Patent Grant
8032310
The Sequence Listing submitted on compact disc (2 copies) as part of the second preliminary amendment to the application is incorporated herein by reference. The file name is “11177647 Seqence Listing.ST25”. The CD was created on Jul. 27, 2006 and the file contains 44,716 bytes.
A portion of the disclosure of the patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure as it appears in the U.S. patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
1. Field of the Invention
The present invention is relevant to a variety of automated selection systems including automated sub-sequence selection systems for usage with any method group of nucleic acid or protein sequences generated by alternative methods. The system is configured to automatically select sets of sub-sequences from incomplete nucleotide sequence data obtained from resequencing DNA microarrays, according to parameters predetermined by the system or determined by a user, for selecting sequence subsets that are optimally suitable for comparison against a collection of predetermined database sequences using one or more similarity search algorithm(s). Embodiments of the invention also enable the further analysis and presentation of relevant results returned by a similarity search resulting from submission of one or more subsequences. Aspects of the invention described herein distinguish between combinations of sequence signatures that arise from a mixture of multiple sequence targets (e.g. microbial organisms) or from a rearrangement of sequences within a single target. Embodiments of the method are also capable of assigning relative abundances of mixed target sequences based on relative signal intensity values from the DNA microarray itself. Moreover, an aspect of the invention is an integral component of an iterative process for designing resequencing DNA microarrays using “prototype” sequence tiles to represent a range of related target sequences (e.g. pathogens).
2. Description of the Related Art
The convergence of biology with engineering and computer science has led to the emergence of biotechnology and bioinformatics which, among many other goals, aim to rapidly obtain and analyze genomic and proteomic sequence information for diagnosis of disease. The experimental viability and widespread availability of such methodologies are due in no small part to the emergence of DNA microarrays (Stenger et al., 2002).
Generally, microarray fabrication applies methods of microprocessor manufacturing to create “gene chips” capable of rapidly and reliably identifying sequences of DNA or proteins that are present in a biological sample. Here, the term “microarray” refers to any type of planar substrate or alternative matrix presenting a high multiplicity (102 to 106) of individual sites, each presenting probes (immobilized nucleic acids or antibodies) designed to selectively capture complementary strands of a target (i.e. gene or gene transcript) analytes in solution. By design, DNA microarrays enable the simultaneous interrogation of thousands of gene or gene transcript elements.
In using a resequencing DNA microarray for genetic analysis, a solution containing amplified and fluorescently-tagged genetic targets are passed across the microarray comprised of a plurality of oligonucleotide probes in a “tiled” format (Kozal et al., 1996). Complementary sequences in the sample bind to the corresponding probes contained on the microarray. The microarray is then analyzed using, for example, a laser scanner that records the intensity of light emission from the microarray's probes. The recorded intensities are then analyzed by array-specific software used to make “base calls,” which is a term describing an algorithmic method of identifying to a certain degree of probabilistic certainty the sequence of nucleic acids (adenine; A, thymine; T, cytosine; C, or guanine; G) contained in the biological sample of interest. A broader IUPAC definition code is also used to describe less precise base calls (see U.S. Provisional Application Ser. No. 60/590,931 filed on July 2, 2004 entitled “Resequencing Pathogen Microarray”, supplemental data, Appendix J “gdas_manual pdf” page 255). If the target sequence is sufficiently homologous to the appropriate tile region of the resequencing microarray (fewer than 1-2 base substitutions per 25 bases) then a complete resequencing of the target is possible. However, the hybridization to the tile region is interrupted when the target sequence contains insertions, deletions, or base substitutions at frequencies of greater than 2 substitutions per 25 bases of target sequence. This results in the “no [base] call” (N) being made from the corresponding sequences on the microarray tile region. N calls also result when the concentration of the target nucleic acid in solution is low or when there are interfering level of competing background nucleic acids in the hybridization solution. Incomplete biological sequence information can also be generated by a number of other nucleic acid and protein sequencing technologies.
The primary intended application of resequencing microarrays is to detect low probability single nucleotide polymorphisms (SNPs) or mutations within a limited range of target sequences. However, although not conventionally performed currently in industry, sequence output of the microarray can also be compared against sequence databases to allow identification of target sequences. The most prevalent comparison method, or similarity search algorithm, for sequence data currently in use is Basic Local Alignment Search Tool, commonly known as and referred to herein as “BLAST.” Numerous variants exist, including Washington University BLAST (WU-BLAST), NCBI-BLAST, FASTA, MPsrch, Scanps, and BestFit (Korf, Yandell & Bedell, 2003). Such comparisons generally yield a number of possible matches in terms of certainty (measured probabilistically) that the tested sample includes the matched biological subject for which a sequence is known. The sequence output by the intensity analysis of the microarray is then often compared to a database that includes known sequences of biological subjects which could include pathogenic microbes. However, one normally skilled in the art of molecular biology would not be capable of visually determining the best sequence sections from a tiled region containing A, C, T and G base calls punctuated and in some cases dominated by varying numbers of no-calls (N).
The use of microarrays for the purposes of genetic sequencing and identification has drastically increased the capability of even a single researcher to extract a large amount of sequence data from a biological sample for comparison against an even larger number of previously sequenced organisms and biological substances. However, the researcher is unable to utilize the information in a time-effective manner. Ambiguous results are also problematic for a researcher submitting sample sequences for comparison due to excessive wait times and poor (inconclusive or conflicting) results associated with attempts to match ambiguous subsequences. Accordingly, a widely-practiced method of obtaining more relevant results from sequence comparison is for a researcher to review sequence output searching for subsequences that appear to have a higher probability of returning a relevant result. In particular, many researchers often find themselves manually and subjectively selecting, or visually parsing, certain subsequences for comparison against those in the sequence database. As a result, a researcher expends time and resources for relatively slowly and subjectively optimizing the sequence data for submission to the similarity search. Thus, the current solution for the above-noted resource utilization problem leads to additional time and resource requirements demanded of the researcher. Moreover, as the current solution is subjective as well as time-intensive, the net gain with respect to facilitating the advancement (and acceleration) of genomic research is ambiguous at best.
However, as noted above the vast repositories of known biological sequences are often contained in shared computing resources. These shared computing resources require vast amounts of data storage capacity, as well as robust and powerful tools with which to compare a submitted sequence to those contained within the database. As the amount of sequence data produced (and submitted) by researchers increases with the improvement and increasing availability of microarrays for general research use, the burden placed on shared databases (and associated systems) in terms of bandwidth and processing requirements increase dramatically. In other words, the increase in data made possible by widespread use of microarrays often leads to less efficient utilization of shared bioinformatics computing resources.
For example, if sequences containing a large percentage of ambiguous sequence data (Ns) are submitted, the sequence database's computing resources will be spent trying to find matches for inherently ambiguous sequences, resulting in all possible similarity search results with low certainty values.
