Bead-free deterministic DNA barcoding using vacuum-driven loading of aqueous oligonucleotides to microwell arrays

Area of Science:

• Biotechnology
• Genomics
• Microfluidics

Background:

• High-throughput experiments require precise sample indexing, often limited by random bead deposition in current droplet and microwell systems.
• Existing bead-based methods are costly due to bead synthesis and offer limited control over barcode distribution.

Purpose of the Study:

• To develop a deterministic, bead-free DNA barcoding strategy for arrayed microwells.
• To enable efficient and reproducible sample indexing for high-throughput single-cell analyses.

Main Methods:

• Utilized a multi-layer, vacuum-driven microfluidic network for deterministic loading of DNA oligonucleotides into 512 arrayed microwells.
• Employed a Combinatorial Dual Indexing (CDI) scheme (i5, i7) for unique barcoding of each microwell.
• Integrated tagmentation and on-chip PCR to generate ATAC-seq libraries.

Main Results:

• Achieved uniform barcode patterning across microwell arrays (∼20% CV) with reduced reagent use (8-16 µL vs. 10-50 µL).
• Demonstrated reasonable barcode loading times (30-40 min per step) and low cross-contamination (∼4%).
• Successfully generated high-quality ATAC-seq libraries from MCF7 breast cancer cell line nuclei.

Conclusions:

• The deterministic, bead-free DNA barcoding strategy offers an efficient alternative for microwell array barcoding.
• This method enhances precision and reproducibility in sample indexing for single-cell genomics applications.
• The platform facilitates high-throughput analyses with reduced reagent consumption and cost.