Genomic medicine 6. Why personalised medicine? Why now? EC support to personalised medicine 9. Why nano and regenerative medicine? Victor Maojo - UPM Data collection F. La Laguna, Personalized virtual medicine p-Health 6thth March CATAI: , pp. Special issue on Nanomedicine. The central role of EHR From data collection to medical decision making Methods Inf Med. PMID: You just clipped your first slide!
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Whitmire, W. McDermott, P. Grenier, D. Yonezawa, H. Nakamura, S. Henderson, I. Ayala, G. Shah, B. Chitcholtan, K. Negrete-Abascal, E. Patrick, S. Hozbor, D. Donato, G. Allan, N. Yokoyama, K. Wai, S. Kouokam, J. Balsalobre, C. Ricci, V. Tan, T. Vipond, C. Dutta, S. Chi, B. Rosen, G. Hong, G. Boardman, B. Khandelwal, P.
However, subsequent deprotection steps may successfully remove at least some of the protecting groups which had previously improperly remained, causing extension to resume, and creating signals from nascent molecules and continue to be out of phasic synchrony with the rest of the population. Those of ordinary skill in the art will appreciate that other factors that contribute to IE may exist and thus are not limited to the examples provided above.
The systems and methods of the presently described embodiments of the invention are directed to the correction IE errors that may arise from any such single or combined causes or mechanisms. For instance, the correction of IE errors caused by a coupling of incomplete deprotection and subsequent successful deprotection is one object of the present invention. With respect to the problem of CF 5 there may be several possible mechanisms that contribute to CF that may occur alone or in some combination.
For example, one possible mechanism may include excess nucleotide species remaining from a previous cycle. This can occur because the washing protocol performed at the end of a cycle will remove the vast majority, but not necessarily all, of the nucleotide species from the cycle. In the present example a result could include a small fraction of an "A" nucleotide species present in a "G" nucleotide species cycle, leading to extension of a small fraction of the nascent molecule if a complementary "T" nucleotide species is present at the corresponding sequence position in the template molecule.
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In the present example, as described above with respect to IE a preparation of 3'-O protected nucleotide species maybe employed where some fraction of the nucleotide molecules will lack a protecting group, or have lost the protecting group. Loss of the protecting group may also occur during the sequencing process prior to the intended deprotecting step. Any such lack of a deprotecting group will cause some nascent molecules to be extended by more than one nucleotide species at a time.
Such improper multiple. The systems and methods of the presently described embodiments of the invention are directed to the correction of CF errors that may arise from any such single or combined causes or mechanisms. For example, the correction of CF errors that arise due to a lack of protecting groups is one object of the present invention.
Further, the systems and methods of the presently described embodiments of the invention are directed to the correction of both IE errors and CF errors, wherein both types of errors may occur in some combination for a population in the same sequencing reaction. For example, IE and CF may each arise from single or combined causes or mechanisms as described above.
Those of ordinary skill will appreciate that a potential for both IE and CF errors may occur at each sequence position during an extension reaction and thus may have cumulative effects evident in the resulting sequence data. For example, the effects may become especially noticeable towards the end of a series of sequencing reactions, which is also sometimes referred to as a "run"- or "sequencing run".
Further, IE and CF effects may impose an upper limit to the length of a template molecule that may be reliably sequenced sometimes referred to as the "read length" using SBS approaches, because the quality of the sequence data decreases as the read length increases. While the overall sequencing throughput with Phred 20 quality for the SBS method is significantly higher than that of sequence data generated by what is known to those in the art as Sanger sequencing methods that employ a capillary electrophoresis technique, it is currently at the cost of substantially shorter read lengths for the SBS method Margulies et al.
Thus increasing the upper limit of the read lengths by avoiding or correcting for degradation of the sequence data produced by IE and CF errors would result in an increase in the overall sequencing throughput for SBS methods. A number of references are cited herein, the entire disclosures of which are incorporated herein, in their entirety, by reference for all purposes.
Further, none of these references, regardless of how characterized above, is admitted as prior art to the invention of the subject matter claimed herein. Embodiments of the invention relate to the determination of the sequence of nucleic acids. More particularly, embodiments of the invention relate to methods and systems for correcting errors in data obtained during the sequencing of nucleic acids by SBS.
An embodiment of method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises a detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; b generating a value for the signal; and c correcting the value for the phasic synchrony. Also, an embodiment of a method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises a detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; b generating a value for the signal; c incorporating the value into a representation associated with a sequence of a template molecule; d repeating steps a - c for each sequence position of the template molecule; e correcting each value for the phasic synchrony error in the representation using a first parameter and a second parameter; and f generating a corrected representation using the corrected values.
