Virtual microfluidics : a novel single-cell technology based on diffusion-restricted reaction that makes high-quality low-input genomic research accessible

Abstract

The extensive genomic diversity of complex systems, such as the human gut microbiome and the evolution of human cancer, has been revealed with advances in DNA sequencing. But we are still at an early stage in understanding this genomic diversity to expand our knowledge in biology and for biomedical applications. Taking the diverse human gut microbiome as an example, little is known about the rapid exchange of antibiotic resistance genes and virulence factors as part of the mobile gene flow between the microbes in the gut. Understanding such heterogeneous systems often involves studying the nature and behavior of the individual cells that constitute the system and their interactions. However, it is technically challenging to probe the genomic material of cells, the smallest unit of life and amplify single genomes for sequencing. Current single-cell technologies require complex instrumentation and the data quality is often confounded by biased genome coverage and chimera artifacts. We address these challenges with a new single-cell technology paradigm to make high-quality low-input genomic research accessible to scientists. We developed hydrogel-based virtual microfluidics as a simple and robust platform for the compartmentalization of nucleic acid amplification reactions. We applied whole genome amplification (WGA) to purified DNA molecules, cultured bacterial cells, human gut microbiome samples, and human cell lines in the virtual microfluidics system. We demonstrated whole-genome sequencing of single-cell WGA products with excellent coverage uniformity and markedly reduced chimerism compared with traditional methods. Additionally, we applied single-cell sequencing to identify horizontally transferred genes between the microbes in the gut and revealed human population activities' selective pressure in shaping the mobile gene pools. Altogether, we expect virtual microfluidics will find application as a low-cost digital assay platform and as a high-throughput platform for single-cell sample preparation. This work offers a significant improvement in making high-quality low-input genomic research accessible to scientists in microbiology and oncology.