# Patent

## Title
Methods and systems for digitally counting features on arrays

## Abstract

Methods, systems and platforms for digital imaging of multiple regions of an array, and detection and counting of the labeled features thereon, are described.

## Background
CROSS-REFERENCE
The present application is a continuation of U.S. application Ser. No. 14/508,911, filed Oct. 7, 2014, which claims the benefit of U.S. Provisional Application No. 61/887,853, filed Oct. 7, 2013, the content of the priority applications is incorporated herein by reference in its entirety.


REFERENCE TO SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence_Listing_BDCRI_007C1.txt, created Dec. 12, 2016, which is 255,871 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND
Array technologies have been widely used in biomedical studies for the detection of biomolecules and profiling of gene expression levels, etc. Arrays are typically comprised of immobilized probes which can bind to or hybridize with target molecules in a sample. Detection of binding or hybridization events is often achieved through the use of optical labels (e.g. fluorophores) and scanning or imaging techniques (e.g. fluorescence scanning or imaging). A feature on an array is a small region of immobilized probes that are specific for a given target molecule, e.g. probes that hybridize to specific DNA or RNA sequences. Identifying the pattern of labeled features on a hybridized array thus provides information about specific molecules, e.g. DNA or RNA molecules in the sample, which in turn can provide valuable data in biomedical studies. Two important engineering requirements for providing high quality, quantitative data for biomedical investigations are (i) to correctly image the hybridized arrays, and (ii) to correctly analyze the images to extract quantitative data. Existing optical imaging systems typically image one region of an array at a time, which can be a slow process if a number of different regions need to be imaged. In addition, current methods of image analysis typically determine a signal intensity level (i.e. an analog quantity) for each array feature. Intensity level measurements are often subject to a variety of instrumental drift and analysis errors, therefore improved methods for determining whether or not target molecules are bound to a given array feature, and improved methods for transforming that data into quantitative measures of the number of target molecules present in a sample, are of great importance to expanding the use of array technologies in biomedical applications.
SUMMARY
The methods, systems, and platforms of the present disclosure provide means for digital counting of labeled features on arrays, and thereby enable quantitative determination of the number of target molecules present in a sample through the use of stochastic labeling techniques.
Disclosed herein is an imaging platform comprising: (a) an optical instrument configured to generate an image of one or more regions of an array, wherein the array comprises a plurality of features, and wherein the plurality of features comprise a set of oligonucleotide probes, and wherein the oligonucleotide probes are complementary to a set of labels; and (b) a processor configured to perform image analysis, wherein the image analysis comprises: (i) reading the image generated by the optical instrument; (ii) locating the features of the array within the image; (iii) measuring a signal intensity for each feature; (iv) measuring a local background intensity for each feature; (v) calculating a local background corrected signal intensity for each feature using the signal intensity and local background intensities; (vi) analyzing the local background corrected signal intensities for the complete set of features to determine a dynamic signal intensity threshold for discriminating between labeled and non-labeled features; and (vii) calculating a number of target molecules present in a sample based on the number of labeled and non-labeled features detected and the predictions of the Poisson distribution. In some embodiments, the image generated by the optical instrument is a fluorescence image. In some embodiments, the image generated by the optical instrument is a phosphorescence image. In some embodiments, the image generated by the optical instrument is a transmitted light, reflected light, or scattered light image. In some embodiments, the image analysis further comprises reading an image that has been previously acquired and stored in a memory device. In some embodiments, locating the features of the array within the image comprises identifying predefined fiducial features on the array. In some embodiments, the calculation of a local background corrected signal intensity is performed by (i) centering a predefined analysis window on each feature within the image, (ii) calculating an intensity value statistic for signal and background pixels according to a predefined pattern of pixels within the feature, and (iii) utilizing the signal and background intensity value statistics to calculate a local background corrected signal intensity for each feature. In some embodiments, the intensity value statistic used for calculating a local background corrected signal intensity for each feature is selected from the list including, but not limited to, the mean, the median, or the ratio of signal to background intensities. In some embodiments, the analyzing of local background corrected signal intensities for the complete set of features to determine a dynamic signal intensity threshold comprises performing one or more statistical analyses selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or an empirical analysis. In some embodiments, the analyzing of local background corrected signal intensities for the complete set of features to determine a dynamic signal intensity threshold comprises fitting a model function to the intensity data by varying model parameters. In some embodiments, the analyzing of local background corrected signal intensities for the complete set of features to determine a dynamic signal intensity threshold comprises maximizing a quality metric relating to a statistical difference between intensities above the threshold and below the threshold.
In some embodiments, an array reader system comprising an output unit for calculating an absolute number of target molecules in a sample is described, wherein the array reader system is configured to read an array comprising a plurality of labeled and non-labeled features. In some embodiments, the array reader system may further comprise an optical imaging system. In some embodiments, the calculation of absolute number of target molecules in a sample is based on transforming optical image data produced by the optical imaging system into a count of the number of labeled and non-labeled features on an array. In some embodiments, the output unit comprises a digital processor and executable software, wherein the executable software comprises computer code for transforming optical image data into a count of the number of labeled and non-labeled features. In some embodiments, the array comprises a microarray, microscope slide, or microwell plate.
In some embodiments of the disclosed array reader system, the optical imaging system has a magnification of less than 1, equal to 1, or greater than 1. In some embodiments, the optical imaging system comprises a fluorescence imaging system. In some embodiments, the optical imaging system comprises a phosphorescence imaging system. In some embodiments, the optical imaging system comprises an imaging system that operates in a transmitted light, reflected light, or scattered light imaging mode, or combinations thereof. In some embodiments, the optical imaging system comprises one or more image sensors, wherein the one or more image sensors have a resolution of at least 320×240 pixels. In some embodiments, the one or more image sensors comprise CCD image sensors. In some embodiments the one or more image sensors comprise CMOS image sensors. In some embodiments, the one or more image sensors comprise one or more circuit boards. In some embodiments, the optical imaging system further comprises one or more components selected from the group including, but not limited to, a microscope objective, a camera lens, a finite-conjugate lens, an infinite-conjugate lens, a plano-convex lens, a double convex lens, a plano-concave lens, a double concave lens, an achromatic cemented doublet, or a bandpass filter. In some embodiments, the optical imaging system comprises a fluorescence imaging system that is designed for use with fluorescein, Cy3, Cy5, or phycoerythrin fluorophores. In some embodiments, the optical imaging system further comprises an illumination system including at least one light source, wherein the at least one light source is an LED or LED assembly. In some embodiments, the at least one light source is electronically synchronized with the image sensor, the at least one light source being turned on when the image sensor is acquiring an image and turned off when the image sensor is not acquiring an image.
In some embodiments of the disclosed array reader system, the illumination system is an off-axis illumination system that satisfies the Scheimpflug condition. In some embodiments, the illumination system is an off-axis illumination system does not satisfy the Scheimpflug condition. In some embodiments, the illumination system is an off-axis illumination subsystem comprising a Kohler illumination system. In some embodiments, the illumination system is an off-axis illumination system comprising an Abbe illumination system. In some embodiments, the illumination system is an epi-illumination system comprising a Kohler illumination system. In some embodiments, the illumination system is an epi-illumination system comprising an Abbe illumination system. In some embodiments, the illumination system is a trans-illumination system comprising a Kohler illumination system. In some embodiments, the illumination system is a trans-illumination system comprising an Abbe illumination system.
In some embodiments of the disclosed array reader system, the optical imaging system further comprises a translation stage, wherein the translation stage is a single-axis translation stage, a dual-axis translation stage, or a multi-axis translation stage.
In some embodiments of the disclosed array reader system, the optical imaging system and output unit are combined within a single, stand-alone instrument. In some embodiments, the optical imaging system and output unit are configured as separate instrument modules.
In some embodiments of the disclosed array reader system, executable software automatically locates features of the array within the acquired image. In some embodiments, the executable software also performs local background correction by (i) centering a predefined analysis window on each array feature within an image, (ii) calculating an intensity value statistic for signal and background pixels according to a predefined pattern of pixels within the feature, and (iii) utilizing the signal and background intensity value statistics to calculate a background corrected signal intensity value for each feature.
In some embodiments of the disclosed array reader system, executable software performs a k-means clustering analysis of the background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array. In some embodiments, the executable software also performs a k-medoids clustering analysis of the background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array.
In some embodiments, executable software performs a mixture model statistical analysis of the background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array. In some embodiments, executable software also performs an empirical analysis based on sorting of background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array. In some embodiments executable software performs an empirical analysis based on sorting of pairwise differences in background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array. In some embodiments, an executable software module performs one or more statistical analyses of the background corrected signal intensity values for the complete set of array features, thereby determining a dynamic signal intensity threshold for discrimination between labeled and non-labeled features of the array, and wherein the one or more statistical analyses are selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or an empirical analysis.
In some embodiments of the disclosed array reader system, executable software calculates the absolute number of target molecules in a sample based on the number of labeled and non-labeled features detected and the predictions of the Poisson distribution. In some embodiments, executable software also calculates a confidence interval for the number of target molecules.
Also disclosed herein is a digital imaging platform comprising: (a) an optical instrument configured to generate an image of one or more regions of an array, wherein the array comprises a plurality of features comprising oligonucleotide probes, and wherein the oligonucleotide probes are complementary to a set of labels; and (b) a digital processor, wherein the digital processor is configured to perform image analysis comprising: (i) transforming background corrected signal intensities for a plurality of features to produce binary output data that determines the number of labeled and non-labeled features in the one or more regions of the array; and (ii) calculating a number of target molecules present in a sample based on the number of labeled and non-labeled features detected within the one or more regions of the array. In some embodiments, the image analysis further comprises automatically locating the features of the array within the image. In some embodiments, the image analysis further comprises correcting a signal intensity for each feature for a local background intensity. In some embodiments, the image analysis further comprises performing one or more statistical analyses of the corrected signal intensities for a plurality of features to define one or more dynamic signal intensity thresholds for the one or more regions of the array, where the statistical analyses are selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or an empirical analysis. In some embodiments, the calculation of the number of target molecules present in a sample is based on both the number of labeled and non-labeled features detected within the one or more regions of the array and on the predictions of the Poisson distribution.
Also disclosed herein is a non-transitory computer readable medium storing a program that calculates a number of labeled features on an array, wherein the array comprises a plurality of feature sets, and wherein individual features of a feature set comprise a set of oligonucleotide probes that are capable of hybridizing to a set of labels, the non-transitory computer readable medium comprising: (a) computer code that locates individual features of the array within a digital image of the array; (b) computer code that performs a local background correction of a signal intensity for one or more features; (c) computer code that analyzes the corrected signal intensity data for the complete set of features and determines a corrected signal intensity threshold; and (d) computer code that transforms the corrected signal intensity for the features into binary output data, thereby providing a count of the number of labeled features on the array. In some embodiments, the computer code for locating individual features of the array within the digital image comprises identifying predefined fiducial features on the array. In some embodiments, the computer code for performing a local background correction of signal intensity for each feature comprises a calculation utilizing a statistic for signal and background intensities selected from the list including, but not limited to, the mean, the median, or the ratio of signal to background intensities. In some embodiments, the computer code for analyzing corrected signal intensities for the complete set of features to determine a corrected signal intensity threshold comprises performing one or more statistical analyses selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or an empirical analysis.
Also disclosed herein is a computer implemented method for performing local background correction of array signal intensity data, the method comprising: (a) centering a predefined data analysis window on a feature within a digital image of the array; (b) calculating an intensity value statistic for signal and background pixels according to a predefined pattern of pixels within or around the array feature; and (c) utilizing the signal and background intensity value statistics to calculate a background corrected signal intensity for the array feature. In some embodiments, the computer implemented method further comprises automatically locating the array feature using, e.g., a predefined set of fiducial features on the array. In some embodiments, the intensity value statistic used for calculation of a background corrected signal intensity is selected from the list including, but not limited to, the mean, the median, or the ratio of signal to background intensities.
Disclosed herein is a computer implemented method for determining a dynamic image intensity threshold for use in discriminating between labeled and non-labeled features on an array comprising a plurality of labeled and non-labeled features, the computer implemented method comprising: (a) measuring image intensity data for each feature of the array; (b) performing a local background correction on the image intensity data for each feature on the array; and (c) performing one or more statistical analyses of the background corrected image intensity data for the complete set of array features, thereby determining a dynamic image intensity threshold for discrimination between labeled and non-labeled features of the array, and wherein the one or more statistical analyses are selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or an empirical analysis.
Also disclosed is a mechanism comprising: (a) a closure; (b) a housing which magnetically holds the closure in a first position; and (c) a translation stage which magnetically holds the closure in a second position. In some embodiments, the mechanism further comprising a gasket positioned between the closure and the housing. In some embodiments, the gasket is attached to the closure. In some embodiments, the gasket is attached to the housing. In some embodiments, the closure and housing are substantially opaque, and the gasket creates a substantially light-tight seal between the closure and the housing in the first position. In some embodiments, one or more magnets are positioned to hold the closure onto the housing in the first position. In some embodiments, one or more magnets are positioned to hold the closure onto a first surface of the translation stage in the second position. In some embodiments, two or more pairs of mating locating features to align the closure with the translation stage in the second position. In some embodiments, two or more pairs of mating locating features to align the closure with the housing in the first position. In some embodiments, the pairs of mating locating features comprise conical pins and conical holes. In some embodiments, the housing comprises an optical instrument. In some embodiments, the translation stage includes a sample holder. In some embodiments, the sample holder is designed to hold a microscope slide, a microarray, or a microwell plate. In some embodiments, the closure is not hinged. In some embodiments, the closure is not attached to either the housing or the translation stage through the use of fasteners such as screws or clips. In some embodiments, the closure is not attached to either the housing or the translation stage through the use of an adhesive. In some embodiments, the closure does not use a latch or mechanical lock.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIGS. 1A-1G show one example of an optical system, and components thereof. FIG. 1A depicts an isometric projection of the exemplary optical system. FIG. 1B depicts a top view of the optical system. FIG. 1C depicts a dimetric view of the optical system. FIG. 1D depicts a front view of the optical system. FIG. 1E depicts a side view of the exemplary optical system comprising a single axis stage, an imaging system, and an illumination system. FIG. 1F depicts a back view of the optical system. FIG. 1G depicts components that control the operation of the optical system.
FIG. 2 shows an exemplary layout of lenses in an imaging system.
FIG. 3 shows an exemplary layout of lenses in an illumination system.
FIG. 4A shows an array image acquired from the optical instrument.
FIG. 4B shows a histogram of intensities for individual features.
FIG. 5 shows a feature intensity distribution observed for hybridization of array probes with labeled target molecules in atitration experiment.
FIG. 6A shows a noisy array image. FIG. 6B shows the intensity distribution before background adjustment. FIG. 6C shows the intensity distribution after background adjustment.
FIGS. 7A-7D show external views of instrument designed for digital counting of features on arrays.
FIG. 8 shows an internal view (front view; 3D CAD model) of an instrument designed for digital counting of features on arrays.
FIG. 9 depicts an internal view (rear view; 3D CAD model) of an instrument designed for digital counting of features on arrays.
FIG. 10 shows a photograph of a system with the sample loading stage in the extended (loading) position, having pulled the door away from the front panel. A Pixel16 array assembly is shown in the loading tray.
FIG. 11A shows an exploded view of a door assembly that utilizes a magnetic mechanism for positioning a door on a sample compartment stage. FIG. 11B shows another exploded view of the door assembly that illustrates conical locator features for ensuring proper alignment of the door with the stage.
FIG. 12 depicts an exploded view of an upper stage assembly with magnets which mate with a corresponding pair of magnets on the door.
FIG. 13 shows an exploded view of a front panel assembly with magnets which mate with a corresponding pair of magnets on the door.
FIG. 14 shows the viewing reference orientation for array production and analysis in one embodiment of an array, showing the 16 array locations on a glass substrate. Nominal dimensions are shown (in millimeters).
FIG. 15 shows the layout of features on one embodiment of an array. Nominal dimensions are shown (in millimeters).
FIG. 16 shows the layout of an array designed for digital counting of target molecules in a sample, including the positions of positive controls (fiducials), negative controls, and index spots.
FIG. 17A shows an example of an array image after transformation to the reference orientation. FIG. 17B the image size (in pixels) and a schematic of feature positions for the two-array image.
FIGS. 18A-B depict software workflows for performing an experiment on an instrument designed for digital counting of features on arrays. (A) Workflow for a single-axis system with manual sample loading, and (B) workflow for a dual-axis system with automatic sample tray loading.
FIG. 19 depicts an analysis window comprising a 12×12 pixel area associated with each feature in the array.
FIG. 20 depicts a map of the pixel designations within the analysis window for each feature in the array.
FIG. 21 depicts a scatter plot (upper) of intensity data obtained from an image of an array that illustrates the different categories of features identified by the analysis software, and a histogram (lower) of the feature intensity data. Dashed lines indicate examples of intensity thresholds determined by the software that are used to discriminate between labeled (“on”) and non-labeled (“off”) features.
FIG. 22 depicts a scatter plot (upper) and histogram (middle) of array feature intensity data that illustrate the use of an intensity threshold (dashed lines) that discriminate between labeled (“on”) and non-labeled (“off”) features of an array. In one embodiment of the presently described analysis methods, the threshold is determined from the maximum slope of a plot of sorted intensity data (lower).
FIG. 23 depicts the results of fitting a 3-component distribution model used to determine an intensity threshold in one embodiment to a 128-bin feature intensity histogram.
FIG. 24A illustrates the deviance calculations for fitting one normal distribution to histograms of array feature intensity data. FIG. 24B illustrates the deviance calculations for fitting two normal distributions to histograms of array feature intensity data. In some embodiments, deviance measurement may be used as a quality metric.
FIG. 25 depicts the uncertainties calculated for various methods of combining output data from replicate experiments.
FIG. 26 shows dilution series data for using digital counting of labeled features on an array to measure the number of target RNA molecules in a sample.
FIG. 27 shows a screenshot of the output data provided by the system software for a dilution series experiment. For each array used in the dilution series experiment, the software displays a histogram of feature intensity data with a blue line indicating the value of the threshold used for counting, overlaid on a digital representation of the array.