The submitted sequence(s) 103 when compared to database records, 109 might or might not return statistically significant or meaningful results. Here, by definition, to “compare” means to perform a similarity search of a query sequence against a database of sequence records using any one of a large number of algorithms for determining similarity (e.g. BLAST). Sequendes that are said to be “comparable” have a sufficient degree of similarity to at least one sequence in a database to result in the return of at least one statistically significant (user defined) result. It is straightforward for an end user to visually identify and select contiguous stretches of nucleotide base calls (comprised of only A, T, C, or G residues) or amino acids that might be comparable. However, as the number or percentage of “Ns” contained within target sequences increases, it becomes exponentially more difficult for the end user to visually determine the comparability of either the entire sequence or subsequences within it.
The results 111 include high probability matches 111a, lower probability matches 111b, and a significant number of statistically insignificant results 111c that can be attributed to a chance match with the database. Ns are treated as “aNy” (wild card) characters by similarity search algorithms meaning the N could be any of the four base residues or gap when the default parameters are used. In the case of a resequencing DNA output, an N indicates the resequencing algorithm could not resolve the call and can correspond to any of the four base residues (A, T, C or G) or to empty space (Korf et al., 2003). In the case that too many non-calls (Ns) are included in the submitted sequence, then the similarity search (e.g. BLAST) will calculate E (expect) values higher than the acceptable E (expect) value (e.g. 1.0e−9) indicating the chances are greater that the returned sequence is not unique. Similarly, shorter sequences may have higher E values indicating their lack of use to the end user in determining the presence of unique DNA material. The results 111, including the numerous ambiguous results 111c, are then left to be analyzed 113 by the researcher.
In the case of
b) illustrates this alternative case often found in practice in the industry that is problematic with regard to researcher time consumption. In contrast to the previously illustrated case, the sequence data 103 is altered in a cut and paste operation 119 performed manually by a human researcher. More specifically, the human researcher often visually scans the raw data output and subjectively copies and pastes subsets of the raw data output 119 that appear to contain fewer “Ns” and submits these subjective selections 121 for comparison 109. However, as the selection of subsets is performed subjectively and repetitively for a large amount of raw data, the human-selected submissions 121 often include comparable 121a and non-comparable 121b data. Consequently, the results from the BLAST comparison 123 still include a wide array of possible matches, ranging from high probability matches 123a, to low probability matches 123b which are often caused by selections in which there are to many non-calls 123c as opposed to the anticipated result of a low probability match caused by a less similar sequence match.
As discussed above,
A variety of different factors can contribute to the inability of a resequencing DNA microarray to make non-ambiguous base calls. In pure target samples, the hybridization patterns necessary for base calling (Cutler et al., 2001; Kozal et al., 1996) are interrupted whenever a stretch of target sequence is sufficiently dissimilar from the probe sequences that are tiled on the microarray surface. This results in the introduction of N calls into the interrupted positions of the resequencing microarray output file. The same effect occurs when the target molecules are present in low concentration and/or when the target sample is not pure but contains varying amounts of other nucleic acid molecules that can bind non-specifically to the tiled probes with low affinity, resulting in a lowered signal-to-noise ratio of hybridization (fluorescence) signals across the probe sets. To illustrate how these factors can determine whether sequences are comparable or non-comparable data,
Overall, the above-noted problems with the current state of industry practice are fundamentally related to researcher time consumption and shared resource allocation. More specifically, the increased amount of subsequence data obtained from samples results in rapid increases in the utilization of shared resources such as sequence comparison databases. Such rapid increases necessitate efficient use for supporting a growing community (in terms of researchers and data). With the aim of using shared resources more effectively, researchers are now often faced with the need to devote time and resources to subjectively and manually selecting sequence subsets for comparison.
As stated above, there is a critical need for advanced diagnostic systems that can rapidly detect both known and unanticipated sequences. More particularly, there remains a critical demand for DNA microarray techniques that reduce the need for human work input and increase the efficiency of shared resource utilization, especially in the case of shared similarity search databases and systems.
In addition to the above-described problems in the industry regarding more effective use of researcher and shared computing resources, the evolution of world events and the emergence of infectious disease and bioterrorism in mainstream society have led to a growing sentiment amongst the scientific community and lay people alike that new, rapid, and accurate techniques for threat identification and eradication must be developed. The concept of broad-spectrum pathogen identification has considerable and obvious appeal to both medical practice and national defense. It is within this framework that the present inventors have endeavored. Furthermore, there remains a need for more ready and robust determination of mixtures and recombinants in biological samples from biological sequence data, regardless of the source of the sequence data.
A first aspect of the present invention provides a computer-implemented biological sequence identifier (CIBSI) system and method for selecting a subsequence from biological sequence data according to at least one selection parameter. The at least one selection parameter corresponds to a likelihood of returning a meaningful result from a similarity search.
One embodiment of the present invention provides a system for automatic selection of optimal sequences or subsequences for comparison against a predetermined set of known sequences. Optionally, the system automatically takes a highly fragmented sequence interspersed with Ns, and selects comparable subsequences that are likely to return a “meaningful” result from a similarity search.
Optionally, the system utilizes a sliding window-type algorithm for selecting subsequences. Subsequently, the system automatically returns outputs from a similarity search to the end user that allow probabilistic assignments of the likelihood that a given set of base calls correspond to a particular predetermined sequence.
In an additional embodiment of the present invention, sequence information derived from a base-calling algorithm, as applied to a microarray hybridization pattern, is used to identify individual biological entities present in a test sample. Optionally, the sequence of target sequences determined by resequencing probes of the microarray is used to query a database using a similarity search algorithm. Similarity search algorithms include, but are not limited to, commonly used local alignment (e.g. Smith-Waterman, BLASTN) sequence alignment algorithms to statistically determine the probability that a given target sequence corresponds to a specific sequence in a database record (Korf et al., 2003).
Further, an additional embodiment of the present invention presents results of the similarity search to a user regarding whether at least one target sequence is present in the sample.
In another embodiment of the present invention, signal intensity data (for example, obtained from a microarray) is handled by the system and associated with the sequence data. The output of a similarity search is assembled or distilled for presentation to a user to communicate whether a biological entity (including, but not limited to, a pathogen) is present or not. Further, intensity data is correlated with the above output to ascribe relative abundances of a present biological entity (including, but not limited to, a pathogen). Optionally, the system provides an end user with an estimate (quantization) of the relative amount of pathogen that was detected in a resequencing microarray assay.
A second aspect of the present invention identifies mixtures of sequences and sequences indicating a recombination event. In one embodiment, the system automatically detects overlapping or homologous sequence fragments on different tiled regions of a microarray, allowing inference of a mixture of sequences. In an additional embodiment, the system determines that the sequence outputs from different tiled regions are not overlapping but correspond to a contiguous sequence that may be used to infer a genetic recombination event. Optionally, the system discriminates between mixtures of different and genetic recombination between different sequences.
A third aspect of the present invention provides a method of designing “prototype” regions (see U.S. Provisional Application Ser. No. 60/590,931) of resequencing DNA microarrays. In this case, a set of related target sequences are compared using a multiple sequence alignment algorithm such as ClustalW or Clustal_X (Thompson et al., 1997; Thompson, Higgins & Gibson, 1994) or another method of searching for sequence databases for partially conserved regions such as HMMer (Eddy, 1998) the outputs of which are used to create a consensus sequence comprised of the most frequent nucleotides at a given residue position in an alignment column. The consensus sequence consists of a mixture of consensus base calls and no-calls (Ns) where no consensus can be achieved for each of the residue positions in the alignment columns.