Additionally, an embodiment of a system for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises a computer with program code stored for execution thereon that performs a method that comprises a generating a value for a signal detected in response to an incorporation of one or more nucleotides in a.
Even further, an embodiment of a system for correcting error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises a computer with program code stored for execution thereon that performs a method that comprises a generating a value for a signal detected in response to an incorporation of one or more nucleotides in a sequencing reaction; b incorporating the value into a representation associated with a sequence of a template molecule; c repeating steps a - b for each sequence position of the template molecule; d correcting each value for the phasic synchrony error in the representation using a first parameter and a second parameter; and e generating a corrected representation using the corrected values.
The advantages achieved by embodiments of the present invention include but are not limited to: a the quality of the sequence data is increased, resulting in lesser depth of sequence coverage being required to achieve a desired level of accuracy of the consensus sequence; b the useful sequence read length is extended, which means that more high- quality sequence data can be obtained from a single run; c because the useful sequence read length is extended, fewer runs will be needed to achieve a given depth of sequence coverage; d because the useful sequence read length is extended, fewer sequences are needed to assemble a sequence contig spanning a given region; and e the resulting increased read lengths facilitate the assembly of overlapping reads, particularly in repetitive sequence regions.
The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings. In the drawings, like reference numerals indicate like structures, elements, or method steps and the leftmost digit of a reference numeral indicates the number of the figure in which the references element first appears for example, element appears first in Figure 1.
All of these conventions, however, are intended to be typical or illustrative, rather than limiting. Figure 1 is a simplified graphical representation of one embodiment of a mathematical model for converting a "perfect" theoretical flowgram to a "dirty" observed flowgram;.
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Figure 2 is a simplified graphical representation of one embodiment of an inversion of the mapping model of Figure 1 ; Fig. Embodiments of the presently described invention are based, at least in part, upon the discovery that a theoretical or "perfect" flowgram can be converted into a real life observed "dirty" flowgram by a mathematical model of IE and CF. The term "flowgram" as used herein generally refers to a representation of sequencing data generated from a sequencing run that may, for instance, include a graph representation of the sequencing data.
For example, a perfect or theoretical flowgram represents data from generated from a sequencing run that has no error from the CAFIE mechanisms described above or other types of background error. Along the same lines a dirty or observed flowgram represents data generated from a sequencing run that includes the CAFIE and background error factors. In the present example, some or all of the error factors may be accurately approximated and applied to the perfect flowgram model to provide a representation of real data obtained from an actual sequencing run.
Importantly, the presently described invention is also based, at least in part, upon the discovery that an inversion of the mathematical model described above can serve to approximate a perfect theoretical flowgram from a dirty observed flowgram. Thus, continuing the example from above an approximation of error may be applied to actual sequencing data represented in an observed flowgram resulting in a perfect or substantially perfect theoretical flowgram representation of the actual sequence data with all or substantially all of the error factors removed.
Those of ordinary skill in the related art will appreciate that the accurate removal of error from data provides for a more efficient and accurate interpretation of said data. Thus, for instance, removing error from data generated in a sequencing run results in more accurate production of calls identifying each nucleic acid species in a sequence generated from a.
Some embodiments of the presently described invention include systems and' methods for analyzing data generated from SBS sequencing runs on a sequencing apparatus. Some examples of SBS apparatus and methods may employ what may be referred to as a pyrophosphate based sequencing approach that may, for instance, comprise one or more of a detection device such as a charge coupled device CCD camera, a microfluidics chamber, a sample cartridge holder, or a pump and flow valves.
Taking the example of pyrophosphate based sequencing, embodiments of an apparatus may use chemiluminescence as the detection method, which for pyrophosphate sequencing produces an inherently low level of background noise. In the present example, the sample cartridge holder for sequencing may include what is referred to as a "picotiterplate" formed from a fiber optics faceplate that is acid-etched to yield hundreds of thousands of very small wells each enabled to hold a population of substantially identical template molecules.
In some embodiments, each population of substantially identical template molecule may be disposed upon a solid substrate such as a bead, each of which may be disposed in one of said wells. Continuing with the present example, an apparatus may include a reagent delivery element for providing fluid reagents to the picotiterplate holders, as well as a CCD type detection device enabled to collect photons emitted from each well on the picotiterplate.
