearly holds a great potential also to relieve the problem of forensic mixture.…”

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

“…More recently, LC WGS has also been successfully used to assess CNAs but again mostly using at least 100 ng of input DNA with coverage of 0.1–3× [6, 8, 9]. A very recent study used a complex cell-sorting and single tube Ion Torrent amplicon-based library preparation method to obtain LC WGS CN profiles from 126–300 cells without extracting DNA [8, 9, 20]. To open CN analysis to samples with very limited DNA extracted by standard methods and compatible with the common Illumina sequencing platforms, we evaluated methods to reduce the required DNA input for LC WGS.…”

DEPArray™ system: An automatic image‐based sorter for isolation of pure circulating tumor cells

“…B). DEP cages are able to trap and move cells of different type and size ranging from small sperm cells to large epithelial cells . This electronic structure is integrated within an innovative microfluidic architecture that includes three micro‐chambers in fluidic connection (Fig .…”