DETAILED DESCRIPTION OF THE INVENTION
Array technologies have been widely used in biomedical studies for the detection of biomolecules and profiling of gene expression levels, etc. Arrays are typically comprised of immobilized probes which can bind to or hybridize with target molecules in a sample. Detection of binding or hybridization events is often achieved through the use of optical labels (e.g. fluorophores) and scanning or imaging techniques (e.g. fluorescence scanning or imaging). A feature on an array is a small region of immobilized probes that are specific for a given target molecule, e.g. probes that hybridize to specific DNA or RNA sequences. Identifying the pattern of labeled features on a hybridized array thus provides information about the presence of specific molecules, e.g. DNA or RNA molecules in the sample, which in turn can provide valuable data in biomedical studies. Two important engineering requirements for providing high quality, quantitative data for biomedical investigations are (i) to correctly image the hybridized arrays, and (ii) to correctly analyze the images to extract quantitative data. Existing optical imaging systems typically image one region of an array at a time, which can be a slow process if a number of different regions need to be imaged. In addition, current methods of image analysis typically determine an analog signal intensity level (i.e. a signal that can have any value between some minimum and maximum values that are determined by various instrumental and experimental parameters) for each array feature. Analog intensity level measurements are often subject to a variety of instrumental drift and analysis errors, therefore improved methods for determining whether or not target molecules are bound to a given array feature, and improved methods for transforming that data into quantitative measures of the number of target molecules present in a sample, are of great importance to expanding the use of array technologies in biomedical applications.
The advantages of the methods, systems, and platforms disclosed herein include: (i) simultaneous imaging of multiple regions of an array for higher throughput image acquisition, and (ii) improved methods for reduction of image data to a digital determination of the presence or absence of bound target molecules (or target molecule labels) for each feature of an array, thereby providing for improved quantitation in some types of array experiments, for example, those utilizing a set of stochastic labels for quantifying the number of target molecules present in a sample. The use of stochastic labeling techniques is described in U.S. Pat. No. 8,835,358 and PCT application US2011/065291, which are incorporated in their entirety herein by reference. In addition to providing a means for more quantitative detection of target molecules, the use of stochastic labeling techniques allows for mitigation of amplification bias in assays involving nucleic acid amplification.
Accordingly, disclosed herein are methods, devices, systems, and platforms for digital counting of labeled features on arrays comprising: (i) optical instruments configured to form images of one or more regions of an array, (ii) arrays comprising a plurality of features further comprising a plurality of probes, and wherein one or more regions of an array may comprise one or more sub-arrays, and wherein the arrays or sub-arrays are designed for use with sets of stochastic labels, and (iii) computer implemented methods for receiving input image data; locating array features within array images; correcting the signal intensity values associated with each feature for local background intensity values; determining dynamic signal intensity thresholds for the one or more array regions by performing statistical analyses of the corrected signal intensity data for a plurality of features; counting the number of labeled and non-labeled features on the one or more regions of the array by comparing corrected signal intensity data for the features to signal intensity thresholds; and calculating the number of target molecules in a sample, for one or more target molecule species, from the number of labeled and non-labeled features detected on the one or more regions of the array.
In some embodiments, systems are described which comprise: (i) an optical instrument (or reader) configured to form images of one or more regions of an array, (ii) a digital processor configured to perform executable instructions and store data in memory devices, and (iii) computer code for performing image analysis in order to transform image data into a digital count of the number of labeled and non-labeled features on the one or more regions of the array. In some embodiments, the computer code further comprises performing a calculation of the number of target molecules in a sample, for one or more target molecule species, from the number of labeled and non-labeled features detected on the one or more regions of the array.
In some embodiments, platforms are described which comprise: (i) arrays designed for use in stochastic labeling experiments, wherein the arrays comprise a plurality of features further comprising a plurality of probes, and wherein one or more regions of an array may comprise one or more sub-arrays, and wherein the arrays or sub-arrays are designed for use with sets of stochastic labels, (ii) an optical instrument (or reader) configured to form images of one or more regions of an array, (iii) a digital processor configured to perform executable instructions and store data in memory devices, and (iv) computer code for performing image analysis in order to transform image data into a digital count of the number of labeled and non-labeled features on the one or more regions of the array. In some embodiments, the computer code further comprises performing a calculation of the number of target molecules in a sample, for one or more target molecule species, from the number of labeled and non-labeled features detected on the one or more regions of the array.
In some embodiments, software applications (or computer code products) are described that determine the number of labeled features on an array, wherein the software application includes code for performing one or more of the following computer implemented methods: (i) receiving input image data, (ii) locating array features within array images, (iii) correcting the signal intensity values associated with each feature for local background intensity values, (iv) determining dynamic signal intensity thresholds for the one or more array regions by performing statistical analyses of the corrected signal intensity data for a plurality of features, (v) counting the number of labeled and non-labeled features on the one or more regions of the array by comparing corrected signal intensity data for the features to signal intensity thresholds, and (vi) calculating the number of target molecules in a sample, for one or more target molecule species, from the number of labeled and non-labeled features detected on the one or more regions of the array.
In some embodiments, computer implemented methods are described for performing local background correction of array signal intensity data, the methods comprising: (i) centering a predefined data analysis window on each array feature within a digital image of the array, (ii) calculating mean or median intensity values for signal and background pixels according to a predefined pattern of pixels within or around each array feature, and (iii) subtracting the mean or median background intensity from the mean or median signal intensity to determine a background corrected signal intensity value for each array feature.
In some embodiments, computer implemented methods are described for determining dynamic image intensity thresholds from the corrected image intensity data for a plurality of features on an array, the methods comprising: (i) collecting image intensity data for each feature of the array, (ii) optionally performing a local background correction on the image intensity data for each feature on the array; and (iii) performing one or more statistical analyses of the background corrected image intensity data for the complete set of array features, thereby determining a dynamic image intensity threshold for discrimination between labeled and non-labeled features of the array. In some embodiments, the one or more statistical analyses are selected from the list including, but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, or empirical analyses based on sorting of image intensity values or pairwise differences in image intensity values. As used herein, the term “dynamic intensity threshold” refers to a parameter that is determined based on an analysis of data derived from the experiment in progress. The use of a dynamic image intensity threshold for discrimination between labeled and non-labeled features of an array helps to minimize or eliminate errors in data processing that may arise from instrumental drift or experimental procedure.
Definitions
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.
As used herein, the terms “system” and “platform” are used interchangeably. Similarly, the terms “image sensor”, “imaging sensor”, “sensor chip”, and “camera” are used interchangeably to describe two dimensional photosensors used for imaging purposes, and the use of the terms “image intensity” and “signal intensity” are also used interchangeably in describing data analysis methods. Finally, unless otherwise stated, the terms “software”, “software application”, “software module”, “computer program”, and “computer code” are also used interchangeably.
Stochastic Labeling Methods
The use of stochastic labeling techniques is described in U.S. Pat. No. 8,835,358 and PCT application US2011/065291, which are incorporated in their entirety herein by reference.
Briefly, high-sensitivity single molecule digital counting may be achieved through the stochastic labeling of a collection of identical target molecules. Each copy of a target molecule is randomly labeled using a large, non-depleting reservoir of unique labels. The uniqueness of each labeled target molecule is determined by the statistics of random choice, and depends on the number of copies of identical target molecules in the collection compared to the diversity of labels. The size of the resulting set of labeled target molecules is determined by the stochastic nature of the labeling process, and analysis of the number of labels detected then allows calculation of the number of target molecules present in the original collection or sample. When the ratio of the number of copies of a target molecule present to the number of unique labels is low, the labeled target molecules are highly unique (i.e. there is a very low probability that more than one target molecule will have been labeled with a given label), and the digital counting efficiency is high. This stochastic methodology transforms the problem of counting molecules from one of locating and identifying identical molecules to a series of yes/no digital questions regarding detection of a set of predefined labels. In some embodiments, the labeled products are detected by means of DNA sequencing. In other embodiments, the labeled products for one or more target molecules of choice are detected with high specificity using the array readout systems described herein.
Arrays and Features
Disclosed herein are arrays designed for use in stochastic counting of one or more target molecules in a sample. Arrays provide a means of detecting the presence of labeled target molecules, wherein the labels comprise a large and diverse set of unique labels.
In many embodiments, arrays comprise a plurality of features (or spots) on the surface of a substrate, wherein each feature further comprises a plurality of attached probes. In some embodiments, the array may comprise one or more regions, each of which may comprise a plurality of features or sub-arrays. For example, an array may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more regions, or alternatively, an array may comprise 15, 20, 25, 30, 35, 40, 45, 50 or more regions. In some embodiments, an array may comprise 60, 70, 80, 90, 100 or more regions. In other embodiments, an array may comprise hundreds, thousands, or tens of thousands of regions.
Non-limiting examples of arrays include microtiter plates, microwell plates, 16-well microscope slides, spotted microarrays, or microarrays fabricated by in situ solid-phase synthesis. A region of an array may comprise one well of a 16-well microscope slide, one well of a glass-bottomed 96-well plate, or one well of a glass-bottomed 384-well plate. Alternatively, a region of an array may comprise more than one well, for example, in some embodiments, a region may comprise 2 adjacent wells, 4 adjacent wells; or a larger number of wells positioned in close proximity to each other. In some embodiments, the arrays may comprise high-density oligonucleotide arrays with more than 1,000 features per square millimeter, and a region on the array may comprise a selected area of the array substrate surface, for example, an area of approximately 1 mm×1 mm.
As indicated previously, in many embodiments, the set of probes attached to a set of features of an array are selected for detection of a specific set of unique labels designed for use in stochastic labeling studies. The attachment of the probes to the array substrate may be covalent or non-covalent, and permanent or temporary. A probe may be a sequence of monomers including, but not limited to, for example, deoxy-ribonucleotides, ribonucleotides, amino acids, or synthetic monomers, or they may be a sequence of oligomers, including, but not limited to, for example, oligonucleotides (e.g. DNA or RNA sequences) or peptide sequences. In some cases, a probe may be a macromolecule, including but not limited to, for example, antibodies or antibody fragments. Each feature on an array corresponds to a small area of the array substrate comprising immobilized probes having the same molecular sequence that bind to or hybridize with the same target molecule. Two or more features on the array may be identical, similar, or different. In many embodiments, arrays will include one or more fiducial marks used for alignment or orientation purposes, as well as positive and negative control features in addition to feature sets used for detection of a stochastic label set. Positive control features may comprise probes that bind to or hybridize with molecules known to be always present in a sample, or probes that bind to or hybridize with molecules spiked into a sample in a controlled fashion. Negative control features may comprise probes that are specific for molecules that are known to be absent from a sample, or they may comprise features having no probes attached to the substrate surface at all.
In many embodiments, the array substrate, also called a support, may be fabricated from a number of materials. The materials may be solid. The materials may be semi-solid. Examples of materials that may be used to fabricate array substrates include, but are not limited to, glass, fused silica, silicon, polymer, or paper.
In some embodiments, the present disclosure also describes arrays for use in stochastic labeling studies. In particular, arrays are described wherein the arrays comprise a plurality of features having immobilized probes thereon that are complementary to a set of labels designed for use in stochastic labeling experiments, and wherein there is at least one feature on the array for every label in the label set. Some embodiments include an array comprising: (a) a plurality of features, optionally organized into a plurality of sub-arrays, wherein the plurality of features comprise: (i) one or more fiducial features comprising oligonucleotide probes of a defined fiducial sequence; (ii) one or more positive control features comprising oligonucleotide probes of one or more defined positive control sequences; (iii) one or more negative control features having no oligonucleotide probes; and (iv) a plurality of label set features comprising oligonucleotide probes, wherein each individual feature comprises a unique sequence selected from a set of label sequences designed for stochastic labeling of one or more target molecules. In some embodiments, the arrays described in the present disclosure comprise oligonucleotide probe sequences comprising 25-mers, wherein the 5′ terminus may optionally be labeled with a 6 carbon atom amino-modifier. In some embodiments, the arrays described in the present disclosure further comprise oligonucleotide probes comprising the set of 960 unique oligonucleotide sequences listed in Table 1. In some embodiments, the arrays described in the present disclosure comprise a set of oligonucleotide probes that are 70% homologous, 80% homologous, 85% homologous, 90% homologous, or 95% homologous with the set of sequences listed in Table 1. In some embodiments, the array described in the present disclosure comprise a set of oligonucleotide probes that includes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the sequences listed in Table 1.