Optionally, an embodiment of the present invention searches for candidate regions to tile onto a resequencing DNA microarray by determining those regions having the acceptable balance of conserved and variable nucleotides to allow hybridization of target to the resequencing microarray tile region but still allow for enough sequence variability. This will allow sequence similarity search-based identification of the target sequences comprising the alignment used to generate the consensus.
In alternative embodiments, nucleotide or amino acid sequences come from an alternate form of sequence generator, including those described in (Shendure et al., 2004) and partial amino acid sequences that may be assembled to form a protein sequence. Selected embodiments of the system handle amino acid or protein sequence data in which relative position is conserved.
In other selected embodiments, the nucleotide sequences include a ribonucleic acid (RNA) transcript that codes for protein synthesis. In a manner similar to that described for determining mixtures versus recombination events in target nucleic acids, a mixture of RNA transcripts can be hybridized and thus resequenced on a tiled microarray to produce raw data that can be analyzed using the present invention to determine relative quantities of different RNA transcripts as compared to recombination through transcript editing and alternative splicing (Leipzig, Pevzner & Heber, 2004).
Additional embodiments of the present invention are applicable to nucleotides, transcriptional products, amino acids, or any mixture thereof. Further, the present invention is also applicable to various types of sequence databases and similarity search algorithms to the extent that is well known in the art. Moreover, embodiments of the present invention are suited or adaptable for a wide range of methods and/or devices that generate a sequence data, including, but not limited to, manual or automated Sanger sequencing, shotgun sequencing, conventional microarrays, resequencing microarrays, microelectrophoretic sequencing, sequencing by hybridization (SBH), Edman degradation and variants thereof, Cyclic-array sequencing on amplified molecules, Cyclic-array sequencing on single molecules, and non-cyclical, single-molecule, real-time methods such as nanopore sequencing (Shendure et al., 2004).
The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention. Other systems, methods, features, and advantages of the present invention will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present invention, and be protected by the accompanying claims.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
a) is an exemplary flowchart illustrating the process currently in use by the industry;
b) illustrates an alternative case often found in practice in the industry;
c) is a schematic drawing of a general system layout for interaction with a comparison database and server;
d) shows an example of a sequence output from analyzing a microarray demonstrating poor hybridization (the four sequences are, in order, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7);
a) is an exemplary schematic drawing of an embodiment of the system;
b) is an exemplary schematic drawing of an embodiment of the Resequencing Pathogen Identifier (REPI);
c) is an exemplary screen shot of an interface for an ASP;
a) is an exemplary flowchart of the extract subsequence step;
b) is an example of a sliding window according to one embodiment of the system (SEQ ID NO.8);
a) is an exemplary flowchart describing in more detail the actions of the system within the Analyze Step;
b) illustrates an exemplary output of an embodiment of the invention (the four sequences are, in order, SEQ ID NO.9 and SEQ ID NO.10);
The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views. Herein, “meaningful” relates generally to a predetermined level of statistical significance or certainty of a result. Alternatively, meaningful indicates a predetermined level of usefulness to a user for drawing a conclusion regarding the presence of a specific biological entity or group of entities. For example, BLAST returns an E-value (corresponding to a probability), where the product of the E-value with the number of known sequences (probability times opportunities) within the database corresponds to the total number of sequence database records that could return the same similarity value (bit score) for the queried subsequence. If a reshuffling of the submitted test sequence returns the same E-value, the original result was not meaningful. Further, the term “Comparable” used herein refers generally to data that includes a sufficient amount (for example, a system or user-defined threshold number or percentage) of actual base calls (non-Ns) to return meaningful results from a similarity search. Additionally, the term “Comparable” can be used interchangeably with respect to the usefulness of results returned to a user from a similarity search using the data. Conversely, the term “non-Comparable” refers generally to data that includes a sufficient amount (in number or percentage) of non-base calls (Ns) to cause less meaningful or ambiguous results from a similarity search.
The system, including the REPI (Resequencing Pathogen Identifier), is designed to automatically and algorithmically parse an output of a incomplete nucleotide or polypeptide sequences by selecting and editing sequence data into subsequences more suitable for sequence similarity searches. To accomplish this objective, the system includes several functional steps, or filters, to modify the data as little as possible while extracting Comparable data from the sequence data. As described above, due to the nature of the resequencing microarray, the sequences often contain large amounts of non-base calls (Ns). Similarity searches such as BLAST typically return ambiguous results for sequences with a large amount of non-base calls. Examples of ambiguous results include, but are not limited to low bit scores and expect (E) values that do not predict a unique similarity match. Therefore an embodiment of the present system extracts those portions, or subsequences, of original sequences that are most likely to return meaningful results from a similarity search.
a) is an exemplary schematic drawing of an embodiment of the present invention. Sequence data 203 are first processed by an Automated Subsequence Parsing module (ASP) 209 of the system 208. The ASP 209 filters the sequence data 203 and selects only those subsets which are likely to result in predetermined probability (e.g. a BLAST expect value of <1.0E−9) matches 211a. These subsets are then compared to a sequence database 109 using, for example, a similarity search algorithm, and the results are returned to the system for further analysis and summarization 214. The resulting search summary 213 is then presented to a user for more in depth analysis 215. Because sequence subsets inherently unlikely to return statistically relevant (e.g. a BLAST expect value of <1.0e−9) search results are removed by the ASP, the results 213 returned to the user generally include a higher proportion of significant (e.g. a BLAST expect value of <1.0e−9) matches 213a than the conventional method of submitting an unparsed sequence. In particular embodiments using a BLAST or BLAST-like similarity search algorithm, expect values between 1.0e−150 to 2 are preferable. More preferably, expect values are between 1.0e−5 and 0.5. Even more preferably, embodiments involving BLAST include a bit score and expect value that allows the unique identification of a single pathogen database record. Optionally, the system 208: further filters results in the summary to display only those subsequence matches meeting criteria set by a user or predetermined by the system. These criteria can include but are not limited to: bit score, expect value (chance that another sequence could result in an identical score), or another score derived from the relative positions of subsequences or the microarray signal intensities of regions used to make base calls. Also illustrated in
b) is an exemplary embodiment of REPI, itself an embodiment of the system. Sequence data 203′ are first processed by an Automated Subsequence Parsing module (ASP) 209′ of the REPI 208′. The ASP 209′ filters the sequence data 203′ and selects only those subsets which are likely to result in high probability matches 211a′. These subsets are then compared to a sequence database 109′ using a similarity search algorithm, and the results are returned to the REPI for further analysis and summarization 214′. The resulting search summary 213′ is then presented to a user for more in depth analysis 215′. As sequence subsets inherently unlikely to return statistically relevant search results are removed by the ASP, the results 213′ returned to the user generally include a higher proportion of significant matches 213a′ than the conventional method of submitting an unparsed sequence. Optionally, the REPI 208′ further filters results in the summary to display only those subsequence matches meeting criteria set by a user or predetermined by the system. Also illustrated in
c) is an exemplary screen shot of an interface (a graphical user interface in this case) for an embodiment of the system in software form. This particular embodiment can interface with network and local BLAST servers (either or both), and enables the modification of several common BLAST parameters 251. Additionally, as described in more detail below, parameters specific to the parsing algorithm 253, such as the window size, are optionally made available to a user.
