With HeliScopeCAGE, the OSC research team has adapted the existing CAGE protocol for use with the revolutionary HeliScopeTM Single Molecule Sequencer. Our mission is to enable research breakthroughs by helping customers harness the power of True Single Molecule Sequencing. Our sequencing services. The HeliScope™ Single Molecule Sequencer is the first genetic analyzer to harness the power of direct DNA measurement, enabled by Helicos True Single.
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Sample preparation does not require ligation or PCR amplification, avoiding the GC-content and size biases observed in other technologies. DNA is simply sheared, tailed with poly A, and hybridized to a flow cell surface containing oligo-dT for sequencing-by-synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. Gene expression measurements can be done using 1 st -strand cDNA-based methods RNA- Seq or using a novel approach that allows direct hybridization and sequencing of cellular RNA for the most direct quantitation possible.
A diverse array of applications have been successfully performed including genome sequencing for accurate variant detection, ChIP-Seq using picogram quantities of DNA, copy number variation studies from both fresh tumor tissue and FFPE tissue samples, sequencing of ancient and degraded DNAs, small RNA studies leading to the identification of new classes of RNAs and the direct capture and sequencing of RNA from cell quantities as few as cells.
Because most next generation sequencing technologies require amplification and a specific size range of target molecules, DNAs not meeting those criteria cannot be sequenced in a reliable manner.
Single-molecule sequencing does not suffer from those limitations as no amplification is necessary and degraded or modified molecules can be used directly as templates. DNA molecules targeted for sequencing are hybridized in place on disposable glass flow cells. Samples are loaded onto the flow cells using the Helicos Sample Loader in which the temperature can be adjusted for optimal hybridization. Each of the 25 channels on one standard flow cell can be addressed individually for addition of sample and any other needed sample preparation steps.
The Analysis Engine processes the images from each physical location and builds sequence reads from those images. Once the run is complete, the images processed, and strand formation complete; the data are downloaded to a compute cluster for reference alignment or assembly as needed. Two protocols will be described. Basic protocol 1 is for shearing genomic DNA so that it is ready for tailing. This step may not be required for all samples.
Basic Protocol 2 is for tailing and blocking samples so they can hybridize to the sequencing flow cell and sequence properly. When supplying samples to a core sequencing facility, samples are generally provided at this stage or after an optional sample concentration determination, depending on the facility.
The Helicos Genetic Analysis System http: If starting with nucleic acids longer than nt, it is generally heliacope to shear the nucleic acids to an average length of nt so that more sequence information can be generated from the same mass of nucleic acids. Not sesuencing sonicators provide equivalent results so shearing with an alternative instrument should only be done after testing to ensure that the resulting DNA is not overly damaged.
For some applications, rather than shearing, it is desirable to cleave with restriction endonucleases or other specific cutters.
In other cases, as with DNA from most ChIP, FFPE, and ancient or degraded samples, shearing is completely unnecessary as the starting material is already sufficiently short that further cleavage is not beneficial. DNA and RNA samples are hybridized to a primer immobilized on a flow cell for sequencing so it is usually necessary to generate a nucleic acid with an end compatible for hybridization to those surfaces.
The target sequence attached to the flow cell surface could, in theory, be any sequence which can be synthesized, but, in practice, the standard commercially-available flow cell is oligo-dT Other sequences have been used successfully when there is a specific tag on the nucleic acid molecules of interest.
Since most work being carried out at the present time is with the oligo-dT surfaces, the discussion here will be restricted to that type of flow cell for simplicity. Because the fill and lock step see Figure 1 will fill in excess As but not excess Ts, it is desirable for the A tail to be at least as long as oligo-dT on the surface. It is not clear how long an A-tail can be before it is an issue so it is generally advisable to keep tails less than nt.
Few natural sequences contain an internal A-stretch long enough to be stably hybridized. Overview of steps required for sequencing. Later steps are generally carried out in a sequencing facility and require training for successful completion. Furthermore, most ligases have substantial sequence specificities that can cause ligation to occur much more efficiently at some sequences than at others. There can be a length dependence or a base composition dependence; but, in any case, differential ligation can lead to biases in the sequences observed.
