(A) Clockwise from top left: (i) genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands; (ii) fragments are bound to beads under conditions which favor one fragment per bead, the beads are captured in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet, resulting in beads each carrying ten million copies of a unique DNA template; (iii) the emulsion is broken, the DNA strands are denatured, and beads carrying single-stranded DNA clones are deposited into wells of a fibre optic slide; (iv) smaller beads carrying immobilized enzymes required for pyrophosphate sequencing are deposited into each well. (B) Microscope photograph of emulsion showing both droplets containing a bead and empty droplets. The thin arrow points to a 28 μm bead, the thick arrow points to an approximately 100 μm droplet. (C) SEM photograph of portion of a fibre optic slide, showing fibre optic cladding and wells prior to bead deposition.

The sequencing instrument consists of the following major subsystems: a fluidic assembly (A), a flow chamber that includes the well-containing fibre optic slide (B), a CCD camera-based imaging assembly (C) and a computer that provides the necessary user interface and instrument control.

Nucleotides are flowed in the order T, A, C, G. The sequence is shown above the flowgram. The signal value intervals corresponding to the various homopolymers are indicated on the right. The first four bases (in red, above the flowgram) constitute the “key” sequence, used to identify wells containing a DNA-carrying bead.

(A) Read length distribution for the 306,178 High Quality Reads of the M. genitalium sequencing run. This distribution reflects the base composition of individual sequencing templates. (B) Average read accuracy, at the single read level, as a function of base position for the 238,066 mapped reads of the same run.