Hybridization and Detection
In many embodiments of the disclosed methods, systems, and platforms, samples may be processed prior to placing them in contact with the immobilized probes on arrays. For example, target molecules in the samples may be labeled with fluorescent dye molecules and/or stochastic labels during the sample preparation step. Prior to hybridization with oligonucleotide probes, for example, target DNA or RNA molecules may be covalently linked to fluorescent dye molecules including, but not limited to, fluorescein, Cy3, or Cy5. Alternatively, target molecules may be labeled after binding or hybridizing to probes on the array. For example, target molecules may be covalently linked to biotin prior to binding or hybridization with probes on the array. Following the binding or hybridization step, the immobilized target molecules may then be labeled with streptavidin conjugated to optical tags including, but not limited to, phycoerythrin, quantum dot nanoparticles, gold nanoparticles, or blue latex beads. There are many methods for labeling target molecules, either before or after binding or hybridization to the array, and many possible choices for suitable optical labels or tags.
Once a sample has been contacted with an array, the array (or one or more regions of the array) may comprise one or more labeled features. Each region of an array that has been contacted with a sample comprising labeled target molecules (where the target molecules are labeled either before or after contact with the array) may, for example, comprise zero, one, two, or more labeled features. Alternatively, a region of an array that has been contacted with a sample may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more labeled features. In some embodiments, a region of an array that has been contacted with a sample may comprise 15, 20, 25, 30, 35, 40, 45, 50, or more labeled features. In high-density arrays, a region of an array that has been contacted with a sample may comprise more than 100 labeled features, more than 1,000 labeled features, more than 10,000 labeled features, more 100,000 labeled features, or more than 1,000,000 labeled features.
Optical Instruments
The methods, systems, and platforms described herein may comprise an optical instrument used for finite-conjugate digital imaging of one or more regions of an array, wherein the instrument typically includes an illumination system, an imaging system, and a translation stage. In some embodiments, the instrument operates as a “macroscope” having a magnification of less than one. In other embodiments, the instrument operates as a “microscope” having a magnification of greater than one. In still other embodiments, the instrument operates as a “contact imager” having a magnification equal to one. The choice of magnification will typically depend on the field of view required to image the region of interest, and on the size of the image sensor.
By way of non-limiting example, if a region of an array comprises a single well of a 16-well microscope slide, or a single well of a glass-bottomed 96-well plate, the dimensions of the region to be imaged may be approximately 7 mm×7 mm, and the pitch (center-to-center distance between two adjacent regions of the array may be approximately 9 mm. In some embodiments, the optical instrument may be used to take an image of one well at a time, or an image of 2 adjacent wells simultaneously, or an image of 4 (2×2) adjacent wells simultaneously, and the required field of view, or region to be imaged, may be adjusted accordingly. Similarly, the optical instrument may form an image of 6 (3×2 or 2×3), 8 (4×2 or 2×4), 9 (3×3), 10 (5×2 or 5×2), or 12 (6×2, 4×3, 3×4, or 2×6) adjacent wells simultaneously.
By way of another non-limiting example, if a region of an array is a single well of a glass-bottomed 384-well plate, the dimensions of the region to be imaged may be approximately 3 mm×3 mm, and the pitch between two adjacent regions of the array may be approximately 4.5 mm. Again, in some embodiments, the optical instrument may be used to take an image of one well at a time, or an image of 2 adjacent wells simultaneously, or an image of 4 (2×2), 6 (3×2 or 2×3), 8 (4×2 or 2×4), 12 (4×3 or 3×4), or 16 (4×4) adjacent wells simultaneously.
In another non-limiting example, the optical instrument may be used to image high-density oligonucleotide arrays, for example arrays having more than 1,000 features per square millimeter, and a region on the array may be approximately 1 mm×1 mm in area, for example.
Imaging System
One main component of the optical instrument is an imaging system. The imaging system may include one or more lenses in addition to a CCD or CMOS camera. Typically the CCD or CMOS camera will have a resolution between a few hundred thousand and a few million pixels. A high resolution camera may have tens of millions of pixels, or more.
The imaging system may be configured to magnify the image of the array. The required magnification of the imaging system can be determined by the required field of view and by the size of the CCD or CMOS sensor. By way of a non-limiting example, if the optical instrument is used to take an image of 2 adjacent wells of a 16-well microscope slide simultaneously, the required field of view is approximately 16 mm×8 mm. If the light-sensitive area of the CCD or CMOS sensor is about 4.8 mm×3.6 mm, the instrument is a macroscope and a magnification of about 0.3 is required. In this case, only data from the central 4.8 mm×2.4 mm of the sensor would be used.
By way of non-limiting example, an appropriate imaging system with a magnification of 0.3 may be constructed using an achromatic cemented doublet lens with a focal length of 85 mm and an infinite-conjugate camera lens with a focal length of 25 mm. If a spectrally selective emission filter is used (for example, a single-band interference filter, multi-band interference filter, longpass interference filter, or longpass colored glass filter), and this filter is typically located between the achromatic cemented doublet lens and the camera lens. Additional configurations of an imaging system with a magnification of 0.3 are possible. For example, the achromatic cemented doublet lens can be omitted, and a finite-conjugate camera lens can be used instead of an infinite-conjugate camera lens. In this case, the spectrally selective emission filter is preferably located on the long-conjugate side of the camera lens.
A sensor with a light-sensitive area of 4.8 mm×3.6 mm is known as a 1⁄3-inch format sensor. If a sensor of different size is used, the required magnification will be different. By way of a non-limiting example, if the required field of view is 16 mm×8 mm and a sensor having a light-sensitive area of 6.4 mm×4.8 mm (known as a 1⁄2-inch format sensor) is used, then the required magnification is 0.4. An appropriate imaging system with a magnification of 0.4 can be constructed using, for example, an achromatic cemented doublet lens with a focal length of 85 mm and an infinite-conjugate camera lens with a focal length of 35 mm.
As another non-limiting example, if the dimensions of a region are about 0.66 mm×0.66 mm and a sensor with a light-sensitive area of 8.8 mm×6.6 mm (known as a 2⁄3-inch format sensor) is used, then the instrument is a microscope and the required magnification is about 10. In this case, only data from the central 6.6 mm×6.6 mm of the sensor will be used. An appropriate imaging system with a magnification of 10 can be constructed using, for example, an infinite-conjugate microscope objective with a focal length of 20 mm and a microscope tube lens with a focal length of 200 mm, with a spectrally selective emission filter typically located between the microscope objective and the tube lens. Alternatively a finite-conjugate 10× microscope objective can be used and the microscope tube lens can be omitted. In this case the spectrally selective emission filter can be located on the long-conjugate side of the microscope objective.
An imaging system of any required magnification can be constructed using a combination of off-the-shelf and custom optical elements that does not necessarily include either a camera lens or a microscope objective. The optical elements may have various combinations of spherical, flat, aspheric, or diffractive surfaces.
Illumination System
Another main component of the optical instrument is an illumination system. The purpose of the illumination system is to illuminate the array within the field of view of the CCD or CMOS camera. To reduce sensitivity to edge effects and to misalignment, it may be desirable for the illuminated area to be slightly larger than the camera's field of view. By way of a non-limiting example, if the field of view is about 16 mm×8 mm, a reasonable illuminated area may be about 18 mm×10 mm. The types of illumination may be Abbe, Kohler, or neither Abbe nor Kohler illumination. Abbe illumination and Kohler illumination are well known and are described in, for example, Chapter 14 of Optical System Design, Second Edition by Robert E. Fischer et al., SPIE Press, McGraw-Hill, NY, 2008.
In some embodiments, the illumination system may be used for off-axis illumination. In other embodiments, the illumination system may be used for trans-illumination or epi-illumination. If the illumination system is used for off-axis illumination or trans-illumination, then the illumination system and the imaging system are separate from each other, with no shared optical components. If the illumination system is used for epi-illumination, then the illumination system and the imaging system may share a beamsplitter and possibly one or more lenses. The beamsplitter may be a plate beamsplitter or a cube beamsplitter. If the optical instrument is used for fluorescence imaging, the beamsplitter is typically a single-edge or multi-edge longpass dichroic beamsplitter.
Often the illumination system may contain a square or rectangular aperture so that the illuminated area has the same shape as the region that is imaged by the CCD or CMOS camera. In embodiments where off-axis illumination is used, the aperture may be trapezoidal in shape instead of square or rectangular. An off-axis illumination system may or may not satisfy the Scheimpflug condition. The Scheimpflug condition is described in, for example, Modern Optical Engineering, Second Edition by Warren J. Smith, McGraw-Hill, NY, 1990.
In some embodiments, the illumination system may contain one or more of the following: spherical lenses, aspheric lenses, a solid homogenizing rod with a rectangular or trapezoidal cross section, a hollow homogenizing light tunnel with a rectangular or trapezoidal cross section, a microlens array or a pair of microlens arrays, a stationary or rotating diffuser, a compound parabolic concentrator, a non-imaging optical element other than a compound parabolic concentrator (e.g., a free-form catadioptric element), an optical fiber, a fiber bundle, or a liquid light guide.
The illumination system may contain one or more light sources, selected from the group including, but not limited to, one or more LEDs, one or more lasers, a xenon arc lamp, a metal halide lamp, or an incandescent lamp, or a combination thereof. The illumination system may also contain a spectrally selective excitation filter selected from the list including, but not limited to, a single-band interference filter, a multi-band interference filter, or a shortpass interference filter. If the illumination system contains two or more light sources, they may be the same (by way of non-limiting example, two or more LEDs with peak emission wavelengths of about 525 nm for excitation of Cy3 dye, mounted as close together as possible on a circuit board) or different (by way of non-limiting example, an LED with a peak excitation wavelength of about 525 nm for excitation of Cy3 dye, and an LED with a peak excitation wavelength of about 625 nm for excitation of Cy5 dye, mounted as close together as possible on a circuit board). Two-color or multicolor LED assemblies are available from, for example, LED Engin, Inc. (San Jose, Calif.) and Innovations in Optics, Inc. (Woburn, Mass.).
In some embodiments, a light source in the illumination system may be controlled electronically. By way of a non-limiting example, a light source may be synchronized with the CCD or CMOS camera so that the light source turns on when the CCD or CMOS camera begins an exposure and turns off when the camera finishes an exposure. If the illumination system contains two or more light sources, they may optionally be controlled together or independently of each other.
In some embodiments, a light source may be left on continuously. In this case, the illumination system may contain an electronically controlled shutter, and the shutter may be synchronized with the CCD or CMOS camera so that the shutter opens when the CCD or CMOS camera begins an exposure and closes when the camera finishes an exposure.
In some embodiments, the optical instrument may contain a single illumination system. In other embodiments, the instrument may contain two or more illumination systems that are identical. In yet other embodiments, the instrument may contain two or more illumination systems that are different. By way of non-limiting examples, an optical instrument for detecting fluorescence from Cy3 and Cy5 may contain one illumination system for Cy3 excitation and another illumination system for Cy5 excitation, or it may contain a single illumination system that is used for both Cy3 and Cy5 excitation.
Translation Stage
Yet another main component of the optical instrument may be one or more translation stages. One purpose of the translation stage may be to move sample holders in and out of the field view of the imaging system. Another purpose of the translation stage system may be to move the imaging system, components of the imaging system, the illumination system, or components of the illumination system relative to the sample or relative to one another, for obtaining the best possible image.
In many embodiments of the presently disclosed systems, the translation stage may further comprise a sample holder. By way of non-limiting examples, if the optical instrument is used to take images of 16-well microscope slides, the translation stage contains a slide holder. If the optical instrument is used to take images of 96-well plates or 384-well plates, and it contains a plate holder. The slide holder, plate holder, or other array support holder may be mounted on the translation stage system in any of a variety of ways known to those skilled in the art.
The translation stage may have one or more axes of motion. By way of a non-limiting example, if the support is a 16-well microscope slide and the instrument takes images of 2 adjacent wells simultaneously, a single axis of motion may be sufficient. By way of another non-limiting example, if the support is a 96-well plate and the instrument takes images of 2 adjacent wells simultaneously, then at least 2 axes of motion would be required. Additional axes of motion for adjustment of focus and tilt may also be added. If the instrument can take an image of all of the regions on the support in a single exposure, then the translation stage may be omitted in some embodiments of the optical instrument.
Housing
The systems and devices described herein can include features for insuring that the sensors of the device detect appropriate signal. For example the systems and devices can include light excluding features. The light excluding features generally reduce unintended signal from reaching light sensitive sensors. In many embodiments, one or more of the imaging system, illumination system, translation stage, and other components of the instrument are surrounded by a housing. The housing can be opaque. The housing can, in some instances, act as a faraday cage. In some instances a single housing is sufficient to exclude light from systems. The single housing can also provide external protection of the system. Alternatively, multiple housings may individually contain one or more components of the instrument. In some instances the housings are nested housings. In various embodiments, the housing can be gas and/or liquid tight.
The housing may have an access point which can exclude light from the interior of the housing. The access point may comprise materials that absorb light in the spectrum relevant to the sensors within the housing, e.g. vantablack in the visible spectrum. The access point may comprise a closure device. The closure device may be opaque. The closure device may be, e.g., a door. The closure device may be substantially light-tight in a closed position. The closure may be light-tight in a closed position.