Further, the systems and methods of the presently described embodiments of the invention may include implementation on a computer readable medium stored for execution on a computer system. For example, several embodiments are described in detail below to process and correct error in signals detected using SBS systems and methods implementable on computer systems.
A computer may include any type of computer platform such as a workstation, a personal computer, a server, or any other present or future computer. Computers typically include known components such as a processor, an operating system, system memory, memory storage devices, input-output controllers, input-output devices, and display devices.
It will be understood by those of ordinary skill in the relevant art that there are many possible configurations and components of a computer and may also include cache memory, a data backup unit, and many other devices. An interface controller may also be included that may comprise any of a variety of known or future software programs for providing input and output interfaces. For example, interfaces may include what are generally referred to as "Graphical User Interfaces" often referred to as GUI's that provide one or more graphical representations to a user.
Interfaces are typically enabled to accept user inputs using means of selection or input known to those of ordinary skill in the related art. In the same or alternative embodiments, applications on a computer may employ an interface that includes what are referred to as "command line interfaces" often referred to as CLI's. CLI's typically provide a text based interaction between an application and a user. Typically, command line interfaces present output and receive input as lines of text through display devices.
For example, some implementations may include what are referred to as a "shell" such as Unix Shells known to those of ordinary skill in the related art, or Microsoft Windows Powershell that employs object-oriented type programming architectures such as the Microsoft. NET framework. Those of ordinary skill in the related art will appreciate that interfaces may include one or more GUI's, CLI' s or a combination thereof.
For example, a multi-core architecture typically comprises two or more processor "execution cores". In the present example each execution core may perform as an independent processor that enables parallel execution of multiple threads. In addition, those of ordinary skill in the related will appreciate that a processor may be configured in what is generally referred to as 32 or 64 bit architectures, or other architectural configurations now known or that may be developed in the future. An operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages.
An operating system, typically in cooperation with a processor, coordinates and executes functions of the other components of a computer. An operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. System memory may include any of a variety of known or future memory storage devices. Examples include any commonly available random access memory RAM , magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, or other memory storage device.
Memory storage devices may include any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard disk drive, USB or flash drive, or a diskette drive. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. In some embodiments, a computer program product is described comprising- a computer usable medium having control logic computer software program, including program code stored therein.
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The control logic, when executed by a processor,. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts. Input-output controllers could include any of a variety of known devices for accepting and processing information from a user, whether a human or a machine, whether local or remote. Such devices include, for example, modem cards, wireless cards, network interface cards, sound cards, or other types of controllers for any of a variety of known input devices.
Output controllers could include controllers for any of a variety of known display devices for presenting information to a user, whether a human or a machine, whether local or remote. In the presently described embodiment, the functional elements of a computer communicate with each other via a system bus. Some embodiments of a computer may communicate with some functional elements using network or other types of remote communications.
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Also a computer may include one or more library files, experiment data files, and an internet client stored in system memory. For example, experiment data could include data related to one or more experiments or assays such as detected signal values, or other values associated with one or more SBS experiments or processes. Additionally, an internet client may include an application enabled to accesses a remote service on another computer using a network and may for instance comprise what are generally referred to as "Web Browsers".
Also, in the same or other embodiments an internet client may include, or could be an element of, specialized software applications enabled to access remote information via a network such as a data processing application for SBS applications. A network may include one or more of the many various types of networks well known to those of ordinary skill in the art. A network may include a network comprising a worldwide system of interconnected computer networks that is commonly referred to as the internet, or could also include various intranet architectures.
For example, firewalls may comprise hardware or software elements or some combination thereof and are typically designed to enforce security policies put in place by users, such as for instance network administrators, etc. Examples of SBS embodiments typically employ serial or iterative cycles of nucleotide species addition to the template molecules described above.
These cycles are also referred to herein as "flows". For example, in each flow either one of the four nucleotide species, A, G, C or T is presented e. Continuing with the present example, a flow may include a nucleotide specie complementary to the nucleotide specie in the template molecule at the sequence position immediately adjacent to the 3' end of the nascent molecule being synthesized, where the nucleotide specie will be incorporated into the nascent molecule.
After each iteration of a flow of a nucleotide specie, a wash method is implemented to remove the unincorporated excess of the nucleotide specie and reagents. Upon completion of the washing stage, the next iteration of a flow presents another nucleotide specie, or mix of nucleotide species, to the template-polymerase complex.
