Genome sequencing in microfabricated high-density picolitre reactors

Abstract

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Figure 1. Sample Preparation.

(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.

Figure 2. Sequencing Instrument.

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.

Figure 3. Flowgram of a 113 base read from an M. genitalium run.

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.

Figure 4. M. genitalium Data.

(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.