The quest to directly sequence individual strands of DNA has challenged genomic technologists for decades. All traditional and “next-generation” sequencing technologies require DNA samples to be amplified numerous times to make many copies of the original molecules, which, only then, can be detected and sequenced. Eliminating these copying steps for genetic analysis would not only reduce the costs and complexity of sample preparation, but would also avoid the errors and biases inherent in the amplification and image analysis process, producing data more closely reflective of the actual biology under study.
As evidenced by previous failed attempts at single-molecule sequencing, the direct measurement of single DNA molecules poses numerous technical hurdles. A single fluorophore on a single nucleotide must be bright enough against a very low background surface to be detected. Reagent contamination must be eliminated. Enzyme fidelity must be exact. Optics must be sensitive and precise. Analysis computers must be powerful enough to manage the immense data output and translate the images into DNA sequence information.
The HeliScope™ Single Molecule Sequencer (Helicos BioSciences Corp., Cambridge, MA) is truly a “DNA microscope” capable of sequencing billions of single DNA molecules at a time. As part of the complete Helicos™ Genetic Analysis system, it is designed to deliver unprecedented sequence data throughput and output, as well as direct analysis of native DNA samples.
Single-molecule sequencing chemistry
Figure 1 - HeliScope Single Molecule Sequencer. (Reproduced with permission from Helicos.)
Figure 2 - tSMS process. a) Sample preparation: DNA samples are sheared into shorter fragments, denatured to single strands, and tagged with a 3′ poly(A) tail and a terminal fluorescent adenosine. DNA strands are then hybridized to the surface of a flow cell via poly(T) capture sites, which also prime the sequencing reaction. b) Captured templates are imaged to map their locations, and the fluorescent tags are removed. c) Sequencing-by-synthesis: fluorescent nucleotides (C, G, T, or A) are added one base per cycle and incorporated into the complementary strand in a template-dependent manner. Nonincorporated nucleotides are washed away, and the strands are illuminated and imaged to determine base addition and DNA sequence. The fluorescent tags are then cleaved, and the next base is added to continue the cycle. (Reproduced with permission from Helicos.)
The HeliScope Single Molecule Sequencer (Figure 1) harnesses the power of proprietary True Single Molecule Sequencing (tSMS)™ technology to perform single-molecule sequencing-by-synthesis. DNA samples are fragmented and captured on a specialized surface within a flow cell, where they then serve as single-molecule templates for sequencingby- synthesis reactions (Figure 2). Fluorescent nucleotides are added one base at a time, and the sequencer records each incorporation event to determine the sequence of the individual DNA strands (Figure 3).
Figure 3 - Image series illustrating templatespecific base incorporation. (Adapted with permission from Harris, T.D., et al. Science 2008, 320, 106–9, and reproduced with permission from AAAS.)
To enable this process, the company’s scientists and engineers developed a series of technical and manufacturing advances. On the chemistry front, a high-fidelity DNA polymerase was combined with proprietary Virtual Terminator™ fluorescent nucleotides, providing rapid and accurate baseby- base synthesis. An imaging reagent was developed to enhance emission intensity and improve fluorophore detection by an order of magnitude. In addition to enhancing signalto- noise, it also reduces photobleaching and fluorophore “blinking.” Together, these specialized reagents ensure accurate and precise single-molecule sequencing chemistry.
Another challenge was to minimize even low-level background fluorescence. Since the sequencer can detect the presence of single fluorophores, it is vital to reduce all nonspecific emission sources, which could produce errors in the sequence. Engineering designs avoid the use of materials that autofluoresce, and strive to minimize the adsorption of stray fluorescent molecules. Reagents, including all enzymes, nucleotides, and buffers, are formulated to eliminate impurities that less sensitive molecular biology applications may tolerate. Helicos worked with its partners to develop quality control assays that ensure reagents meet these standards of purity. The proper selection of single- molecule- grade reagent and surface materials, together with diligent handling of flow cells during manufacture and storage, and optimized surface rinsing protocols, collectively produce images that are nearly free of spurious incorporation events.
At each step of the sequencing-by-synthesis process, the HeliScope Sequencer records the incorporation of fluorescently labeled nucleotides in the growing DNA strands. The instrument maximizes run efficiency by processing two 25-channel flow cells at once, performing strand synthesis in one flow cell while simultaneously imaging the other (Figure 4).
Figure 4 - HeliScope Single Molecule Sequencer optics configuration. (Reproduced with permission from Helicos.)
To ensure the integrity needed to perform single-molecule sequencing, reagents and flow cells are prepackaged in ready-to-use kits that simplify handling and facilitate correct loading. The packaging design incorporates seals at both the top and bottom of the bottles to provide reagent access, protection against contamination, and venting for proper flow. The reagent storage system on the sequencer has both refrigerated and ambient temperature locations to optimize on-board stability of the various components. When the bar-coded reagent cartridge or bottle is scanned, the sequencer’s graphical user interface verifies the appropriateness of the kit and indicates the proper location for placement.
Reagent delivery is driven by the appropriate application script and supports both the chemistry and imaging conditions on a flow cell. For each step in the sequencing- by-synthesis reaction, the required reagents are metered, prepared, and delivered to the flow cell. The fluidics design provides “just-in-time” mixing to tailor the exact conditions needed for each step and provide flexibility for future applications. In addition, the sequencer accurately controls the temperature in the flow cell for optimum reaction conditions. This high level of flexibility in the fluidics architecture supports continuous improvement in performance and throughput, without the need for expensive instrument upgrades as existing and new chemistries are introduced and optimized.
To accurately detect the incorporation of fluorescently labeled nucleotides into single strands of DNA, two primary attributes are required. First, the imaging system must have adequate resolution to discriminate individual strands tethered to the surface in nonordered locations. Second, there must be sufficient signal-to-noise ratio to effectively “see” the fluorescent labels and discriminate them from the background.