Thus, while ligation of a poly-dA tail is sometimes readily achievable, we recommend not using ligases for applications that require very quantitative results.
The DNA is denatured and snap cooled prior to tailing but, depending on the complexity of the sample and intramolecular folding, some tailing biases could arise.
If there is sufficient DNA to measure both mass and average length, it is possible to determine the proper amount of dATP to be added to generate poly dA tails nucleotides long. If there is insufficient sample to determine mass and length, there is an alternative, low sample mass technique that can be used to generate tails of the proper length. Helicos Single Molecule Sequencing is carried out on a glass flow cell with 25 channels for the same or different samples.
The system can be run with either one or two flow cells at a time. Samples are inserted into the flow cell via the sample loader included with the overall system. Each channel is individually addressable and sample is applied using a vacuum. Generally, samples for sequencing are prepared in such a way that the polyA tail is longer than the oligo-dT50 on the surface of the flow cell.
To avoid sequencing the unpaired A residues, a fill and lock protocol is carried out on either the Sample Loader or the HeliScope Sequencing System. Virtual Terminator nucleotides incorporate opposite the complementary base and prevent further incorporation because of the chemical structure appended to the nucleotide. The hybridized molecule is locked in place when the polymerase encounters the first non-A residue and inserts the appropriate Virtual Terminator nucleotide.
If flow cells with a specific sequence are used instead of oligo-dT50, the fill step is omitted and all four Virtual Terminator nucleotides are used so that each hybridized DNA is locked in place and becomes labeled.
If not already loaded on the HeliScope Sequencing System, the flow cells are inserted and the first template picture is taken. Because every DNA molecule should now have a dye attached, an image will include all molecules capable of nucleotide incorporation. Also, because the label could correspond to any base, no sequence information is obtained at this stage. Thus, for most molecules, sequencing commences with the second base of the original molecule.
In order to sequence the hybridized DNAs, it is first necessary to cleave off the fluorescent dye and terminator moieties present on the Virtual Terminator nucleotides. The current generation of nucleotides is synthesized with a singlr linkage that can be rapidly and completely cleaved. Following cleavage, the now separated fluorescent dyes are washed away and then new polymerase and a single fluorescent nucleotide are added.
After excitation of the fluorescent moiety by heliscole system laser, another heilscope is taken and, on mooecule standard sequencing run, this cyclic process is repeated times. The number of sequencing cycles is user-adjustable and can be modified depending on user needs for run time and length of read.
Though single molecules are visualized, multiple photon emissions are registered for each molecule with the time spent at each FOV dependent on the brightness of the dye in the particular nucleotide as well as camera speed and detection efficiency. At the present time, the imaging process is the rate determining step and run time could be reduced at the expense of throughput by reducing the number of FOV per channel.
Similarly, improvements in camera technology or improved dyes could reduce the run time by lowering the amount of time spent with each image. At the other end of the spectrum, up to FOVs are possible per channel so it is possible to get increased output but this comes at the expense of increased run time. Schematic view of the optical path of the HeliScope Genetic Analysis System and its two flow cells published with permission.
Massively parallel DNA sequencing has revolutionized many fields of biology by allowing the generation of sequence information on an unprecedented scale Kahvejian et al. The incredible sequence output has been used for many purposes, but primarily for whole genome sequencing of a myriad of species and individuals.
The genome information has been complemented by a host of ancillary applications making use of sequencing technologies to shed light on epigenetics, transcription, protein binding, and diagnosis of various medical and other conditions. Most of the sequence data generated thus far has been achieved with amplification-based sequencing systems reviewed in Metzker but single-molecule, non-amplification based sequencing is now possible including at the scale of resequencing whole human genomes Pushkarev et al.
