Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing

Abstract

An efficient, nanoliter-scale microfabricated bioprocessor integrating all three Sanger sequencing steps, thermal cycling, sample purification, and capillary electrophoresis, has been developed and evaluated. Hybrid glass-polydimethylsiloxane (PDMS) wafer-scale construction is used to combine 250-nl reactors, affinity-capture purification chambers, high-performance capillary electrophoresis channels, and pneumatic valves and pumps onto a single microfabricated device. Lab-on-a-chip-level integration enables complete Sanger sequencing from only 1 fmol of DNA template. Up to 556 continuous bases were sequenced with 99% accuracy, demonstrating read lengths required for de novo sequencing of human and other complex genomes. The performance of this miniaturized DNA sequencer provides a benchmark for predicting the ultimate cost and efficiency limits of Sanger sequencing.

Fig. 1.

Integrated nanoliter-scale nucleic acid bioprocessor for Sanger DNA sequencing. (A) Top view of the assembled bioprocessor containing two sets of thermal cycling reactors, purification/concentration chambers, CE channels (black), RTDs (red), microvalves/pumps (green), pneumatic manifold channels (blue), and surface heaters (orange). (B) Expanded view, showing microdevice layers. Rim colors indicate the surface on which the respective features are fabricated. The top two glass wafers are thermally bonded and then assembled with a featureless PDMS membrane and manifold wafer.

Fig. 2.

Bioprocessor components. (A) Photograph of the microdevice, showing one of two complete nucleic acid processing systems. Colors indicate the location of sequencing reagent (green), capture gel (yellow), separation gel (red), and pneumatic channels (blue). (Scale bar, 5 mm.) B–F correspond to the following component microphotographs. (B) A 250-nl thermal cycling reactor with RTDs. (Scale bar, 1 mm.) (C) A 5-nl displacement volume microvalve. (D) A 500-μm-diameter via hole. (E) Capture chamber and cross injector. (F) A 65-μm-wide tapered turn. (Scale bars, 300 μm.) All features are etched to a depth of 30 μm.

Fig. 3.

False-color fluorescence images of the capture, purification, and injection steps. Cooler colors indicate lower intensity; warmer colors indicate higher intensity. Fluidic channels are outlined in white. Relative electric potentials are indicated by + and −. (Scale bars, 300 μm.) (A) Thermally cycled dye-terminator sequencing reagent is pumped into the buffer-filled upper chamber (a). Simultaneously, an electric field drives the sequencing mixture into the lower, capture-gel-filled chamber. (B) Fluorescently labeled DNA extension fragments selectively hybridize and concentrate at the gel/buffer interface (b). (C) Desired extension fragments (c) are bound to the capture gel interface, and contaminants (d) continue to electrophorese out of the gel. (D) Purified and concentrated sample is ready for injection. (E) Extension fragments are released from the capture gel at 70°C and injected into the channel junction (e). Pinching potentials constrain the plug size. (F) A ≈1-nl sample plug is injected onto the separation capillary at (f), and reverse potentials pull away uninjected material (g).

Fig. 4.

High-quality sequence data generated on the integrated bioprocessor. Sanger sequencing extension fragments from a 750-bp pUC18 PCR amplicon are resolved at 73°C, 167 V/cm with linear polyacrylamide separation gel (3.5% linear polyacrylamide/5% DMSO/3 M urea/1× TTE buffer) in 34 min. Automatic base calls by the program phred and base numbers are indicated above the electropherogram. Elapsed time in minutes from injection is indicated parenthetically every 50 bases. The base position where no call could be made is assigned “N.” Compared with the known pUC18 sequence, total read length is 556 bases with an accuracy of 99%. Black arrows indicate base call errors; correct calls are given below. X indicates an inserted base.

Fig. 5.

Base-call accuracies and sequence read length as predicted by phred. Percent accuracy is related to the phred quality score by: 100 × (1 − P_e), where P_e, the probability that the base call is incorrect, is equal to 1/10^Q/10. A one-in-a-hundred error rate is indicated by the dashed line. The gray line plots phred quality scores at each base position. The black line charts predicted read accuracy at each base position: 100 × (Base_i − ΣP_ei)/Base_i.