and emPCR Genomic DNA was fractionated into smaller fragments (300-800 base pairs) and polished (made blunt at each end). Short adaptors were then ligated onto the ends of the fragments. These adaptors provided priming sequences for both amplification and sequencing of the sample-library fragments. One adaptor (Adaptor B) contained a 5'-biotin tag for immobilization of the DNA library onto streptavidin-coated beads. After nick repair, the non-biotinylated strand was released and used as a single-stranded template DNA (sstDNA) library. The sstDNA library was assessed for its quality, and the optimal amount (DNA copies per bead) needed for emPCR is determined by titration. The sstDNA library was immobilized onto beads. The beads containing a library fragment carried a single sstDNA molecule. The bead-bound library was emulsified with the amplification reagents in a water-in-oil mixture. Each bead was captured within its own microreactor where PCR amplification occurs. This resulted in bead-immobilized, clonally amplified DNA fragments. Sequencing Single-stranded template DNA library beads were added to the DNA Bead Incubation Mix (containing DNA polymerase) and were layered with Enzyme Beads (containing sulfurylase and luciferase) onto a PicoTiterPlate device. The device was centrifuged to deposit the beads into the wells. The layer of Enzyme Beads ensured that the DNA beads remained positioned in the wells during the sequencing reaction. The bead-deposition process was designed to maximize the number of wells that contain a single amplified library bead. The loaded PicoTiterPlate device were placed into the Genome Sequencer FLX Instrument. The fluidics sub-system delivered sequencing reagents (containing buffers and nucleotides) across the wells of the plate. The four DNA nucleotides were added sequentially in a fixed order across the PicoTiterPlate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads were sequenced in parallel. When a nucleotide complementary to the template strand was added into a well, the polymerase extended the existing DNA strand by adding nucleotide(s). Addition of one (or more) nucleotide(s) generated a light signal that was recorded by the CCD camera in the instrument. This technique was based on sequencing-by-synthesis and called pyrosequencing. The signal strength was proportional to the number of nucleotides; for example, homopolymer stretches, incorporated in a single nucleotide flow, generated a greater signal than single nucleotides. However, the signal strength for homopolymer stretches was linear only up to eight consecutive nucleotides, after which the signal fell off rapidly. Data were