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

Achieving high-throughput and cost-effective next-generation sequencing requires sample (samples, cells/beads) pooling. A key approach to pooling samples while maintaining unambiguous sample identification is to employ DNA barcoding, which assigns each specimen a unique nucleotide (index) sequence. For barcoding in droplets and microwells, one widely used approach is to randomly seed a unique oligo-coated bead into each compartment. Synthesis of oligonucleotide-coated beads (e.g., split-pooling) is exceptionally intensive and random seeding can result in errors. 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, the oligonucleotide solutions are designed using a Combinatorial Dual Indexing (i5, i7) scheme with the deterministic loading. Deterministic loading of the solutions is achieved by sequentially mating two bifurcated and complementary microchannel-network layers to the microwell array. Vacuum-assisted flow and dead-end channel design yields uniform barcode patterning in the microwell array (∼20% CV), reasonable barcode loading times (30 - 40 min per one step), and reduced reagent use (∼ 8-16 µL at 25 µM oligos vs. 10-50 µL at 100 µM for bead systems). Cross-contamination occurred in ∼4% of the microwells, well within the acceptable range. 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, with off-chip quality control of the amplified breast cancer DNA. Overall, we describe a deterministic and bead-free DNA barcoding strategy for efficient barcoding of widely used microwell arrays.