In addition to its flexibility with respect to sequence data formats, the system accepts sequence data from a variety of different source types. As described above, these types include, but are not limited to, manual or automated Sanger sequencing, shotgun sequencing, conventional microarrays, resequencing microarrays, microelectrophoretic sequencing, sequencing by hybridization (SBH), Edman degradation and variants thereof, Cyclic-array sequencing on amplified molecules, Cyclic-array sequencing on single molecules, and non-cyclical, single-molecule, real-time methods such as nanopore sequencing. Alternatively, the raw sequence S301 can be comprised of transcript nucleic acids (messenger ribonucleic acid (mRNA) or intermediate phase sequences used for viral transcription and translation. For example, in an embodiment of the invention directed towards RNA transcripts, RNA can either be hybridized directly to an array after it is fragmented (as done with Affymetrix gene expression arrays) or converted to DNA using reverse transcriptase. Tile regions are constructed from exon (protein coding sequences) regions of the genome and a resequencing array is used to analyze which of the sequences made it into transcripts. In alternative embodiments, the process described in
In a resequencing microarray, the structure of the overall gene sequence is suggested by the position of the partial sequences within the overall structure of the resequencing tile. For example, the resequencing array may only give reads of 5-10 consecutive base calls at a time, each of which are separated by a consecutive series of Ns.
The process for formatting the sequence data extracted from a resequencing microarray is depicted in the flowchart shown in
Each identified partial sequence is then compared against a stored reference sequence S2409 to determine whether the partial sequence corresponds to one of the stored reference sequences. This comparison will yield a statistical value indicating the similarity between the stored reference sequence and the partial sequence. Then, if the statistical value is beyond a predetermined threshold, the partial sequence is stored S2413 to be combined with other partial sequences SS2415. Alternatively, if the statistical value is below a predetermined threshold, the partial sequence is discarded. This process continues until all the partial sequences are compared, thus generating a composite set of data to be submitted to the CIBSI, as discussed below.
The system then performs a Control Check S303 before extracting a candidate subsequence S305. After extraction of a candidate subsequence, the system then trims the non-calls (Ns) from the beginning and end of the candidate subsequence S307. Then the system checks the length of the trimmed candidate subsequence S309 to determine whether an alternative sequence or subsequence should be selected S311, the candidate subsequence is ready to go to a similarity search server or be added to a similarity search queue S315, or whether an additional check of the percentage or proper base calls in the subsequence meets an acceptable threshold for sending it to be compared (using a similarity search). The selection of an alternative sequence or subsequence S311 is accomplished in one embodiment through the use of a sliding window algorithm. For those sequences that are sent to be compared S315, the system gathers the results returned from the similarity search server, performs statistical analysis on these results and filters them for the user according to user preferences S317. Optionally, the system can simply return similarity search results for the submitted subsequences. Also, as will be made more evident given the exemplary embodiments described below, the steps of the algorithm can be rearranged or modified in alternative embodiments. Further, as also described in more detail below, the behavior of the system is predetermined by the system or optionally defined by the user.
The following paragraphs describe each of the main functional steps shown in
The calculated percentage is then compared to a First Jump Threshold parameter, A (for example, 25%), which can either be predetermined by the system or selected by a user S507. If the calculated percentage of actual base calls within the window does not satisfy the criteria defined by the First Jump Threshold, A, the system advances the window a number of base calls S509 according to a First Window Jump parameter, X, that may also be predetermined by the system or selected by the user, but is preferably between one and Z, the Window Size parameter. Advancement of the window at this or any other point can occur in any direction (for example, towards the end of the sequence). In the case of the calculated percentage of actual base calls within the window satisfies the criteria defined by the First Jump Threshold A, the system marks the beginning of a candidate subsequence S511 at the beginning of the sliding window. The window is then moved incrementally a number of base calls S513 according to a Second Window Jump parameter, Y, and at each slide increment, the percentage of actual base calls within the window is calculated S515. If the calculated percentage of actual base calls within the candidate subsequence fails to meet the criteria set by a Second Jump Threshold B, the system marks the end of the candidate subsequence at the base call of the sequence corresponding to the base call at the end of the window S519. For each sequence, the system searches for the largest continuous string(s) of usable data. As the Window Jump parameters X and Y increase, the module becomes more permissive with respect to selecting candidate subsequences with a greater number of non-base calls. As the Jump Thresholds A and B increase ceteris paribus, the module becomes less permissive.
b) is an example of a sliding window algorithm according to an embodiment of the present invention. The beginning and end positions of the sliding window 551 determine the length and contents of the candidate subsequence 553. This subsequence is then trimmed in a Trim function described below.
The next function of the system is a length evaluation S309.
In addition to receiving results from the similarity search regarding submitted subsequences, the system optionally provides further analysis of the submitted subsequences.
The analysis performed and statistics calculated by the system include, but are not limited to, the selected subsequence length as a percentage of the sequence and the subsequence length in base calls, which together can be used to indicate what portion of the target biological entity gene was identified. The subsequence length and percentage of subsequence base calls allow a researcher to monitor the system's algorithms and functional steps. Further, in the case of resequencing microarrays, threshold parameters for base calling algorithms including, but not limited to, GDAS, can be monitored. In an alternative embodiment, the system accepts and also formats statistical results returned from the similarity search, allowing the user to manipulate and organize results using a provided graphical user interface.
Optionally, the system can store all of the outputs returned by a similarity search and analysis described above S907, including, for example, BLAST results. The system also optionally displays S909 all of the results or a subset of the results returned by a similarity search and/or calculated by this system to the user. Certain embodiments of the present invention then allow these results to be sent and saved for archiving or transfer S911.
The following table illustrates exemplary ranges and preferable subranges for several exemplary parameters and thresholds described above.
Preferred subranges:
In yet another embodiment of the system, the search (comparison) output can be analyzed for Optimization Parameters, J, S913. As described above, Parameters and Thresholds of the system, including but not limited to A, B, X, Y, E, F, and H, are set by the user or predetermined by the system. Alternatively, an embodiment of the system enables the optimization of one or several of these variables by the system itself or through the use of a complementary functional module. For example, optimization of a Parameter or Threshold could be performed in accordance with well-known optimization (for example, SIMPLEX linear programming) or Artificial Intelligence (including state space search methods such as uniform or heuristic search) techniques by analyzing system performance recorded over the course of multiple uses or “passes” of system operation. Corresponding Optimization parameters, J, can be used, for example, to automatically alter the various parameters and thresholds used in the previous steps and run the process again using these new optimized parameters S917. Alternatively, instead of starting again from the raw data file, the Optimization Parameter J can be used to refine output of selected subsequences S915 by using the Optimization Parameters J to alter parameters related to, for example, the behavior or function of a similarity search algorithm used in conjunction with the system. The Optimization Parameters can be adjusted by a user or the system itself to enhance system performance with respect to, for example, speed or relevant/meaningful similarity search results.