The closure device can be opened, e.g., for insertion and removal of a 16-well slide, 96-well plate, 384-well plate, or other array support. A sensor (for example, a photointerrupter) may be used to determine whether the closure device is open or closed. The instrument's software or electronic hardware may prevent the light source in the illumination system from turning on when the closure device is open, may prevent power from being applied to the image sensor, and/or may prevent the translation stage from moving when the closure device is open.
In some embodiments, the housing may further comprise a mechanism for automated opening and closing of the closure device, as illustrated in FIGS. 10-13. The closure device can provide access to the interior of the housing. The closure device can provide access for the array to be loaded in and out of the instrument. This operation can be performed automatically. In some instances, the closure device can exclude ambient light during imaging, while opening reliably to permit loading.
In some instances the closure device does not comprise pivoting parts. In some instances the closure device does not interact with pivoting parts or latches. In some embodiments of the disclosed systems and platforms, the closure device is held by magnets to the housing. Magnets can hold the closure device to the housing in a closed position. Magnets can hold the closure device to a loading device, e.g. a tray, in an open position. During a transition from an open to closed position the closure device can transition from being primarily magnetically attached to a loading device to being primarily magnetically attached to the housing. During a transition from a closed to open position the closure device can transition from being primarily magnetically attached to the housing to being primarily magnetically attached to the loading device. In some instances the transition between the open and closed state is magnetically unstable, such instability causing the closure device to move from the transition state to either the more stable open or closed position.
The closure device can comprise a self-locating function provided by conical features on the door. The thicknesses of the parts which support the magnets on each side of a mating pair, and the depth of retaining pockets within those parts, defines the spacing between magnets in each mating pair, and thus the holding forces. The design geometry is matched to the power of the motors to provide enough retaining force, without requiring high motor torque. The system is further designed such that the motor current and speed (and hence torque) can be controlled to improve the performance, and avoid creating a safety hazard. Two of the four magnet pairs are used to temporarily hold the door to the front of the sample tray, when the tray moves outward for loading an array assembly, as depicted in FIG. 10. The other two magnet pairs are used to hold the door closed against the front panel, after the tray has moved inwards (and separated the other two magnet pairs in the process). The respective allocation of magnets is shown in FIG. 11A. The mating magnets on the front of the stage are shown in FIG. 12. The locations of the mating magnets in the front panel are shown in FIG. 13. To provide for secure grip (and therefore reliable operation), rare earth magnets provide high strength (e.g. neodymium magnets). Some embodiments of the design call for disc magnets approximately 8 mm in diameter and 3 mm thick, with the magnetic field parallel to the axis. In some embodiments, it is sufficient to replace one magnet from each pair with a weaker magnet, or with a piece of magnetic material such as iron or mild steel.
In some embodiments of the systems and platforms disclosed herein, a mechanism for providing for automated door or lid closure on one or more instrument compartments is provided, wherein the mechanism comprises: (a) a closure; (b) a housing which magnetically holds the closure in a first position; and (c) a translation stage which magnetically holds the closure in a second position. In some embodiment, the mechanism further comprises a gasket positioned between the closure and the housing. In some embodiments of the mechanism, the gasket is attached to the closure. In other embodiments, the gasket is attached to the housing. In some embodiments, the closure and housing are substantially opaque, and the gasket creates a substantially light-tight seal between the closure and the housing in the first position. In some embodiments of the mechanism, one or more magnets are positioned to hold the closure onto the housing in the first position. In some embodiments of the mechanism, one or more magnets are positioned to hold the closure onto a first surface of the translation stage in the second position. In some embodiments, the mechanism further comprises two or more pairs of mating locating features to align the closure with the translation stage in the second position. In some embodiments, the mechanism further comprises two or more pairs of mating locating features to align the closure with the housing in the first position. In some embodiments of the mechanism, the pairs of mating locating features comprise conical pins and conical holes. In some embodiments, the housing comprises an optical instrument. In some embodiments, the translation stage includes a sample holder. In some embodiments, the sample holder is designed to hold a microscope slide, a microarray, or a microwell plate. In some embodiments, the closure is not hinged. In some embodiments, the closure is not attached to either the housing or the translation stage through the use of fasteners such as screws or clips. In some embodiments, the closure is not attached to either the housing or the translation stage through the use of an adhesive.
Image Data
The methods, systems, and platforms described herein for counting one or more labeled features on an array may comprise data input, or use of the same. The data input may comprise imaging information and/or images of one or more regions of arrays. The images comprise pixel data, wherein each unit of pixel data may be encoded in, by way of non-limiting examples, 4, 8, 12, 14, 16, 32, 64, 128, 256, or more bits. An image may encompass one or more regions of an array. The spatial resolution of an image may be determined by the spatial resolution of the optical instrument, but in some embodiments of the disclosed methods and systems, spatial resolution may be enhanced by digital image processing schemes based on, by way of non-limiting examples, interpolations, extrapolations, modeling, and/or transforms.
The methods, systems, and platforms described herein for counting one or more labeled features on an array may comprise acquisition and analysis of images of one, two, or more distinct regions on an array. In some embodiments, two or more regions to be imaged may overlap, partially overlap, or not overlap at all. Furthermore, two or more regions to be imaged may be adjacent, or non-adjacent.
The methods, software, systems, and platforms described herein for counting one or more labeled features on an array may comprise acquisition and analysis of images of all or a portion of an array. In some embodiments, the region of an array that is imaged may comprise at least about 1% of the total area of the array. In some embodiments, the region of the array that is imaged image may comprise at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of the total area of the array. In other embodiments, the region of the array to be imaged may comprise at least about 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or more of the total area of the array. In still other embodiments, the region of the array to be imaged may comprise at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of the total area of the array. In some embodiments, the region of the array to be imaged may comprise at least about 75%, 80%, 85%, 90%, 92%, 95%, 97% or more of the total area of the array.
The methods, software, systems, and platforms described herein for counting one or more labeled features on an array may comprise acquisition and analysis of images of all or a portion of the features of an array. In some embodiments, the image may encompass between 10% and 100% of the total number of features on the array. In some embodiments, the image may encompass at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the total number of features on the array. In some embodiments, the image may encompass at most 95%, at most 90%, at most, 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, at more 10%, or at most 5% of the total number of features on the array. The number of features encompassed by the image may fall within any range bounded by any of these values (e.g. from about 15% to about 90% of the total number of features of the array).
Image Acquisition
The methods, systems, and platforms described herein comprise software for acquiring images from an optical instrument. In some embodiments, e.g. for optical instruments comprising two or more image sensors, the image acquisition may operate in a parallel mode, i.e. where two or more images are acquired simultaneously. Alternatively, the image acquisition may operate in a serial mode, where two or more images are acquired sequentially. In general, image acquisition may be performed in a continuous fashion (i.e., wherein the image is acquired within a single exposure time period) or intermittently (i.e., wherein the image is acquired in a discontinuous fashion, e.g. using two or more separate exposure time periods, wherein in some embodiments two more images are combined for signal averaging purposes).
In a non-limiting example, an array may comprise 16 wells where an image is formed for each well. The image acquisition module may sequentially read the 16 images. Reading the 16 images can be completed in a continuous time period; or, the system may read a first image followed by analyzing the first image, and then the procedure of image reading and image analysis repeats till the 16th image is analyzed. Alternatively, the image acquisition module may read a pair of images at once, and repeat the reading till all the 16 images are acquired. The 16 images may be read sequentially in a single time period. In some applications, a pair of images may be read, followed by immediate image analyses.
Image Analysis
In general, one of the objectives in performing image processing and analysis is to improve signal-to-noise ratios and quantitation. In an ideal array experiment, labeled features comprising bound target molecules and/or labels would produce a uniform, non-saturated signal level when imaged and non-labeled features would appear uniformly dark, with a signal level of close to zero. In reality, a variety of artifacts due to instrumental and/or assay procedural issues including, but not limited to, stray light, background fluorescence (in the case of fluorescence-based imaging), particulate contaminants, and non-specific binding of assay components, can produce images that hinder one's ability to extract quantitative signal intensity data and make definitive calls as to which features of the array are labeled. Accordingly, the methods, systems, and platforms disclosed herein may comprise software for performing a variety of image processing tasks including, but not limited to, feature location, image orientation correction, background correction, intensity measurement, data scaling, data thresholding, and data analysis functions.
Image orientation and location of features. In some embodiments, fiducial features incorporated into the design of an array are used to orient the image and locate features in an automated fashion using custom image analysis software. By way of non-limiting example, the microarray pattern shown in FIGS. 15 and 16 consists of a 32×32 array of features, where fiducial features in the top and bottom rows permit location of the array in the digital images. The fiducial features are typically arranged in an asymmetric pattern whose orientation is readily identifiable, e.g. fiducial features located in the top row of features in an array such as that depicted in FIG. 16 may comprise a distinctive pattern for which the left and right ends of the row are asymmetric, while the pattern of fiducial features in the bottom row is typically different from that in the top row. This permits easy manual and automatic identification of incorrect placement of the array, and also facilitated detection of imaging problems. In some embodiments, the image may be transformed also transformed so that the apparent orientation of the images corresponds to the orientation as viewed by a user, often referred to as the viewing reference orientation, as shown in FIG. 14 for a specific embodiment of an array designed for use with the methods, systems, and platforms disclosed herein.
Refinement of feature locations. In some embodiments, the measured location of each feature is refined so as to account for array fabrication errors, which can produce offsets of several image pixels. The locations of features obtained during the initial image orientation and feature location step may be used to subdivide the array or array region into analysis windows, for example an array may be divided into 32×32 analysis windows, wherein each analysis window comprises an image area of 12×12 pixels centered on each feature, as shown in FIG. 19. The size of the analysis window used is dependent on the size of the features on the array, and may be any size that is necessary to correctly locate and distinguish between features and background regions on the array. By way of non-limiting examples, the analysis window may be defined as a 5×5, 7×7, 9×9, 15×15, 51×51, or 101×101 pixel area that is centered on the array feature. The position of the feature within the window may be determined on the basis of the signal intensity distribution and clustering of the pixels within the analysis window. The refined location of the feature is calculated as an offset in coordinates X and Y from the site predicted by a perfect rectilinear grid. In some embodiments, distortion of the feature location results due to defects such as dust is avoided by making use of the correlation between printing artifacts between different arrays on the same substrate. Since the printing artifacts are typically consistent, the correction relative to a hypothetical rectilinear grid is also consistent. The feature location optimization results for a given feature are combined across all of the arrays being analyzed, and the median offset is used for subsequent analysis, which greatly decreases noise in the final experimental results.
Local background correction. Once the feature pixel set “S” and background pixel set “B” have been defined for each location in the array (for example, see FIG. 20), the local background is removed via a calculation involving signal intensity and background intensity statistics. Examples of suitable signal and background intensity statistics for use in local background correction calculations include, but are not limited to, the mean, the median, or a ratio of signal-to-background. In some embodiments, following feature location refinement performed as described above, the pixels within the analysis window are assigned to be signal pixels, background pixels, or transitional pixels, i.e. pixels to be disregarded, in subsequent calculations of signal and background intensity statistics (see FIG. 20). In some embodiments of the disclosed methods, local background correction is performed via subtraction in logarithm space, i.e. a calculation that is closely related to a signal-to-background ratio calculation, as illustrated in a non-limiting example below:
Given the 16-bit pixel data measurements for a defined feature and background area, on next calculates a single value S for the signal pixels and a value B for the background pixels respectively. One useful statistic is the median value for each set of pixels, i.e.
S=the median of the pixel values for the set of pixels “S”
B=the median of the pixel values for the set of pixels “B”
Various other statistics could be used in this situation, such as the mean of the set of values, or a nominated percentile within the set. It is not necessary, and may not be optimal, to use the same statistic for both S and B. For example, low data noise and strong separation between “on” and “off” data points can be obtained by using:
S=the median of the pixel values for the set of pixels “S”, and
B=the 25th percentile of the pixel values for the set of pixels “B”.
As a further enhancement, the particular percentile used can be a pre-stored and re-configurable parameter stored in a settings file.
The background-corrected intensity statistic for each spot is:

I=log2(16S)−log2(16B)

An example of a scatter plot (intensity statistic vs feature number) and histogram of intensity data are shown in FIG. 21. In this example, the background is corrected for by performing a subtraction of logarithms, such that the intensity metric is related to a ratio of S and B. In some situations, a linear subtraction (e.g. I=S−B) is preferable. Once the background-corrected intensity statistics have been calculated for the complete set of features, the next task is to determine which of the features are labeled (i.e. “on”, or “positive”) and which are non-labeled (i.e. “off” or “negative”). This is accomplished by determining a signal intensity threshold value based on a statistical analysis of the local background-corrected feature intensities, and subsequently counting how many features, k, have background-corrected signal intensities that are larger than this threshold level. The signal intensity threshold may be considered a “dynamic” signal intensity threshold in that the threshold is determined through analysis of the data from the current experiment, and thereby eliminates potential errors due to such factors as instrumental drift and variations in assay procedure.

Determination of dynamic signal intensity thresholds. In many embodiments of the methods, systems, and platforms disclosed herein, a dynamic signal intensity threshold is determined for one or more regions of an array by performing one or more statistical analyses of the background corrected signal intensity data for the complete set of features. Any of a variety of statistical (or empirical) analysis techniques may be used, including but not limited to, k-means clustering, k-medoids clustering, mixture model statistical analysis, probe reference distribution methods, or empirical analysis based on sorting of background corrected signal intensity values, sorting of pairwise differences in background corrected signal intensity values, etc. In some embodiments, analyses may utilize spatial and/or temporal information collected across multiple analysis windows, across multiple array regions, or over specified periods of time, or combinations thereof, to improve the quality of the analysis and thereby improve the quantitative aspects of the disclosed methods. In some embodiments, other sources of information, including, but not limited to, for example, locations of probes, frequently occurring artifact patterns, previously derived results, literature reports, array manufacturers' suggestions, human knowledge, and/or human guidance may also be integrated into the analysis.
By way of a non-limiting example of threshold determination, in some embodiments of the disclosed methods, the background corrected signal intensity threshold may be determined using an empirical approach (e.g. the “E-Derivative” approach; see FIG. 22) wherein the background corrected signal intensity data for the complete set of array features constitutes a set I={Ii}. The set I is sorted in increasing order to obtain a set of ordered corrected signal intensity values z={zi}={Sort[yi]}. Next, the differences between each sorted array value are calculated to obtain d={d1, d2, . . . , dm}, where di=zi+1−zi. The intensity differences are then smoothed using a “window” whose width is w, to produce a smoothed, sorted array s:




  
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The threshold is T, the point for which the slope of the smoothed, sorted data is steepest (see FIG. 21):

T=max(sj)

The number of features, k, which are “on” (or labeled) is:

k=Σi=1mI[Ii>T]. 

By way of another non-limiting example of threshold determination, in some embodiments the background corrected signal intensity threshold may be determined by fitting the background corrected feature intensity data to two more assumed distributions (i.e. a “Mixture Model” approach), wherein the assumed distributions comprise normal distributions, uniform distributions, etc. The mixture model approach essentially models the underlying process that generated the data, by assuming that the positive feature intensities are generated from a positive feature distribution with higher average signal intensity, and the negative feature intensities are generated from a negative feature distribution with lower average signal intensity. This approach additionally models the variability in the feature intensities generated by each distribution, which can be useful in cases where the negative feature intensities tend to be much less variable, while the positive feature intensities tends to be much more variable. The choice of the distributions is determined by the shape of the data curve in a background corrected feature intensity histogram. The parameters of the model, e.g. the estimated average intensities for “on” and “off” features and their corresponding variance, are estimated from the data using a method such as the Expectation Maximization algorithm.
By way of another non-limiting example of threshold determination, in some embodiments the background corrected signal intensity threshold may be determined by fitting the background corrected feature intensity data to a model function comprising three assumed distributions (i.e. a “3-Component Model” approach), wherein the assumed distributions comprise a log-normal distribution, Dist1, for the “off” spots, a normal distribution, Dist2, for the “on” spots, and a flat offset FlatLevel. Adjustable parameters for the model include: (i) the number of bins in the starting histogram, (ii) Dist1 amplitude, (iii) Dist1 position, (iv) Dist1 standard deviation, (v) Dist2 amplitude, (vi) Dist2 position, (vii) Dist2 standard deviation, and (viii) FlatLevel. An example fit to histogram data is shown in FIG. 23. One non-limiting example of a method to determine the threshold after fitting feature intensity data to such a distribution is as follows: (i) fit the 3-component distribution to the histogram data, and (ii) set the threshold T by calculating the following values: (1) the intensity tlow where the high-intensity side of the fitted log-normal distribution component drops below 1 (or a defined parameter for comparison), (2) the intensity tsubflat where the high-intensity side of the fitted log-normal distribution component drops below the fitted FlatLevel result, (3) the intensity tsubnorm where the high-intensity side of the fitted log-normal distribution component drops below the value of the fitted normal distribution at that histogram bin, and (4) choosing T=min[tlow, tsubflat, tsubnorm]. Alternative approaches for determining a threshold using a 3-component model approach will be apparent to those of skill in the art. It can be beneficial to calculate starting values of model parameters, to improve the speed and reliability of the modelling process, which can be achieved using methods such as a coarse search to identify the dominant peaks in the histogram, or based on assumptions derived from typical historical data sets.
By way of another non-limiting example of threshold determination, in some embodiments the background corrected signal intensity threshold may be determined using a “Peak Split Fiducials” approach. This approach, which copes well with low-quality data, is described as follows. An initial split of the feature intensity data into high and low intensity groups is made using the scale defined naturally by the spread between “on” (label present) and “off” (label absent) features in the fiducial rows. Then, the histogram peak (after optionally smoothing the data using standard methods such as a moving average filter) is found for each group. The threshold is then determined by examining the spread in the intensity data around the low-intensity group peak. Define upper and lower bounds of fiducial intensity: (i) Foff=[median of OFF fiducials], (ii) Fon=[median of ON fiducials], and (iii) Frange=Fon−Foff. Perform an initial split of the data based on the fiducial scale, at the level Splitvalue=Foff+PeakSplit×Frange, where the parameter PeakSplit is a percentage of Frange. Find 2 peaks: (i) Peak1=the intensity peak for which the histogram is a maximum, for all features of intensity less than Splitvalue, (ii) Peak2=the intensity peak for which the histogram is a maximum, for all features of intensity greater than Splitvalue. Calculate the standard deviation, Stdev1, of all the features in the neighbourhood of Peak1, defined as all index features from the lowest intensity up to Peak1+PeakOffsetFraction×(Peak2−Peak1), where PeakOffsetFraction is an adjustable parameter. Set the threshold to the lesser of Tpsf and TLocMin, which are calculated as follows: (i) Tpsf=Peak1+StdevMultiple×Stdev1, where StdevMultiple is a parameter, OR TLocMin=the intensity corresponding to the minimum of a smoothed histogram curve between Peak1 and Tpsf. Similar approaches using different methods for determining the spread around either peak can also be used.
The methods and systems disclosed herein may comprise detecting one or more labeled features within one or more regions on an array. In some embodiments, detecting a labeled feature within a region may comprise comparing the background corrected signal intensity for a feature with a dynamic signal intensity threshold derived through statistical analysis of the background corrected signal intensities for the complete set of features. When the background corrected signal intensity for a given feature is above the threshold, the feature may be classified as a labeled feature. Alternatively, if the background corrected signal intensity for a given feature is below the threshold, the feature may be classified as non-labeled. Application of a background corrected signal intensity threshold to the corrected signal intensity data for the complete set of features thus constitutes a binary transformation of the data to a digital output wherein features are classified as either labeled (“on”) or non-labeled (“off”). Those of skill in the art will recognize that there are many possible variations in the type and order of analysis steps that may be applied to achieve this binary transformation.
Calculation of the absolute number of target molecules in a sample. The absolute number of target molecules in a sample, wherein the target molecules have been labeled in a stochastic fashion as described previously, may be determined using arrays comprising feature sets comprising probes that are specific for the labels in the stochastic label set. Following hybridization or binding of the target molecules or labeled target molecules to the array, the array is imaged and processed as described above, and the number of target molecules, N, in the sample is determined from the number, k, of labeled features based on Poisson distribution statistics:




  N
  =
  
    
      -
      m
    
    *
    
      log
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        1
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where m is the total number of features (i.e. the total number of unique labels in the set of stochastic labels).

Quality metrics. In some embodiments, it is beneficial to include a numerical measure of the quality of the data, to help to gauge the success of an experiment. In some embodiment, this quality measurement may be based on statistics from the feature-by-feature intensity data. One simple quality measurement QSep1 is simply the difference between the means of the positive and negative features intensities, after background correction and scaling, i.e. QSep1=(mean intensity of features having an intensity above the signal intensity threshold)−(mean intensity of features having an intensity below the threshold). In some embodiments, this metric may also incorporate the spread in the intensities of the feature distribution(s) by scaling the difference between means by the standard deviation of each distribution, e.g. QSep2=QSep1/(standard deviation of intensities for feature having intensities below the threshold intensity). Other quality measurements can be constructed based on the separation and breadth of modelled distributions which are fitted to the experimental data. In some embodiments, deviance measurement may be used for a quality metric (FIG. 24); this is a calculation based on the degree of separation between two fitted normal distributions. In some embodiments, it is preferable to empirically determine a dynamic intensity threshold by setting the threshold to a value which maximizes a quality metric.
Confidence intervals. In some embodiments of the methods disclosed herein, it is beneficial to define confidence intervals (see Dube, et al. (2008), PLoS ONE 3(8): e2876 for a more complete description) when specifying estimates of the absolute number of target molecules detected in a sample using the techniques described above. The 95% confidence interval of the estimation of N from stochastic labeling experiments can be derived from k for a single reaction employing a single set of m distinct labels. The 95% confidence interval for N ranges from Nlow to Nhigh, where



  
    
