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.

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if requiredaccording to the journal that you are submitting your paper to) *colston1@llnl.gov ABSTRACT. The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10 6 smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of ~18, twenty cycles earlier than 2 commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.