The description of the present invention is further aided by the following detailed example. The following detailed example corresponds to an exemplary embodiment of the invention and is not intended to set forth restrictions on the invention with regard to system operation, parameter settings, sequence data, etc.
An embodiment of the REPI was used to ascertain which base call subsequences from the CustomSeq/GCOS/GDAS process would likely return significant BLAST results through the use of a customized sliding window algorithm. Subsequently, REPI automatically returns BLAST outputs to the end user that allow probabilistic assignments to the likelihood that a given set of base calls correspond to a particular microbial sequence. Moreover, the REPI automatically links sequence fragments to individual pathogens.
Raw sequence data from the resequencing microarray chips is provided by the Genetic Data Analysis Software version 2.0 (GDAS) packaged with the microarray reader. from Affymetrix. GDAS base calling is based on a previously described base-calling algorithm (Cutler et al., 2001). Each of the FASTA output files containing the base calls obtained from the GDAS software was analyzed using specialized software (an embodiment of REPI) that is an exemplary embodiment of the present invention described herein.
In the case of the present examples, the sequence output of GDAS is most often a scattered mixture of contiguous sequence calls (A, T, C or G) that are interspersed with varying amounts of no-calls (Ns) where the GDAS software does not make a base call due to weak hybridization signal on the chip and/or high background hybridization caused by non-specific binding (Cutler et al., 2001). An example output of the GDAS output (including SEQ ID NO:1) for an Adenovirus 4 (+) clinical sample for the Ad4FIBER tile region is shown below:
In the examples provided, REPI was interfaced to a local BLAST (NCBI nt) database (contained on an Apple G5 single processor (1.8 GHz) computer with 4.5 GB of random access memory) via a CGI (Perl) interface. Displayed results included all database sequences within an expect value (E-value) threshold of 1.0e−9. The E-value represents the number of alignments expected at random giyen the size of the search space, the scoring matrix, and the gap penalties; the lower the E-value the less likely the database sequence similarity matches was in fact a random identification.
The REPI output is comprised of the (Comparable) subsequence and its name, followed by the names, lengths, E-values, and bits scores for each match with that subsequence in descending order of bit scores. The name is reported as the GenBank record's FASTA definition line and includes the sequence length. The score is the normalized score computed from the scoring matrix and gap penalties, the higher the score the greater the similarity.
The REPI output of the example listed above is shown below. (The first subsequence is SEQ ID NO:2 and the second subsequence is SEQ ID NO:3.) For each Comparable subsequence, REPI returns (in descending order of bit score ranking) all GenBank data records having expect values of<the evalue threshold value, currently 1.0 E−9. The highest bit score is achieved for the adenovirus 4 prototype while field strains from Air Force and Navy training sites are suitably distinguished by lower bit scores.
In this detailed example, the REPI parameters were set as follows:
In addition to the embodiments described above, the extensible nature of the system allows for ready adaptation to a number of higher bioinformatics tasks that utilize discontinuous segments of nucleotide or amino acid sequences. Several examples of these supplementary applications are described below.
In the previous examples, the present inventors provided data showing that sequence fragments can be linked automatically to individual target sequences. In several more preferred embodiments, this approach discriminates between a mixture and a recombination within a set of orthologous biological target sequences. Herein, an ortholog is defined generally as a same gene in a different species, usually an indication of common genetic ancestry.
More specifically, the system attempts to perform automatic alignment of sequence calls from different tile regions of the resequencing microarray to detect the presence of homologous sequence fragments on different tiled regions of the array, allowing inference of a mixture of target sequences. Optionally, the system further determines that the sequence outputs from different tiled regions representing orthologous genes are not mixtures of orthologous genes but correspond to a contiguous sequence that may have arisen by a genetic recombination event between different regions of two or more orthologous genes.
In such additional embodiments, the system allows for automatic detection of highly overlapping or homologous sequence fragments on different tiled regions of the array, allowing inference of a mixture of target sequences. Further, the system optionally determines that the sequence outputs from different tiled regions are not highly overlapping but correspond to contiguous sequence that may be registered to positions within known target sequences to infer a genetic recombination event.
Fit among subsequences can be evaluated, for example, through detection of appropriate amounts of overlap among the sequences. In one embodiment, the fit is a quantitative relationship between the length (or bit score) of the homologous overlap region in relation to the non-overlapping sequence(s), and the relationship of each to the overall sequence of the whole gene for a given protein. In addition to traditional methods of evaluating overlapping biological sequences, the system optionally analyzes sequence overlap using synchronization and detection methods applicable to analog and digital communications. Moreover, the issue of identifying overlapping sequences is not unlike that problem posed by initial synchronization in digital communication systems. Accordingly, one method that may be employed according to the present invention is the use of a sliding correlator. In a sliding correlator, two sequences (a data sequence, and a hypothesis sequence), are compared to one another by correlating the two sequences. The two sequences are shifted in position with respect to one another, and shifting stops only when the correlation result is detected to be above a predetermined level. In practice, a sliding correlator is often preceded by some other method for reducing the area of search such as the transmission of a synchronization preamble. Likewise, according the present invention, an already-detected overlap between subsequences may be used as a preamble in order to limit the amount of time required for the synchronization process. This type of synchronization is described in Bhargva, et al. “Digital Communications By Satellite” John Wiley and Sons, Chapter 9, pages 269-291, the entire contents of which being incorporated herein by reference. Likewise, other synchronization or acquisition algorithms may be applied such as those described in §8.22 of Sklar, B. “Digital Communications Fundamentals and Applications”, Prentice Hall, 1988, pages 453, 460, the entire contents of which being incorporated herein by reference. The acquisition criterion used is selected based on a sufficiently low probability of false acquisition. In this case, the acquisition criterion may be the probability of false detection of 10%, although 9%, 8%, 7%, 6% inclusive, down to 0.1% may be employed.
If the subsequences do not exceed a predetermined threshold of fit, the system begins analysis of an alternative subsequence S1207. That is, if two or more potentially homologous or orthologous sub-sequences do not fit a model for either mixture or recombination, the system can proceed to search for other subsequences S1201. The collection of grouped subsequences can be compared to the entire (target) sequence using a similarity search algorithm S1211. In such a case, the level of similarity S1213 between the linked subsequences and a target sequence provides data indicating whether the detected biological sequences are from a mixture of different biological entities or whether the detected sequences indicate a recombination. Alternatively, the system employs a method of check points to evaluate overlapping hybridization between portions of subsequences. The check point method performs such an evaluation at multiple points along the subsequences S1209. In this approach, the number of checkpoints is compared against a threshold S1215 to provide evidence distinguishing between a biological mixture and a recombination.
