Using droplet based microfluidic technology for DNA sequencing

Droplet generation technology enables researchers to conduct large-scale studies with minimal sample sizes, making DNA sequencing more efficient and cost-effective. In addition, it can also help identify small intracellular variations associated with cancer diagnosis. This article discusses the application of droplet generation in DNA analysis and demonstrates the basic setup required for conducting experiments using droplet microfluidics.

1 Opportunities and Challenges of DNA Analysis

Scientists analyze DNA to identify cancer mutations, characterize human antibodies, and differentiate microorganisms. DNA analysis involves various techniques, including sequencing the genome using known sequence probe arrays or using next-generation sequencing (NGS) platforms [1].

The NGS platform utilizes chip arrays to achieve large-scale parallel analysis, which can simultaneously detect hundreds of millions of molecules, thereby reducing costs and improving efficiency [1].

However, the analytical capability of traditional chip arrays is limited and requires additional fluid handling and reagent mixing systems, which increases the complexity of the experimental process and limits the overall throughput. In order to reduce the sequencing cost of each base, it is usually necessary to process multiple samples in parallel, and although barcode labeling can partially alleviate this problem, it increases the time of the experimental process [1].

2 Microfluidic technology based on droplets

Microfluidic droplet technology utilizes immiscible phases (usually water and oil phases) to generate discrete droplets, and the generation of droplets is precisely controlled at high speed through microfluidic pumps [3]. Its main advantages include:

• Low sample volume requirement

• High degree of automation

• high-throughput

• low cost

·Microfluidic devices can quickly and accurately control fluid volume, improving DNA analysis throughput. For example, droplet microfluidics technology can generate and sort thousands of droplets per second.

Each droplet is an independent reaction chamber, suitable for applications such as digital PCR molecular counting, protein crystallization screening, and gene expression analysis. The number of reactions can increase with the increase of droplet generation rate, and the reduction of droplet volume can reduce reagent consumption, making the experiment more flexible [1].

Overall, this technology can perform hundreds of millions of reactions with minimal reagent consumption, greatly improving experimental efficiency and reducing costs. However, unlike fixed point detection based on chip arrays, droplets flow in microfluidic channels and cannot be labeled with positional information to indicate reaction results, thus requiring additional labeling methods [1].

3 High throughput single-cell genome sequencing

Traditional sequencing techniques are often difficult to detect small variations in the genome, so scientists use single-cell whole genome sequencing to identify copy number variations and single nucleotide variations.

High throughput and scalable single-cell sequencing can be achieved through microfluidic technology. For example, a study used barcode labeling technology to analyze the amplified genomic DNA of individual cancer cells. This method can detect cells with pathogenic mutations during cancer remission and reveal the complex clonal evolution process of acute myeloid leukemia (AML) tumors, which cannot be captured by traditional sequencing methods [3]. Therefore, this technology is expected to improve the accuracy of cancer genome heterogeneity analysis and optimize personalized treatment strategies [3].

4 How to start the experiment?

Droplet microfluidic technology provides a high-throughput and low-cost DNA analysis method. If you wish to conduct relevant experiments, the following are the basic equipment required:

2-3 microfluidic pumps(Or one pump with 2-3 channels, such as a 4U pump) - controls the flow of continuous phase (oil phase) and dispersed phase (water phase). It is recommended to use a 4U pressure pump or 2-3 ExiGo microfluidic injection pumps, where the 4U pressure pump can independently control 4 channels to achieve stable and accurate flow control.

• 2-3 piecesflow sensor——Real time monitoring of the flow rates of oil and water phases to ensure fluid stability.

• microfluidic chip——Generate droplets and ensure the controllability of droplet size.

• Stable channel surface chemical modification——Improve droplet stability.

• surfactant——Stabilize the oil-water interface and enhance the stability of droplets.

• oil phase——Improve droplet stability.

• pipeline——Connect the pump to the microfluidic chip.

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References

1. Abate, Adam R et al. “DNA sequence analysis with droplet-based microfluidics.” Lab on a chip vol. 13,24 (2013): 4864-9. doi:10.1039/c3lc50905b

2. Pellegrino, Maurizio et al. “High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.” Genome research vol. 28,9 (2018): 1345-1352. doi:10.1101/gr.232272.117

3. Sohrabi, S., & Moraveji, M. K. (2020). Droplet microfluidics: Fundamentals and its advanced applications. RSC Advances, 10(46), 27560-27574.