High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

Abstract

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (microCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q > or = 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The microCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of microCAE technology should make it the next generation, high performance DNA sequencing platform.

Figure 1

(A) Overall layout of the 96-lane DNA sequencing microchannel plate (MCP). (B) Vertical cut-away of the MCP. The concentric PMMA rings formed two electrically isolated buffer moats that lie above the drilled cathode and waste ports. (C) Expanded view of the injector. Each doublet features two sample reservoirs and common cathode and waste reservoirs. The arm from the sample to the separation channel is 85 μm wide, and the arm from the waste to the separation channel is 300 μm wide. The separation channel connecting the central anode and cathode is 200 μm wide. (D) Expanded view of the hyperturn region. The turns are symmetrically tapered with a tapering length of 100 μm, a turn channel width of 65 μm, and a radius of curvature of 250 μm. Channel widths and lengths are not drawn to scale.

Figure 2

The Berkeley 4-color rotary confocal scanner. Excitation at 488 nm from an argon ion laser is passed through the hollow shaft of a stepper motor. The beam is displaced to a radius of 1 cm and passed through a 20× objective. Fluorescence (gray line) is collected by the objective and passed through the dichroic beam splitter to the 4-color detection system.

Figure 3

Image of processed sequencing data from the 96-lane DNA sequencing microdevice. Each band in a lane represents one base. The DNA bases C, A, G, and T are color coded as blue, green, black, and red, respectively. The data for all lanes were collected in 24 min after injection; 4.5% wt/vol linear polyacrylamide, 50°C, energy-transfer labeled M13mp18 forward sequencing primer.

Figure 4

Processed sequencing output from a lane in Fig. 3 exhibiting average performance. The three panels display the typical quality of data obtained at the start, middle, and end of the run. Base numbers are indicated above the base calls.

Figure 5

The average phred score over the 95 passed lanes as a function of base position (Upper). Miscall probabilities are listed at the right as accuracy. The distribution of phred scores from the 95 passed lanes is shown at six positions in the processed data (Lower). The base position and average score of each distribution is listed above the graphs. Distributions representing the beginning (10 bp), high quality (100, 200, and 300 bp), declining quality (400 bp), and end (450 bp) regions of the separation are shown.