For example, a co-infection of two viruses of the same type might produce a recombinant within a single gene that is identical to one virus except for the 5′ end, which has been substituted with the corresponding section of the second viral gene for the same protein. When this new recombinant virus genome is hybridized on a resequencing microarray, it might produce signals from the corresponding sections of the resequencing tile regions. An embodiment of the present invention includes an assembly algorithm to construct a “model” of the target sequence showing which parts might fit together to form an entire target. If the two have significant overlap (for example, demonstrating homology beyond some threshold value), one might conclude that there is probably a mixture. But if there were little or no overlap, there would remain a possibility that there is a recombinant. The degree of overlap (or lack of) could be affected by low concentrations of target with correspondingly smaller amounts of the tiles being filled in. This same principle can be applied even more readily, and with greater impact, on viruses where the recombination is a steady and recurring event, as in the case of retroviruses, where recombinations between viral genes result in the formation of new viral strains. In fact, such described functionality is essential for the distinction of mixtures of target sequences versus a recombination between target sequences. Moreover, this additional functionality may also be used to more rapidly detect common regions in detected (and possibly) new recombinations and assist in the design PCR primers to assist in broader studies of recombinations detected by the system.
Not only is the present inventive approach able to distinguish between mixtures of biological entities and recombination events within a given entity (described elsewhere herein), an additional embodiment of the system advantageously provides an end user with quantitative estimate of the relative amount of target sequence that was detected in the resequencing microarray assay. Such decision-quality information is of increased utility when, for example, a clinician or clinical laboratory technician attempts to assign cause and effect when multiple pathogen genomic signatures are detected. Further, supplementing data regarding the presence of a biological substance(s) with data regarding the (relative and absolute) abundance of the biological substance(s) provides additional context for decision-making by an end user. Further, an embodiment of the system is configured to automatically analyze and compare such “presence” and “abundance” data to provide an end user with decision-quality information.
Embodiments of the system are configured to utilize two types of data for providing abundance information. The first is the absolute intensity of the hybridization signals on the chip. A non-linear relationship exists between the amount of target in solution and the amount that actually hybridizes and the resulting signal. However, an estimate of the amount of target nucleic acid in the sample could be made by comparison with a standard curve prepared under control conditions. For example, the signal intensity data is readily available from the CEL file in the Affymetrix data hierarchy and is typically used for quantitative assessment of gene expression changes. An embodiment of the system accommodates the inclusion of intensity values within the data it inputs, outputs, and handles. Secondly, the percentage of base calls, both as a percentage of the total tile region size and as a percentage of base calls within a selected subsequence satisfying the sliding window algorithm, can be used as a measure of concentration. Results from tests performed by the inventors show that both of these percentage metrics decrease with decreasing target concentration, although the correct target sequence can still be identified.
In another very preferred embodiment, the system allows for the analysis of transcriptional markers (e.g. RNA) that have been resequenced using the presently described type of microarray (via hybridization of RNA or complementary cDNA). In a method analogous to that described above for inference of genomic recombination events, transcriptional sequences may also be assembled to determine biological entity viability and transcriptional editing events that can serve as markers for infection.
Further, the system optionally is adaptable for use with biological sequences other than nucleic acids and their related transcriptional products such as the amino acids sequences of proteins. Generally, proteomic applications of the present invention are consistent with the system's ability to handle biological sequence data and optimize such data for comparison against known sequences. The studies of gene expression and protein evolution have lead to amino acid sequence databases (and associated similarity search algorithms) similar in scale and accessibility as genetic sequence databases described above. Moreover, the analysis of spectrum data returned from mass spectrometry methods for sequencing proteins lends itself conveniently to even more elaborate embodiments of the invention. For example, protein sequence spectrum data includes intensity data similar to those used in the analysis of microarrays. As described above with respect to other types of sequences, an advanced embodiment of the invention provides for the handling and utilization of such intensity data to provide higher quality information to an end user.
In addition to the use of the system in diagnostic applications, alternative embodiments of the system facilitate the design of more effective and efficient resequencing microarrays for use with diagnostic embodiments of the system. A more effective approach to choosing and designing probes for inclusion on microarrays inevitably leads to more efficient use of the real estate on a given microarray. Consequently, microarrays can be made to accurately detect a larger variety of biological sequences for a given size, or microarrays tailored for specific applications can be made cheaper and more accessible by reducing the required number of probes required on a microarray, which increases the opportunity for size reduction and higher manufacturing yields. One key driver of designing a microarray is resolution. Generally, resolution as described herein refers to the discriminatory power to distinguish between closely related strains of a biological entity. For example, some applications may require a high resolution to distinguish between Air Force and Navy Field Strain of adenovirus, while other applications require only identification of the presence of an adenovirus. The embodiments described below illustrate uses of the system in enabling a microarray designer to objectively and systematically balance resolution and microarray size/density.
Accordingly, the functionality of the system provides accelerated and more effective design of resequencing microarrays compared to a conventional method of subjectively choosing probes.
The above aspects related to design are further illustrated by the following example conducted by the inventors. In this example, a method is described for creating a consensus sequence to be used as a target sequence on a microarray that is capable of identifying those test sequences used to create it.
Beginning with 15 adenovirus hexon gene sequences, these sequences are arranged according to phylogeny using a dendrogram (for example, see
Next, all selected test sequences are subjected to a multiple alignment analysis S1505 such as ClustalW (Thompson et al., 1994), a sample output of which is shown at
Once a candidate consensus sequence is calculated, it is input into REPI (or, alternatively, another embodiment of the present invention) to initially evaluate its potential effectiveness as a target sequence S1509 using an Expect threshold value of 1e−9 for returning prospective matches S1511. At this point, the REPI results are compared to the initial set of test sequences S1513 as, for example, a percentage of the initial set of test sequences present in the REPI results. If this percentage of initial test sequences. identified by the REPI results is greater than a threshold (predetermined by the system or, alternatively, defined by the user), the candidate consensus sequence is the most probabilistically favorable large-scale target sequence, and the process moves to simulating hybridization S1527. In the illustrated example, the above comparison threshold is 100%, corresponding to a condition that all of the initial test sequences must be returned by REPI as exceeding the Expect threshold. Reducing this comparison threshold results in a system more permissive of a target sequence that may misidentify or fail to identify a certain number or percentage of a desired set of test sequences.
Otherwise, if each of the initial set of test sequences are not all present in the REPI results, the missing test sequences are then be evaluated with the candidate consensus sequence individually. The candidate consensus sequence are also evaluated in combination with the multiple alignments created in a previous step to identify any missing sequence segments that are critical for their identification within this consensus. The objective of the following steps is to incrementally add necessary sequence information to the original candidate consensus sequence without losing the original sequence generality of the original.
Accordingly, a multiple alignment is performed S1515 again. This time, the multiple alignment includes the current consensus sequence. Subsequently, gaps in the candidate consensus sequence are identified S1517. The gaps in the alignment of the candidate consensus sequence with the test sequences are possible positions for missing sequence data where adding incremental sequence information to the candidate consensus sequence from the missing test sequences may be beneficial.
