Field of the Invention
The present invention relates generally to instruments for performing sequencing-by-syntheses or other sequencing processes, and more particularly to flowcells and particle separators used in such instruments.
Description of the Related Art
DNA (deoxyribonucleic acid) sequencing instruments are used to determine DNA molecular sequences. Such instruments are useful for clinical studies, diagnostics, so-called “personalized medicine” (medical treatment tailored to an individual's genetic content or the like), and so on. Current instruments for performing DNA sequencing use a variety of technologies to analyze the base pairs that form the DNA sequence. For example, some instruments perform sequencing on a library of cloned colonies of single-stranded DNA molecule fragments (DNA template colonies) that are fixed in place inside a flowcell. The flowcell is essentially a small chamber in which the DNA template colonies are subjected to a series of nucleobase extension processes. Each successive extension is detected to determine the base pair sequence of each DNA template colony. The flowcell provides an environment to hold the DNA template colonies during the extension process, and also during the inspection process to read each extended base pair.
Many sequencing-by-synthesis instruments use an optical system such as a microscope to detect the nucleobase extensions, although non-optical systems are also known. A typical optical instrument uses visible chemical labels to determine the identity of each extended base pair. For example, each nucleobase that makes up the DNA molecule (adenine, guanine, cytosine and thymine) may be labeled with a unique fluorescent probe that is visible through the microscope. The label is read each time the DNA template colony is extended, and then the label is removed to make way for the next base pair extension.
In modern “next-generation” instruments, millions of DNA template colonies may be immobilized in a single flowcell, and processed simultaneously. A variety of flowcell designs have been developed to hold the immobilized DNA template colonies, but they usually include certain common features. A typical flowcell includes a rigid flow channel, an optically transparent cover that encloses the channel, and a fluid inlet and a fluid outlet through which the appropriate reagents are passed to control the growth and extension of the DNA template colonies. Examples of such flowcells are found in U.S. Pat. Nos. 8,481,259, 8,940,481 and 9,146,248 and U.S. Patent Application Publication Nos. 2009/0298131 and 2014/0267669, all of which are incorporated herein by reference.
The DNA template colonies may be secured within the flowcell in various ways. For example, clonal DNA template colonies may be secured to individual beads, and then the beads may be secured in a random pattern to a functionalized surface within the flowcell. This technology is useful, but provides little or no control over the spacing of the beads, and thus the DNA template colonies, which can make data acquisition more difficult. This technology also may use separate processing steps outside the flowcell to amplify the DNA templates. This process also may experience relatively inefficient bead capture properties, which may require a greater amount of amplification when preparing the library of DNA templates.
Another technology uses a flowcell having a pattern of organized microwells to immobilize the DNA template colonies. Each well includes a gel functionalized with primers to capture the DNA templates, and the captured templates are amplified to form DNA template colonies in situ within the flowcell. The gel is placed in the wells by coating the entire substrate surface, and then removing the gel from the interstitial space between the individual wells. This process can have several drawbacks. For example, it can lead to additional operating and material costs due to the need to remove a large proportion of the functionalized gel, and any gel that might remain in place can create undesirable interstitial DNA template seeding sites. Also, the gel can be removed accidentally from the well, decreasing the density of the DNA primers.
The inventors have determined that there continues to be a need to advance the state of the art of flowcells for sequencing instruments and similar devices.