      
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Ratio of the number of copies of a target molecule in two samples. Frequently, researchers seek to compare the expression levels of genes in different samples, by calculating a ratio between gene expression levels in two or more samples. Using calculations such as those described above, it is possible to derive confidence intervals for such ratios where the number of target molecules in each sample are determined using the methods, systems, and platforms as disclosed herein.
Replicate experiments. The benefit of performing replicate experiments, and the proper calculation of associated uncertainties, is illustrated in FIG. 25. While results (blue points) from replicate experiments can simply be combined (blue error bars), calculating the uncertainty from Poisson statistics, wherein one considers the replicates as comprising a larger pool of labels, gives the smaller green error bars illustrated in the figure. The accuracy of this estimation will vary depending on the consistency between replicates, and there is a numerical simplification employed in considering the labels of replicate experiments to be a pool of diverse labels. Therefore, in some embodiments of the disclosed methods, different methods for calculating confidence intervals may be more appropriate at high ratios of k/m.
User Interface
The methods, software, systems, and platforms disclosed herein may comprise a user interface, or use of the same. The user interface may provide one or more inputs from a user. The input from the user interface may comprise instructions for counting the one or more labeled features in a real time mode. The input from the user interface may comprise instructions for counting the one or more features from one or more images. The one or more images may be archived images. The one or more images may be live captured images.
Different platform operators may have their own preferences about the timing to analyze images. One platform operator may want to run the image analyses while live capturing images. Another platform operator may run the image analyses after all the images have been collected. Or, another platform operator may run the image analyses on a set of archived images. These options can be selected via inputs to the user interface.
Digital Processing Device
The methods, software, systems, and platforms disclosed herein may comprise a digital processing device, or use of the same. The digital processing device may comprise one or more hardware central processing units (CPU) that carry out the device's functions. The digital processing device may comprise an operating system configured to perform executable instructions. The digital processing device may be connected to a computer network. The digital processing device may be connected to the Internet such that it accesses the World Wide Web. The digital processing device may be connected to a cloud computing infrastructure. The digital processing device may be connected to an intranet. The digital processing device may be connected to a data storage device.
Suitable digital processing devices may include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, subnotebook computers, netbook computers, netpad computers, set-top computers, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. In some instances, smartphones may be suitable for use in the system described herein. In some instances, select televisions, video players, and digital music players with optional computer network connectivity may be suitable for use in the system described herein. Suitable tablet computers may include those with booklet, slate, and convertible configurations, known to those of skill in the art.
The digital processing device may comprise an operating system configured to perform executable instructions. The operating system may be software, including programs and data, which manages the device's hardware and provides services for execution of applications. Suitable server operating systems may include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Suitable personal computer operating systems may include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. The operating system is provided by cloud computing. Suitable mobile smart phone operating systems may include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.
The digital processing device may comprise a storage and/or memory device. The storage and/or memory device may be one or more physical apparatuses used to store data or programs on a temporary or permanent basis. The digital processing device may be a volatile memory and may require power to maintain stored information. The digital processing device may be a non-volatile memory and may retain stored information when the digital processing device is not powered. The non-volatile memory may comprise flash memory. The non-volatile memory may comprise dynamic random-access memory (DRAM). The non-volatile memory may comprise ferroelectric random access memory (FRAM). The non-volatile memory may comprise phase-change random access memory (PRAM). The storage device may include, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. The storage and/or memory device may be a combination of devices such as those disclosed herein.
The digital processing device may comprise a display. The display may be used to send visual information to a user. The display may be a cathode ray tube (CRT). The display may be a liquid crystal display (LCD). The display may be a thin film transistor liquid crystal display (TFT-LCD). The display may be an organic light emitting diode (OLED) display. The OLED display may be a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. The display may be a plasma display. The display may be a video projector. The display may be a combination of devices such as those disclosed herein.
The digital processing device may comprise an input device to receive information from a user. The input device may be a keyboard. The input device may be a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. The input device may be a touch screen or a multi-touch screen. The input device may be a microphone to capture voice or other sound input. The input device may be a video camera to capture motion or visual input. The input device may be a combination of devices such as those disclosed herein.
Non-Transitory Computer Readable Storage Medium
The methods, software, systems, and platforms disclosed herein may comprise one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked digital processing device. A computer readable storage medium may be a tangible component of a digital processing device. A computer readable storage medium may be optionally removable from a digital processing device. A computer readable storage medium may include, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like. The program and instructions may be permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.
Computer Programs (General)
The methods, software, systems, and platforms disclosed herein may comprise at least one computer processor, or use of the same. The computer processor may comprise a computer program. A computer program may include a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, features, Application Programming Interfaces (APIs), data structures, and the like, that perform particular tasks or implement particular abstract data types. A computer program may be written in various versions of various languages.
The functionality of the computer readable instructions may be combined or distributed as desired in various environments. A computer program may comprise one sequence of instructions. A computer program may comprise a plurality of sequences of instructions. A computer program may be provided from one location. A computer program may be provided from a plurality of locations. A computer program may include one or more software modules. A computer program may include, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.
Web Applications
A computer program may include a web application. In light of the disclosure provided herein, those of skill in the art will recognize that a web application may utilize one or more software frameworks and one or more database systems. A web application may be created upon a software framework such as Microsoft .NET or Ruby on Rails (RoR). A web application may utilize one or more database systems including, by way of non-limiting examples, relational, non-relational, feature oriented, associative, and XML database systems. Suitable relational database systems may include, by way of non-limiting examples, Microsoft® SQL Server, mySQLTM, and Oracle®. Those of skill in the art will also recognize that a web application may be written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. A web application may be written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). A web application may be written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). A web application may be written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®. A web application may be written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, JavaTM, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), PythonTM, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. A web application may be written to some extent in a database query language such as Structured Query Language (SQL). A web application may integrate enterprise server products such as IBM® Lotus Domino®. A web application may include a media player element. A media player element may utilize one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, JavaTM, and Unity®.
Mobile Applications
A computer program may include a mobile application provided to a mobile digital processing device. The mobile application may be provided to a mobile digital processing device at the time it is manufactured. The mobile application may be provided to a mobile digital processing device via the computer network described herein.
A mobile application may be created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications may be written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Featureive-C, JavaTM, Javascript, Pascal, Feature Pascal, PythonTM, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
Suitable mobile application development environments may be available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments may be available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, AndroidTM SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.
Those of skill in the art will recognize that several commercial forums may be available for distribution of mobile applications including, by way of non-limiting examples, Apple® App Store, AndroidTM Market, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.
Standalone Applications
A computer program may include a standalone application, which may be a program that may be run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications may be often compiled. A compiler may be a computer program(s) that transforms source code written in a programming language into binary feature code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Featureive-C, COBOL, Delphi, Eiffel, JavaTM, Lisp, PythonTM, Visual Basic, and VB .NET, or combinations thereof. Compilation may be often performed, at least in part, to create an executable program. A computer program may include one or more executable complied applications.
Web Browser Plug-Ins
A computer program may include a web browser plug-in. In computing, a plug-in may be one or more software components that add specific functionality to a larger software application. Makers of software applications may support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins may enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. Those of skill in the art will be familiar with several web browser plug-ins including, Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. The toolbar may comprise one or more web browser extensions, add-ins, or add-ons. The toolbar may comprise one or more explorer bars, tool bands, or desk bands.
In view of the disclosure provided herein, those of skill in the art will recognize that several plug-in frameworks may be available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, JavaTM, PHP, PythonTM, and VB .NET, or combinations thereof.
Web browsers (also called Internet browsers) may be software applications, designed for use with network-connected digital processing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of non-limiting examples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. The web browser may be a mobile web browser. Mobile web browsers (also called mircrobrowsers, mini-browsers, and wireless browsers) may be designed for use on mobile digital processing devices including, by way of non-limiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google® Android® browser, RIM BlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSPTM browser.
Software Modules (General)
The methods, software, systems, and platforms disclosed herein may comprise one or more software, servers, and database modules, or use of the same. In view of the disclosure provided herein, software modules may be created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein may be implemented in a multitude of ways. A software module may comprise a file, a section of code, a programming feature, a programming structure, or combinations thereof. A software module may comprise a plurality of files, a plurality of sections of code, a plurality of programming features, a plurality of programming structures, or combinations thereof. The one or more software modules may comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. Software modules may be in one computer program or application. Software modules may be in more than one computer program or application. Software modules may be hosted on one machine. Software modules may be hosted on more than one machine. Software modules may be hosted on cloud computing platforms. Software modules may be hosted on one or more machines in one location. Software modules may be hosted on one or more machines in more than one location.
Databases
The methods, software, systems, and platforms disclosed herein may comprise one or more databases, or use of the same. In view of the disclosure provided herein, those of skill in the art will recognize that many databases may be suitable for storage and retrieval of imaging information. Suitable databases may include, by way of non-limiting examples, relational databases, non-relational databases, feature oriented databases, feature databases, entity-relationship model databases, associative databases, and XML databases. A database may be internet-based. A database may be web-based. A database may be cloud computing-based. A database may be based on one or more local computer storage devices.
EXAMPLES
The following illustrative examples are representative of specific embodiments of the methods, systems, and platforms described herein, but are not meant to be limiting in any way.
Example 1—Optical Instrument
FIG. 1 shows one embodiment of the optical instrument. This embodiment was used for simultaneously imaging 2 adjacent wells of a 16-well microscope slide. Each well contained 1024 (32×32) features, also called spots, which may be labeled with fluorescence. A spot diameter was approximately 80 microns. Center-to-center distance between adjacent spots was 161 microns. The purpose of the instrument was to determine the brightness of each spot. FIG. 1E shows the translation stage system 105, imaging system 106, and illumination system 107. The translation stage system contained a single-axis translation stage which was constructed from a Misumi model SSELBW9-170 recirculating ball slide driven by a Haydon Kerk 26000-series linear actuator. A holder for a 16-well microscope slide was mounted on the translation stage. The linear actuator was controlled by a Peter Norberg Consulting model BC2D20-0700 motion controller. FIG. 1G shows a USB hub 112, custom circuit board 113, and a motion controller 114. The motion controller and the CCD camera were USB devices. The USB hub allowed communication between a computer and the instrument to take place over a single USB cable. The custom circuit board contained a Luxdrive model 3021-D-E-1000 LED driver. The custom circuit board contained a logic chip to synchronize the LED with the CCD camera (turn the LED on when the CCD camera starts an exposure and off when the CCD camera finishes an exposure) and to prevent the LED from turning on when the instrument's door is open.
Example 2—Imaging System
An embodiment of an imaging system is illustrated in FIG. 2. Light emitted by dye molecules on a surface of the support 205 was collimated by an achromatic cemented doublet lens 204, filtered by a bandpass filter 203, and focused by a camera lens 202 onto the sensor 201 of a CCD camera. Lens 204 (Edmund Optics model 47640) had a focal length of 85 mm and a diameter of 25 mm. Filter 203 (Semrock model FF01-593/40-25) was an emission bandpass filter for use with cy3 or phycoerythrin dye. Lens 202 (Fujinon model HF25HA-1B) has a focal length of 25 mm. Lens 202 was a multi-element lens, but it is shown in FIG. 2 as an infinitesimally thin single-element lens because the design details of the multi-element lens were proprietary to Fujinon. The adjustable aperture stop of lens 202 was set to 2.8. The CCD camera (Point Grey Research model CMLN-13S2M-CS) had 1296×964 pixels and a pixel size of 3.75 microns. In this embodiment only the central 1280×640 pixels were used. The camera's plastic housing was removed and the camera's circuit board was cooled by a small fan. Lenses 204 and 202 formed a finite-conjugate imaging system with a magnification of 0.3.
Example 3—Illumination System
An embodiment of an illumination system is illustrated in FIG. 3. Light emitted by light source 301 was collimated by lens 302 and then passed through aperture 303, bandpass filter 304, and lenses 305, 306, and 307, before reaching sample 308. Light source 301 (LedEngin model LZ4-40G100) was an LED with a peak emission wavelength of approximately 525 nm. Lens 302 (Thorlabs model ACL2520-A) was an aspheric lens with a diameter of 25 mm and a focal length of 20 mm. Aperture 303 (Fotofab custom part) was a rectangular hole (19 mm×7.5 mm) in a 25-mm-diameter steel disk. Filter 304 (Semrock model FF01-531/40-25) was an excitation bandpass filter for use with cy3 or phycoerythrin dye. Lenses 305, 306, and 307 were plano-convex lenses with diameters of 25 mm and focal lengths of 60 mm. Lenses 301 and 302 formed a finite-conjugate imaging system with a magnification of 3 and imaged light source 301 onto the pupils of lenses 306 and 307. Lenses 306 and 307 formed a finite-conjugate imaging system with a magnification of 1 and imaged aperture 303 onto sample 308. The illumination system was tilted at 45 degrees with respect to sample 308. The illuminated area was approximately 19 mm×10.6 mm, where 10.6 mm (=7.5 mm/cosine (45 degrees)).
Example 4—Reference Probe Preparation
The purpose of this experiment was to illustrate the use of an ad hoc method to count the number of hybridizations taking place on an array. This example used probes that the specific DNA sequences attached to an array. The 32×32 feature arrays used in this experiment contain 960 different measure spots along with 32 positive control probes and 32 negative control probes (see FIGS. 15 and 16). The 32 positive control probes were used to ensure actual binding can occur by using stock oligonucleotide, while the 32 negative control probes contained empty spots with no probes at all. In the image analysis step, we considered a probe to have intensity above the set intensity threshold, then we referred to the probe as a positive probe, while if the probe intensity was below the set intensity threshold, we referred to the probe as a negative feature. Positive probes were assumed to measure whether there was a significant amount of corresponding complementary oligonucleotide in the sample, while negative features represented absent oligonucleotides. The positive probes were otherwise referred to as labels, meaning we can count up to 960 unique labels or barcodes, to measure 960 copies of oligonucleotides in the sample, which can then be further generalized to predict the actual amount of oligonucleotides in the original sample. Depending on the experiment, out of the M total labels, or 960 total labels, we could calculate the total number of copies, N, of the oligonucleotides in the sample, by predicting N from the actual observed unique barcodes or number of positive probes, k.
Example 5—Threshold Computation
The purpose of this experiment was to demonstrate one method to compute a threshold for discriminating between labeled and non-labeled features on an array.