Segments from the missing sequences corresponding to the gaps are then added, or “spliced,” to the original consensus sequence in place of the gaps S1519 to form a “patched” consensus sequence.
After splicing S1519, the patched candidate consensus sequence is again submitted to REPI for evaluating the impact of the above manipulation S1521. Optionally, the system or user determine additional acceptability thresholds corresponding to a number (or percentage) of missing test sequences now correctly identified S1523 or a number (or percentage) of previously identified test sequences now incorrectly identified by the REPI results S1525. Such thresholds generally correspond to a tolerance for improvement or degradation in the effectiveness of a patched consensus sequence with respect to the initial candidate consensus sequence. In the illustrated example, the addition of two sequence segments is enough to add Ad1 and Ad5 to the list of REPI hits without losing any of the previously identified test sequences.
On the other hand, if the splicing operation failed to add the missing adenovirus types to the list of those identified or other sequence hits were lost in the process the new consensus sequence would be abandoned, Ad1 and Ad5 are separated and the remaining sequences are reevaluated S1524. Alternatively, if the splicing operation failed to meet an acceptability threshold, a reevaluation is performed. Accordingly, in cases where the sequence differences among the initial set of test sequences fails to meet an acceptability threshold (for example, those described immediately above), two or more candidate consensus sequences may be necessary to provide target sequences that are able to identify a desired percentage of the initial set of test sequences. Optionally, the system accommodates consideration and evaluation of such additional candidate consensus sequences in parallel.
If all of the original sequences have been identified in the REPI results (see
The sequence shown in
If all of the simulated sequences match their respective sequence as the top score and top “hit” (similarity score based on bit score and/or expect value), then this potential consensus sequence has passed the evaluation process and can be used as a target sequence to identify, by type, those sequences used to create it S1535. Alternatively, if all of the simulated sequences do not match their respective sequence as the top score S1535, or the top score and E-value match multiple sequences S1533, the potential consensus fails the evaluation the sequences are broken up into multiple groups and sent back for reevaluation S1534.
The potential sequences that did not correctly identify their respective sequence (Ad1, Ad50, Ad34, Ad3) are sent back to step one to be reevaluated for one or more consensus S1534. The potential sequences Ad4, Ad21, Ad16, Ad7, Ad5, that did correctly identify their respective sequence as the top score and E-value are grouped together and are sent back through the process described above starting with the creation of a multiple alignment to build a new candidate consensus sequence without the use of those sequences that failed and did not correctly identify themselves S1536. The new candidate consensus sequence (for example, see
In yet another embodiment, the system provides for tracking and analysis of time trends in sequence analysis. By performing and recording analysis similar to that described above iteratively or continuously over time, genetic or proteomic evolution and/or mutation can be tracked more easily than using the conventional method.
In a specific embodiment related to pathogen detection, the invention described herein is used for the routine diagnosis and surveillance of common respiratory pathogens in a clinical setting (at or near point-of-care). Readily obtainable samples (e.g. nasal wash, throat swab, sputum, blood, food, soil, water, or air) are processed in a simple manner to produce nucleic acid isolates that are obtained using an adsorptive process, enriched for pathogen-specific targets, amplified using a non-biased (e.g. total) or multiplexed PCR amplification method, and hybridized on the resequencing microarray for a defined of time prior to washing and imaging. The overall process is sufficiently simple such that a skilled technician (medical technologist level) will be able to perform the assay without a significant interruption in their routine work pattern. Base calls are made using the custom algorithms or using the steps specified by the vendor. REPI, or some variant thereof, is used to automatically parse the base calls made by the microarray, and provide the end-user (e.g., physician, health care provider, public health officer, or other decision-makers) with decision-quality information for management (e.g., diagnostic, treatment, prognostic and outbreak control/containment measures) of the infectious pathogen(s) that are causative of the disease symptoms and complications. This analysis occurs locally through the use of an embedded sequence database that would be queried by REPI (e.g. local dedicated BLAST server). In addition to providing a routine diagnostic functionality, the microarray used in conjunction with this embodiment also carries markers for highly improbable (e.g. avian influenza or bioterrorism) pathogens that would be cause for involvement of others, namely public health officials.
In selected embodiments of the present invention, CIBSI outputs are arranged in multiple layers. In a particular embodiment, CIBSI outputs are arranged in three layers for presentation to a user or data interpreter. A first layer of output provides “species level” information, a second layer of output provides “serotype/strain level” information, and a third layer provides “low level” information. Examples of species include, but are not limited to, Influenza A, Influenza B, Adenovirus, S. pyogenes, B. anthracis, and F. tularensis. Although the species level layer is presented first to a user or data interpreter, the user or data interpreter is able to select and view other layers through, for example, user inputs, predetermined display settings, or prescribed protocol by qualified individuals. Alternative embodiments provide for rules and algorithms to retrieve, organize, and present data corresponding to predetermined levels of detail within each layer.
In one embodiment, a rule for a positive at the first level is that if any one of the multiple tile regions for a species produces a positive (for example, produces a subsequence having a BLAST expect value of 1.0e−9 or less or produces a high bit score for a single pathogen database record), then the result is POSITIVE at the species level. This enables a fully autonomous first layer of detail to be established. The user can produce more information that this first layer through, for example, a human intervention step to access second or third layers of information. The second layer of output including “serotype/strain level” information enables a user to view sequence database (for example, GenBank) record names and identifiers with scores above a predetermined threshold to determine serotype and/or strain. Optionally, text search algorithms can be applied sequence databases without precise naming conventions to automatically provide serotype/strain level information (for example, through searching and parsing of GenBank data). Alternatively, the system provides for addenda (for example, adenovirus 4, AF field strain or influenza A, H3N2, Fujian 411) to be made to the first layer of information by a user or data interpreter by selecting an option next to a display field. The third layer of output includes raw CIBSI output. Optionally, algorithms may be applied to the raw CIBSI output. Other embodiments of the present invention move additional information to the first layer of output.
The computer system 2001 also includes a disk controller 2006 coupled to the bus 2002 to control one or more storage devices for storing information and instructions, such as a magnetic hard disk 2007, and a removable media drive 2008 (e.g., floppy disk drive, read-only compact disc drive, read/write compact disc drive, compact disc jukebox, tape drive, and removable magneto-optical drive). The storage devices may be added to the computer system 2001 using an appropriate device interface (e.g., small computer system interface (SCSI), integrated device electronics (IDE), enhanced-IDE (E-IDE), direct memory access (DMA), or ultra-DMA).
The computer system 2001 may also include special purpose logic devices (e.g., application specific integrated circuits (ASICs)) or configurable logic devices (e.g., simple programmable logic devices (SPLDs), complex programmabl& logic devices (CPLDs), and field programmable gate arrays (FPGAs)).
