Sequencing the genomes of individual cancer cells provides the highest resolution of intratumoral heterogeneity. To enable high throughput single cell DNA-Seq across thousands of individual cells per sample, we developed a droplet-based, automated partitioning technology for whole genome sequencing. We applied this approach on a set of gastric cancer cell lines and a primary gastric tumor. In parallel, we conducted a separate single cell RNA-Seq analysis on these same cancers and used copy number to compare results. This joint study, covering thousands of single cell genomes and transcriptomes, revealed extensive cellular diversity based on distinct copy number changes, numerous subclonal populations and in the case of the primary tumor, subclonal gene expression signatures. We found genomic evidence of positive selection – where the percentage of replicating cells per clone is higher than expected – indicating ongoing tumor evolution. Our study demonstrates that joining single cell genomic DNA and transcriptomic features provides novel insights into cancer heterogeneity and biology. SIGNIFICANCE We conducted a massively parallel DNA sequencing analysis on a set of gastric cancer cell lines and a primary gastric tumor in combination with a joint single cell RNA-Seq analysis. This joint study, covering thousands of single cell genomes and transcriptomes, revealed extensive cellular diversity based on distinct copy number changes, numerous subclonal populations and in the case of the primary tumor, subclonal gene expression signatures. We found genomic evidence of positive selection where the percentage of replicating cells per clone is higher than expected indicating ongoing tumor evolution. Our study demonstrates that combining single cell genomic DNA and transcriptomic features provides novel insights into cancer heterogeneity and biology. ### Competing Interest Statement Authors associated with 10X Genomics are employees. 10X Genomics developed the single cell DNA assay.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

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.

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 x 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 microm, respectively, which provides a relatively large surface area (ca. 3 cm(2)) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instrument's response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.