Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Abstract

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Microfluidic Single‐Cell Omics Analysis

“…A gap was introduced to direct cell‐carried fluid streamlines so that single cells could be isolated along with reasonable trap depth. This type of microdam was widely used for single‐cell in situ analysis, culture, transfer, pairing, and fusion . However, this microarray design was compromised by the low efficiency of cell trapping.…”

“…By optimizing the geometry, packing density and assay time were simultaneously balanced for successful isolation of more cells per unit area . The serpentine‐like structure can also support downstream physical manipulation by releasing cells into another larger chamber or using bypass channel trap . Fatsis‐Kavalopoulos et al proposed a cell assembly generator (CAGE) integrating single‐cell traps and separated clustering chambers (Figure D).…”

“…Perpendicular to the main flow channel, the micropockets were designed asymmetrically to decelerate the flow rate outside the cell‐trapping structures for highly efficient cell capture. Furthermore, the improved flow profile enabled subsequent cell lysis in the sub‐nanoliter cell trap as well as amplification in the outlet wells …”

Single-Cell Omics Analyses Enabled by Microchip Technologies

“…Degenerate-oligonucleotideprimed PCR enabled genome-wide amplification of DNA in single cells, and a multiple annealing and loop-based amplification cycling followed by PCR further improved performance (44,45). Although PCR-based methods have more uniformity compared with MDA, they lack the extensive genome coverage, long DNA product lengths, and sensitive detection of single-nucleotide mutations (44,46). Alternatively, isothermal MDA uses random sequence primers and the high-fidelity strand-displacement properties of the Phi29 polymerase to amplify DNA (46,47).…”

“…Although PCR-based methods have more uniformity compared with MDA, they lack the extensive genome coverage, long DNA product lengths, and sensitive detection of single-nucleotide mutations (44,46). Alternatively, isothermal MDA uses random sequence primers and the high-fidelity strand-displacement properties of the Phi29 polymerase to amplify DNA (46,47). In addition to challenges with uniformity and amplification bias, MDA also experiences nonspecific synthesis from negative primer interactions, such as primer dimers, or DNA contamination that outcompetes the desired template (48)(49)(50).…”