The computer system 2001 may also include a display controller 2009 coupled to the bus 2002 to control a display 2010, such as a cathode ray tube (CRT), for displaying information to a computer user. The computer system includes input devices, such as a keyboard 2011 and a pointing device 2012, for interacting with a computer user and providing information to the processor 2003. The pointing device 2012, for example, may be a mouse, a trackball, or a pointing stick for communicating direction information and command selections to the processor 2003 and for controlling cursor movement on the display 2010. In addition, a printer may provide printed listings of data stored and/or generated by the computer system 2001.
The computer system 2001 performs a portion or all of the processing steps of the invention in response to the processor 2003 executing one or more sequences of one or more instructions contained in a memory, such as the main memory 2004. Such instructions may be read into the main memory 2004 from another computer readable medium, such as a hard disk 2007 or a removable media drive 2008. One or more processors in a multi-processing arrangement may also be employed to execute the sequences of instructions contained in main memory 2004. In alternative embodiments, hard-wired circuitry may be used in place of or in combination with software instructions. Thus, embodiments are not limited to any specific combination of hardware circuitry and software.
As stated above, the computer system 2001 includes at least one computer readable medium or memory for holding instructions programmed according to the teachings of the invention and for containing data structures, tables, records, or other data described herein. Examples of computer readable media are compact discs, hard disks, floppy disks, tape, magneto-optical disks, PROMs (EPROM, EEPROM, flash EPROM), DRAM, SRAM, SDRAM, or any other magnetic medium, compact discs (e.g., CD-ROM), or any other optical medium, punch cards, paper tape, or other physical medium with patterns of holes, a carrier wave (described below), or any other medium from which a computer can read.
Stored on any one or on a combination of computer readable media, the present invention includes software for controlling the computer system 2001, for driving a device or devices for implementing the invention, and for enabling the computer system 2001 to interact with a human user (e.g., print production personnel). Such software may include, but is not limited to, device drivers, operating systems, development tools, and applications software. Such computer readable media further includes the computer program product of the present invention for performing all or a portion (if processing is distributed) of the processing performed in implementing the invention.
The computer code devices of the present invention may be any interpretable or executable code mechanism, including but not limited to scripts, interpretable programs, dynamic link libraries (DLLs), JAVA classes, and complete executable programs. Moreover, parts of the processing of the present invention may be distributed for better performance, reliability, and/or cost.
The term “computer readable medium” as used herein refers to any medium that participates in providing instructions to the processor 2003 for execution. A computer readable medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical, magnetic disks, and magneto-optical disks, such as the hard disk 2007 or the removable media drive 2008. Volatile media includes dynamic memory, such as the main memory 2004. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that make up the bus 2002. Transmission media also may also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications.
Various forms of computer readable media may be involved in carrying out one or more sequences of one or more instructions to processor 2003 for execution. For example, the instructions may initially be carried on a magnetic disk of a remote computer. The remote computer can load the instructions for implementing all or a portion of the present invention remotely into a dynamic memory and send the instructions over a telephone line using a modem. A modem local to the computer system 2001 may receive the data on the telephone line and use an infrared transmitter to convert the data to an infrared signal. An infrared detector coupled to the bus 2002 can receive the data carried in the infrared signal and place the data on the bus 2002. The bus 2002 carries the data to the main memory 2004, from which the processor 2003 retrieves and executes the instructions. The instructions received by the main memory 2004 may optionally be stored on storage device 2007 or 2008 either before or after execution by processor 2003.
The computer system 2001 also includes a communication interface 2013 coupled to the bus 2002. The communication interface 2013 provides a two-way data communication coupling to a network link 2014 that is connected to, for example, a local area network (LAN) 2015, or to another communications network 2016 such as the Internet. For example, the communication interface 2013 may be a network interface card to attach to any packet switched LAN. As another example, the communication interface 2013 may be an asymmetrical digital subscriber line (ADSL) card, an integrated services digital network (ISDN) card or a modem to provide a data communication connection to a corresponding type of communications line. Wireless links may also be implemented. In any such implementation, the communication interface 2013 sends and receives electrical, electromagnetic or optical signals that carry digital data streams representing various types of information.
The network link 2014 typically provides data communication through one or more networks to other data devices. For example, the network link 2014 may provide a connection to another computer through a local network 2015 (e.g., a LAN) or through equipment operated by a service provider, which provides communication services through a communications network 2016. The local network 2014 and the communications network 2016 use, for example, electrical, electromagnetic, or optical signals that carry digital data streams, and the associated physical layer (e.g., CAT 5 cable, coaxial cable, optical fiber, etc). The signals through the various networks and the signals on the network link 2014 and through the communication interface 2013, which carry the digital data to and from the computer system 2001 maybe implemented in baseband signals, or carrier wave based signals. The baseband signals convey the digital data as unmodulated electrical pulses that are descriptive of a stream of digital data bits, where the term “bits” is to be construed broadly to mean symbol, where each symbol conveys at least one or more information bits. The digital data may also be used to modulate a carrier wave, such as with amplitude, phase and/or frequency shift keyed signals that are propagated over a conductive media, or transmitted as electromagnetic waves through a propagation medium. Thus, the digital data may be sent as unmodulated baseband data through a “wired” communication channel and/or sent within a predetermined frequency band, different than baseband, by modulating a carrier wave. The computer system 2001 can transmit and receive data, including program code, through the network(s) 2015 and 2016, the network link 2014, and the communication interface 2013. Moreover, the network link 2014 may provide a connection through a LAN 2015 to a mobile device 2017 such as a personal digital assistant (PDA) laptop computer, or cellular telephone.
The system of certain embodiments of the present invention can be implemented in hardware, software, firmware, or a combination thereof. In the preferred embodiment, the system is implemented in software that is stored in a memory and that is executed by a suitable instruction execution system. If implemented in hardware, as in an alternative embodiment, the system can be implemented with any technology, which is all well known in the art.
Any process descriptions or blocks in the flow charts should be understood as representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps in the process, and alternate implementations are included within the scope of the preferred embodiment of the present invention in which functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those reasonably skilled in the art of the present invention.
It should be emphasized that the above-described embodiments of the present invention, particularly, any “preferred” embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the invention. Many variations and modifications may be made to the above-described embodiment(s) of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and described by the following claims.
The present application claims priority to U.S. provisional Application Ser. No. 60/590,931, filed on Jul. 2, 2004, U.S. provisional Application Ser. No. 60/609,918 filed on Sep. 15, 2004, U.S. provisional Application Ser. No. 60/631,437 filed on Nov. 29, 2004, U.S. provisional Application Ser. No. 60/631,460 filed on Nov. 29, 2004 and U.S. provisional Application Ser. No. 60/691,768 filed on Jun. 16, 2005. This application is also related to U.S. application Ser. No. 11/177,646. The entire contents of these applications are incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6519583 | Koleszar et al. | Feb 2003 | B1 |
| 7024312 | Selifonov et al. | Apr 2006 | B1 |
| Number | Date | Country | |
|---|---|---|---|
| 20070065832 A1 | Mar 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60590931 | Jul 2004 | US | |
| 60609918 | Sep 2004 | US | |
| 60631437 | Nov 2004 | US | |
| 60631460 | Nov 2004 | US | |
| 60691768 | Jun 2005 | US |
