Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

40Characterizing the transcriptome of individual cells is fundamental to understanding complex 41 biological systems. We describe a droplet-based system that enables 3' mRNA counting of up 56peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 84 RESULTS 86Droplet-based platform enables barcoding of tens of thousands of cells 88The scRNA-seq microfluidics platform builds on the GemCode ® technology, which has 89 been used for genome haplotyping, structural variant analysis and de novo assembly of a 90human genome [10][11][12] . The core of the technology is a Gel bead in Emulsion (GEM). GEM 91 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 5 generation takes place in an 8-channel microfluidic chip that encapsulates single gel beads at ~80% fill rate (Fig. 1a-c). Each gel bead is functionalized with barcoded oligonucleotides that 93 consist of: i) sequencing adapters and primers, ii) a 14bp barcode drawn from ~750,000 94 designed sequences to index GEMs, iii) a 10bp randomer to index molecules (unique molecular 95 identifier, UMI), and iv) an anchored 30bp oligo-dT to prime poly-adenylated RNA transcripts 96 (Fig. 1d). Within each microfluidic channel, ~100,000 GEMs are formed per ~6-min run, 97encapsulating thousands of cells in GEMs. Cells are loaded at a limiting dilution to minimize co- 98occurrence of multiple cells in the same GEM. 100Cell lysis begins immediately after encapsulation. Gel beads automatically dissolve to 101 release their oligonucleotides for reverse transcription of poly-adenylated RNAs. Each cDNA 102 molecule contains a UMI and shared barcode per GEM, and ends with a template switching 103 oligo at the 3' end (Fig. 1e). Next, the emulsion is broken and barcoded cDNA is pooled for 104PCR amplification, using primers complementary to the switch oligos and sequencing adapters. Methods, Fig. 1f). Briefly, 98-nt of Read1s were aligned against the union of human (hg19) 123peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/065912 doi: bioRxiv preprint first posted online 6 Based on the distribution of total UMI counts for each barcode (Online Methods), we 124 estimated that 1,012 GEMs contained cells, of which 482 and 538 contained reads that mapped 125 primarily to the human and mouse transcriptome, respectively (and will be referred to as human 126 and mouse GEMs) (Fig. 2a). >83% of UMI counts were associated with cell barcodes, 127indicating low background of cell-free RNA. Eight cell-containing GEMs had a substantial 128 fraction of human and mouse UMI counts (the UMI count is >1% of each species' UMI...

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

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We have fabricated a low-cost disposable polymerase chain reaction thermal chamber that uses buoyancy forces to move the sample solution between the different temperatures necessary for amplification. Three-dimensional, unsteady finite element modeling and a simpler 1-D steady-state model are used together with digital particle image velocimetry data to characterize the flow within the device. Biological samples have been amplified using this novel thermal chamber. Time for amplification is less than 30 min. More importantly, an analysis of the energy consumption shows significant improvements over current technology.