Droplet barcoding for massively parallel single-molecule deep sequencing

Abstract

The ability to accurately sequence long DNA molecules is important across biology, but existing sequencers are limited in read length and accuracy. Here, we demonstrate a method to leverage short-read sequencing to obtain long and accurate reads. Using droplet microfluidics, we isolate, amplify, fragment and barcode single DNA molecules in aqueous picolitre droplets, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read sequencing. We show that this approach can provide accurate sequences of up to 10 kb, allowing us to identify rare mutations below the detection limit of conventional sequencing and directly link them into haplotypes. This barcoding methodology can be a powerful tool in sequencing heterogeneous populations such as viruses.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

“…In Abseq, the usual fluorophore or heavy metal-tagged antibodies are replaced with DNA sequence tags that can be read out at the single-cell level using droplet microfluidic barcoding242526 and DNA sequencing. DNA tags afford a number of valuable advantages for labeling antibodies.…”

Robotic automation of droplet microfluidics

“…Dye-labeled CFPE droplets are generated in a T-junction, paired with reinjected FITC droplets, and coalesced. 32,33 The merged droplets exhibit polydispersity characteristic of electrocoalescence, arising from instability of the emulsion upon collection and merger of too many or few droplets in the electrode region (Fig. 3e).…”

Finding a helix in a haystack: nucleic acid cytometry with droplet microfluidics

“…Barcoding of genomes 44 or transcriptomes 45–48 using droplet microfluidics uniquely tags cellular DNA or RNA with a nucleotide sequence. After sequencing, this tag is used to group reads originating from single cells.…”