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In some embodiments a "flow cycle" may refer the addition of four nucleotide species either iteratively or in parallel where for instance one flow cycle includes the addition of all four nucleotide species. When charted on a flowgram, a value for the detected light or other signal for each flow may be about zero indicating a nucleotide specie in the flow was not complementary to the nucleotide specie in the template at the next sequence position and thus not incorporated , or about one indicating incorporation of exactly one nucleotide specie complementary to the nucleotide specie in the template was detected , or about an integer greater than one indicating incorporation of 2 or more copies of the nucleotide specie presented in the flow complementary two consecutive nucleotide specie in the template were detected.
As described above, a theoretical outcome for an iterative series of flows results in a signal from each flow that should be either exactly zero, or an integer and represented in a perfect flowgram. Through various experimental variations that include CF and IE mechanisms, the actual detected signals tend to fluctuate around these expected theoretical values by varying amounts. The detected signals that include this variation are represented as a dirty or observed flowgram.
The terms flowgram and pyrogram are used interchangeably herein. The terms "perfect flowgram", "clean flowgram" and "theoretical flowgram" are used interchangeably herein. The terms "dirty flowgram", "real-life flowgram" and "observed flowgram" are used interchangeably herein. Further, as used herein, a "read" generally refers to the entire sequence data obtained from a single nucleic acid template molecule or a population of a plurality of substantially identical copies of the template molecule.
A "nascent molecule" generally refers to a DNA strand which is being extended by the template-dependent DNA polymerase by incorporation of nucleotide species which are complementary to the corresponding nucleotide species in the template molecule. The term "completion efficiency" as used herein generally refers to the percentage of nascent molecules that are properly extended during a given flow. The term "incomplete extension rate" as used herein generally refers to the ratio of the number of nascent molecules that fail to be properly extended over the number of all nascent molecules.
Some embodiments of the presently described invention correct the detected signals of each flow to account for the CF and IE mechanisms described above.
For example, one aspect of the invention includes calculating the extent of phasic synchronism loss for any known sequence, assuming given levels of CF and IE. Furthermore, an IE rate of no greater than 0. Table 1. It will be understood that the values presented in Table 1 are for the purposes of illustration only and should not be considered limiting. Those of ordinary skill will appreciate that several factors may contribute to variability of values such as the genomic or reference sequences and other parameters used to formulate predictions.
As described above, correction of CF and IE is desirable because the loss of phasic synchronism has a cumulative effect over the read length and degrades the quality of a read as read length increases. In one embodiment of the presently described invention, values representing both CF and IE are assumed to be substantially constant across the entire read of a substantially identical template molecule population, such as for instance a population of template molecules residing within a single well of a picotiterplate system. This permits numerical correction of each sequence position across the entire read using two simple parameters "incomplete extension" and "carry forward" without any a priori knowledge of the actual sequence of the template molecule.
The system and methods of the presently described embodiments of the invention are useful in determining, and correcting for, the amounts of CF and IE occurring in a population of template molecules. For example, embodiments of-the invention correct the signal value detected from each flow for each population of substantially identical template molecules residing in each well to account for CF and IE. Embodiments of the present invention model the lack of phasic synchronism as a nonlinear mapping: Equation 1 :. A model for such a mapping formula can be generated by, for example, analyzing the errors that are introduced to an observed flowgram q by sequencing a " polynucleotide template molecule having a known sequence.
An illustrative. For example on the left hand side of Figure 1, theoretical flowgram is an illustrative representation of a theoretical perfect or ideal flowgram p , that shows an idealized signal strength value depicted in brackets next to its associated nucleotide specie. Each idealized value of theoretical flowgram is an integer or zero. On the right hand side of Figure 1, observed flowgram is an illustrative representation of a detected signal strength value from an observed or simulated dirty flowgram q.
Similarly, each signal strength value in flowgram is depicted in brackets next to its associated nucleotide specie. Also on the right hand side of Figure 1 is flow that provides a representative number representing the iterative flow sequence associated with a nucleotide specie and signal values e. For instance, flow 1 as illustrated in Figure 1 is associated with the "C" nucleotide specie introduced in said iteration of flow and a corresponds to a signal value for both theoretical flowgram and observed flowgram In the example of Figure 1 the differences in signal strength values between theoretical flowgram and observed flowgram for the each flow iteration is indicative, at least in part, of a loss of phasic synchronicity.
For instance, the signal values represented in observed flowgram are not integers, rather each are typically. Mapping model represented as "M", may be estimated using known values for parameters Parameters may be employed to estimate mapping model and convert the signal values of the theoretical flowgram p into the observed values q In the present example, the error value represented by mapping model accumulates with each iteration of flow , and grows exponentially.