1. Field of the Invention
The present invention relates generally to the field of nucleic acid sequencing and, more specifically, to a method and system for DNA (deoxyribonucleic acid) sequencing by selective excitation of microparticles.
2. Description of the Related Art
FIG. 1 illustrates a conventional method of DNA sequencing with microparticles. The method of FIG. 1 derives DNA sequence data 112 from a microparticle array 102 through cycles of sequencing reactions 104, non-selective excitation 106 of the microparticles, and optical signature detection 108. Each microparticle in the microparticle array 102 typically contains DNA molecules with both unknown sequences to be determined and known sequences that are used in the sequencing reactions. Thousands to millions (to potentially billions or more) of these microparticles are distributed and immobilized on the surface of a glass substrate, as conceptually shown in FIG. 2, which illustrates an example of a microparticle array 102. The microparticle array 102 includes DNA sequencing microparticles 204 distributed and immobilized on a substrate 202. The microparticles 204 can take many forms, such as 1-micron diameter beads covered with DNA molecules amplified by a water-in-oil emulsion PCR (polymerase chain reaction) technique, or clusters of DNA molecules amplified by a bridge amplification technique, or individual unamplified DNA molecules. The microparticles 204 can be distributed either randomly (e.g., irregularly spaced) or in an orderly pattern (e.g., regularly spaced pattern such as a square grid pattern or a hexagonal grid pattern) on the substrate 202. The substrate 202 is typically made of glass and located inside a flow cell, which allows the microparticles 204 to be exposed to a series of reagents to perform sequencing reactions. At the end of each cycle of sequencing reactions, each microparticle takes on an optical signature, often as the result of the incorporation of one of the four fluorophores such as Cy3, Cy5, Texas Red, and a fluorescence resonance energy transfer (FRET) pair, that reveals the corresponding bases adenine (abbreviated “a”), cytosine (abbreviated “c”), guanine (abbreviated “g”) and thymine (abbreviated “t”) of the DNA.
FIGS. 3A-3C illustrate different types of individual sequencing microparticles that can be used for DNA sequencing. FIG. 3A illustrates an individual microparticle 204 formed by a 1-micrometer diameter bead 302 covered with clonal DNA molecules 304 that have been previously amplified by a water-in-oil emulsion PCR technique. The bead 302 is attached directly to the substrate 202 in fluid 306. FIG. 3B illustrates an individual microparticle 204 as a cluster of clonal DNA molecules 304 attached to the substrate 202 and placed in fluid 306. The DNA molecules have been previously amplified by a bridge amplification technique. FIG. 3C illustrates an individual microparticle as a single DNA molecule 304 attached to the substrate 202 and placed in fluid 306. The single DNA molecule 304 is sequenced without amplification.
Referring back to FIG. 1 together with FIGS. 2 and 3A-3C, DNA sequencing with microparticles includes performing a sequencing reaction 104 on the microparticle array 102 to cause each microparticle 204 to take on an optical signature that reveals the DNA sequence information. The microparticle array 102 is exposed to sequencing reagents, which enables each cycle of sequencing reactions to be performed in a massively parallel manner. For example, one cycle of sequencing reaction can be comprised of hybridizing anchor primers and ligating a pool of fluorescently-labeled query primers. At the end of each cycle of sequencing reactions 104, each microparticle takes on an optical signature that reveals the DNA sequence information associated with that microparticle. For example, the optical signature can be the result of the incorporation of one of four fluorophores corresponding to bases “a,” “c,” “g,” and “t” of the DNA 304.
The next step is to optically excite 106 the microparticles 204 and to detect 108 the optical signatures of the microparticles. As will be explained with reference to FIGS. 4A-4C, the conventional optical excitation is non-selective. This cycle of reaction 104, non-selective excitation 106, and optical signature detection 108 is repeated multiples times to sequence the DNA 304 in each microparticle 204. DNA sequence data 112 is output from this process.
Conventional DNA sequencing methods with microparticles suffer from low throughput (measured in bases per second) because the rate at which the optical signatures of the microparticles are detected is limited. This is largely due to the use of conventional non-selective excitation patterns, followed by optical imaging using optical microscopy, as used in conventional DNA sequencing methods. FIGS. 4A-4C illustrate conventional non-selective excitation patterns used to excite the microparticles for subsequent imaging using optical microscopy. Specifically, FIG. 4A illustrates a wide-field excitation pattern 402 used with the microparticles 204 on the substrate 202, where all the microparticles in the field of view (FOV) are illuminated. FIG. 4B illustrates line-scanning excitation, where the microparticles 204 are illuminated by a line of light 402 that scans the substrate 202. FIG. 4C illustrates spot-scanning excitation, where the microparticles 204 are illuminated by a spot of light 406 that scans the substrate 202.
Conventional non-selective excitation patterns can be generated by a variety of means. A wide-field excitation pattern 402 is typically generated by focusing an arc lamp source through the microscope optical train in a Kohler epi-illumination configuration, or by shining a laser source at a steep angle in an off-axis or total internal reflection (TIR) illumination configuration. A line-scanning excitation pattern 404 is typically generated by focusing a spatially-coherent laser through the microscope optical train and incorporating a scanning element. A spot-scanning excitation pattern 406 is typically generated by focusing a spatially-coherent laser source through the microscope optical train in a confocal configuration.
In such conventional DNA sequencing methods, detection of the microparticle optical signatures is typically performed by optical-microscope imaging of the microparticles illuminated with non-selective excitation patterns as shown in FIGS. 4A-4C. The speed of this approach is limited fundamentally for several reasons. First, the field of view (FOV) of an optical microscope is coupled fundamentally to resolution, i.e., the higher the resolution, the smaller the FOV. Similarly, the depth of field (DOF) of an optical microscope is coupled fundamentally to resolution, i.e., the higher the resolution, the smaller the DOF. Because a high-resolution optical microscope is required to resolve the microparticles using conventional sequencing methods, the FOV and DOF are relatively small. Consequently, imaging a microparticle substrate requires the acquisition of hundreds to thousands of smaller images that collectively cover the slide like tiles. Between each image, either the substrate or the optical microscope must be translated and focused precisely with respect to the microscope objective, during which time the optical microscope cannot be acquiring sequence data. Second, a high-resolution image of a microparticle slide is a very inefficient representation of the sequence information contained in the microparticles. For example, in a typical high-resolution image of a microparticle slide, the number of pixels in the image greatly exceeds the number of microparticles in the image. However, assuming that each microparticle can take on one of only four optical signatures, each microparticle carries just 2 bits of sequence information. Consequently, several thousands times more data is acquired than is necessary to generate the sequence information, according to conventional sequencing methods.
Thus, there is a need for a more efficient, faster, and more convenient method of nucleic acid sequencing.
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