The United States Department of Agriculture, Agricultural Research Services (ARS) intends to make a sole source award to Fluidigm, San Francisco, CA for an EP1 System MX/HX Package, which includes a EP 1 Reader, Data collection software, Genotyping Analysis Software (20 seats), Digital PCR Analysis Software (20 seats), IFC Controller MX (48.48/Digital), IFC Controller HX (96.96), Stand-Alone Thermal Cycle and Vacuum Accessory, Electronic manual, 1 year instrument warranty coverage and installation and 2 day training. The purpose of procurement is to fulfill USDA, ARS requirements to acquire a fully integrated system enabling analysis of SNP genotyping, mutation detection, and absolute quantization of nucleic-acid sequences by digital PCR. The ability to precisely calibrate small quantities of DNA greatly enhance productivity in next-generation sequencing by reducing sample input requirements as well as improving emulsion PCR efficiencies. Our laboratory is sequencing multiple samples types on the Illumina GA2X and Roche 454 systems. In calibration of sequencing libraries we are only interested in measuring for ‘amplifiable’ molecules, i.e., those that are appropriately adapted. Here we describe how Fluidigm’s digital PCR strategy fits our needs, and its impact on our sequencing efficiency and comparison to alternate approaches. The digital PCR analysis is unique to theFluidigm package. Digital PCR is a direct measurement of target molecules without the need for reference standards. Using the dynamic arrays, sample enrichment allows for successful detection of target molecules even within a dense background of competing molecules. The precision of digital PCR enables a more exact ratio of DNA copies / emPCR bead. Optimization of this ratio yields higher % Pass Filter and conversely a lower % Dot & Mixed (failed emPCRs). This ability to optimize directly impacts the efficiency and productivity of each sequencing run producing highest number of confirmed bases. Additionally, the sensitivity of the digital array allows for sequencing of samples 1000-fold less than currently required. Roche generally suggests 3-5 ug of DNA for library prep. Fully 25-30% may be consumed in traditional calibration methodologies. It is anticipated that many of the samples will be substantially < 1 ug and digital PCR will enable measurement even for samples < 5 ng. This will benefit metagenomics projects where samples derived from environmental sources will be scarce and immeasurable by alternate means. Because this system must also meet assay requirements for genotyping and gene expression, both critical research needs, the following additional specifications are required by USDA: 1) flexible and user configurable (eg. non-prespotted) arrays 2) arrays that contain between 765-9,000 distinct reaction chambers 3) arrays that are constructed in a format compatible with manual and/well-plate format handling and standard dispensing equipment 4) arrays that assay gene expression with real time PCR reagents), genotyping (end point analysis), and digital PCR 5) assays done in distinct reaction chambers allowing independent control of input sample and assay volumes (nL range) 6) assays that have detection sensitivity of a single copy per chamber (reaction) 7) assays that provide absolute quantification of amplifiable DNA for Next Generation Sequencing libraries (Roche 454, Illumina GA) 8) genotyping assays that produce a genotyping call rate of 99% or greater and a call accuracy of 99.75% or greater 9) integrated system has the capacity to analyze greater than 27,000 reactions per day by a single operator without the use of a liquid handling robot and is easily scalable with additional thermal cyclers 10) software that automatically analyzes the resulting data from the experimental reactions run within the system. Estimated date of award: 09/09/2009

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