Deterministic DNA barcoding using vacuum-driven loading of free oligonucleotides to microwell arrays

Achieving high throughput in experiments requiring sample indexing depends primarily on precise and reproducible reagent deposition prior to analysis. Contemporary droplet and microwell systems utilize random deposition of oligonucleotide-coated beads into reaction chambers, requiring costly bead synthesis and offering limited control over the final distribution of barcoded beads. As an alternative, we present deterministic, aqueous barcoding of 512 arrayed microwells using a multi-layer, vacuum-driven microfluidic network. To uniquely barcode each of the 512 microwells, we deposit DNA oligonucleotide solutions designed using a Combinatorial Dual Indexing (CDI) (i5, i7) scheme via deterministic loading. Deterministic fluid loading is achieved by sequentially mating two bifurcated, orthogonal microchannel networks to a planar microwell array. The microchannel networks actuate fluid flow through a combination of an applied vacuum force and a dead-end channel design. After loading the oligonucleotide solutions, we observed uniform barcode patterning across the arrays of microwells ([~]20% CV), reasonable barcode loading times (30 - 40 min per step), and reduced reagent use ([~] 8-16 {micro}L at 25 {micro}M oligos vs. 10-50 {micro}L at 100 {micro}M for bead systems). We detected cross-contamination in [~]4% of the microwells. Following DNA barcode delivery, on-chip PCR of nuclear DNA from a breast-cancer cell line having the characteristics of the differentiated mammary epithelium (MCF7) was successfully performed, and off-chip quality control of the amplified breast cancer DNA was completed. Overall, we describe a deterministic and bead-free DNA barcoding strategy for efficient barcoding of microwell arrays that are important in single-cell analyses.