Next Generation Sequencing Leaders :: Product Reviews
Throughput and read lengths from amplification-based systems has grown at a prodigious rate, straining the capacity of informatics resources, storage capacity, and the ability of biologists to connect function with much of the sequence. Because the technology has evolved at such a rapid rate, it has been sometimes difficult to fully validate all protocols and assess the limitations of the data generated. While some of these limitations can be overcome by sheer mass of data, there are some sequencing applications for which the amplification of the target sequence is not possible or occurs in such a biased or unpredictable manner that the data quality suffers as a result.
Single molecule sequencing can be used for virtually all sequencing applications, but, in some situations, it is absolutely required to sequence in an amplification-free manner.
To circumvent amplification issues, various methods for single molecule sequencing have been envisioned for many years because of the inherent advantages of examining single molecules rather than ensembles of molecules Efcavitch and Thompson, An early report of single molecule sequencing by synthesis employed FRET to detect incorporation Braslavsky et al.
As the technology advanced, the mode of detection was changed to measure the fluorescence directly from labeled nucleotides Harris et al. While numerous single-molecule approaches are being actively developed, the first commercially-available system was the Helicos Genetic Analysis System. With each sequencing run, this system can generate more than 1,, usable reads with a median read length of about 35 nt. The lack of amplification and ligation in sample preparation and sequencing leads to exquisite quantitative abilities and a virtual lack of GC bias in sequencing.
It is likely that other single molecule systems will be available soon; each with their own set of read yields, error rates, and read lengths. A brief overview of the theoretical background for the technology and selected applications benefitting from single molecule attributes will be described.
For images to be useful, the signal from the molecules of interest must be significantly higher than the background noise level. Any accidental or random source of light emission will be read as a base incorporation and hence appear as an insertion in the sequence.
Thus, background signal must be kept to an absolute minimum. This is accomplished by attention to both reagent purity and through the use of Total Internal Reflectance Fluorescence TIRF to minimize the ability of molecules far from the surface to fluoresce. This technology Axelrod et al.
Single Molecule Sequencing with a HeliScope Genetic Analysis System
By proper choice of light angle, light absorption sequencong thus fluorescence can be restricted to a very narrow layer near the surface of the flow cell where the desired nucleic acid molecules are located. This minimizes the contribution of the solution to emitted light and enhances the signal to noise so that photons from a single molecule can be visualized. However, the DNA length is also limited laterally by neighboring molecules that could potentially overlap.
The current lateral resolution limit of the camera is about nM and the thickness of the evanescent wave allowed by TIRF is about nM. The length of DNA required to approach these limits is dependent upon the DNA persistence length which is affected by many factors. The DNA length limit at which signal is lost has not been determined but lengths of several kilobases have been observed to provide a signal.
Helicos single molecule fluorescent sequencing
After incorporation of the fluorescent nucleotide and rinsing to remove any unincorporated molecules, the flow cells are irradiated with a solid state nm red laser to excite molecules on the surface. The flow cell is mounted on a movable platform helicope that each FOV can be localized under the laser beam and one of four CCD cameras can take pictures via a confocal microscope.
Optical focus on the flow cell, critical for maintaining resolution between molecules, is maintained mlecule a separate laser. Prior to each run, this mollecule goes through a process of focus-finding on one channel so the selected focus channel needs to contain a reliable sample.
This limit is caused by the diffraction limit of light and thus the inability to distinguish molecules that are physically located too close to each other. Ordered surfaces will be capable of sequencing about five times as many molecules without danger of having two molecules within an unresolvable distance because it eliminates the random overlap of molecules that can be created in disordered deposition strategies Schwartz and Quake, Because each cycle of base addition seqiencing a standard run involves saving images in each heliscole 50 channels, a tremendous amount of storage space would be required for both saving and processing the images.
These images are processed in real time by the Singls Analysis Engine which is dedicated to such processing so that sequence data can be ready within an hour or so after the run finishes. Typically, most images are discarded as soon as they are processed.
Two full runs of data can be stored on the machine before a run needs to be deleted. A new run will not start if it is judged to have insufficient data storage for the next run. Spot finding is performed in real time which greatly reduces the amount of data that needs to be saved.