    
    
        I. Set ILL, intensity lower limit, IUL, intensity upper limit, and w, window size.
        II. Obtain a set of feasible threshold intensities, y={yi: ILL<Ii<IUL}
        III. Sort y in increasing order to obtain y*.
        IV. Calculate d={d1, d2, . . . , dm}, where di=yi+1*−yi*.
        V. Calculate a gap statistic for each of the observed intensities:
    
    






  
    x
    j
  
  =
  
    
      
        ∑
        
          i
          =
          
            j
            -
            w
          
        
        
          j
          +
          w
        
      
      ⁢
      
        d
        i
      
    
    
      
        2
        ⁢
        w
      
      +
      1
    
  




    
    
        VI. Identify the threshold c, such that c=max(xj)
        VII. Count the number of spots, k, above the threshold c, where k=Σi=1mI[yi>c].
        VIII. Given a number of simulations desired, nsim, perform the following procedure nsim times: Randomly select m values with replacement from y={y1, y2, . . . , ym} to obtain ysim. Then repeat Step I-VII with ysim to obtain a final count.
        IX. Calculate {circumflex over (σ)}k the standard deviation of the nsim simulated counts.
        X. Calculate the 95% CI for the count as:

[k−1.96{circumflex over (σ)}k+1.96{circumflex over (σ)}k]
        
            Note that in order to obtain the true estimate of the molecule count in the sample, we needed to transform by:
        
        
    
    






  N
  =
  
    
      -
      m
    
    *
    
      log
      (
      
        1
        -
        
          k
          m
        
      
      )
    
  




    
    
        
        
            and similarly, for the 95% CI upper and lower values, where m is the total number of features on the array.
        
        
    
    


Example 6—Detection of Kan Genes
The purpose of this experiment was to determine the count of kan genes in a sample. The sample containing the kan genes was hybridized to an array. FIG. 4A displays a region of the array acquired from the imaging system. The bright intensity at a spot was correlated with a higher probability of a gene being present at the spot. The image analysis software examined the statistics of the intensity distribution, such as deviance, skewness, kurtosis, and median. These statistics provided guidance for the software to automatically choose the best method to detect the presence of kan genes. In this example, a mixture model algorithm was used to determine the intensity threshold to be 6.6, which optimally divided the intensity distribution into “on” and “off” domains, as shown in FIG. 4B.
Example 7—Titration Experiment
The purpose of this experiment was to detect the presence of molecular hybridization in a titration experiment. After obtaining the intensity measurements of a region, the intensity distribution was computed and is shown in FIG. 5. A person with ordinary skill can identify two modes in the distribution; however, it was very difficult to determine the precise value of the threshold. The invented software automated the task of determining the signal intensity threshold, and determined that an intensity value of 6.02 provides the optimal threshold for distinguishing between labeled and non-labeled features.
Example 8—Background Adjustment
The purpose of this experiment was to demonstrate use of one background subtraction method to process images. FIG. 6A shows an acquired image with pronounced artifacts. A systematic background subtraction was performed to reduce noise. We defined an analysis window centered on a spot. The software then calculated the mean spot intensity, S, spot standard deviation {circumflex over (σ)}S, number of spot pixels nS, background mean B, background standard deviation σB, and number of background pixels nB. Then, the software calculated the log 2 background subtracted intensity statistic for each spot:



  
    I
    =
    
      
        
          
            
              log
              2
            
            ⁡
            
              (
              
                16
                ⁢
                
                  S
                  _
                
              
              )
            
          
          -
          
            
              log
              2
            
            ⁡
            
              (
              
                16
                ⁢
                
                  B
                  _
                
              
              )
            
          
        
        
          
            
              
                τ
                S
                2
              
              
                n
                S
              
            
            +
            
              
                τ
                B
                2
              
              
                n
                B
              
            
          
        
      
      ⁢
      
        
      
      ⁢
      where
    
  




  
    
      
        
          
            τ
            S
          
          =
          
            
              
                log
                2
              
              ⁡
              
                (
                
                  16
                  ⁢
                  
                    (
                    
                      
                        σ
                        S
                      
                      +
                      
                        S
                        _
                      
                    
                    )
                  
                
                )
              
            
            -
            
              
                log
                2
              
              ⁡
              
                (
                
                  16
                  ⁢
                  
                    σ
                    S
                  
                
                )
              
            
          
        
      
    
    
      
        
          
            τ
            B
          
          =
          
            
              
                log
                2
              
              ⁡
              
                (
                
                  16
                  ⁢
                  
                    (
                    
                      
                        σ
                        B
                      
                      +
                      
                        B
                        _
                      
                    
                    )
                  
                
                )
              
            
            -
            
              
                
                  log
                  2
                
                ⁡
                
                  (
                  
                    16
                    ⁢
                    
                      σ
                      B
                    
                  
                  )
                
              
              .
            
          
        
      
    
  



FIGS. 6B and 6C show the intensity distributions before and after background adjustment, respectively, demonstrating that background correction enhances the ability of the software to correctly evaluate the presence of the labeled features on the array.

Example 9—Alternative Background Correction
The purpose of this experiment was an alternative way to adjust background. We defined an analysis window centered on a spot. The software then calculated the median spot intensity S and median local background intensity B. Then, the software calculated the log 2 background subtracted intensity statistic for each spot:

I=log2(16{tilde over (S)})−log2(16{tilde over (S)}).

Example 10—Pixel16 Cartridge and Custom Microarray
This example illustrated one embodiment of an array for use with the disclosed methods, systems, and platforms in performing stochastic labeling experiment.
The Pixel16 cartridge consists of (i) an epoxysilane functionalized glass slide serving as an array substrate, (ii) 16 copies of the custom microarray described in FIGS. 14-16, printed on the functionalized surface of the slide, and (iii) a polymer well frame affixed to the printed side of the slide which serves to define 16 wells which are fluidically separate and in register with the array pattern. The well frame is affixed to the slide following array printing using a die-cut double-sided adhesive.
Custom DNA microarray layout. The microarray pattern consists of a 32×32 array of spots as shown in FIGS. 15 and 16. Fiducial spots in the top and bottom rows permit location of the array in the scanned images. Also, the fiducial spots are arranged in an asymmetric pattern whose orientation is readily identifiable: the top row has a distinctive pattern whose ends are distinct, and the bottom row is different from the top row. This permits easy manual and automatic identification of incorrect placement of the Pixel16, and also facilitates detection of imaging problems. The remaining 960 spots are each associated with one of the unique probe sequences listed in Table 1.
Oligonucleotide sequences and solution components. Oligonucleotide solutions are provided for preparation of printing solutions in 96-well microplates. Concentration as supplied is 100 μM in H2O. Dilution prior to printing is performed using the Tecan GenMate. Dilution is 880 μL of stock oligo+1320 μL of buffer. The dilution buffer used is 250 mM sodium phosphate with 0.00833% sarcosyl. Buffer is filtered using a 0.2 μm filter. Three sets of plates are prepared in each probe preparation operation. Tips are discarded after each source plate. The final dispensed solution is 40 μM DNA in 150 mM sodium phosphate with 0.005% sarcosyl. The fiducial oligo is supplied at 500 μM in H2O. The fiducial oligonucleotide sequence is: 5′-/5AmMC6/TCC TGA ACG GTA GCATCT TGA CGAC-3′ (Seq ID No: 1), 25 bases, 5′ Amino Modifier C6, standard desalting; supplied at 500 μM in H2O. The fiducial is diluted by mixing 176 μL of fiducial, 704 μL of water, and 1320 μL of buffer. The final fiducial mixture is 40 μM in 150 mM sodium phosphate with 0.005% sarcosyl.
Table of oligonucleotide sequences. The oligonucleotide sequences for the 960 probe sequences (i.e. the sequences that are complementary to the set of stochastic labeling sequences used in molecular counting experiments) are listed in Table 1.







TABLE 1







Oligonucleotide Probe Sequences for Custom Microarray
















IDT



IDT
Calc'd

Seq


Plate
Ref.
Well
Seq

Mfg
Molec.

ID


Name
#
Pos.
Name
Sequence
ID
Weight
Tm
No


















AJ_P1
85652
A01
AJ_1
/5AmMC6/CCC AAA GGG TAC
10653
8795
61.7
  2



  789


CAG AGC TTA AGG TCA A
 4039





 


AJ_P1
85652
A02
AJ_2
/5AmMC6/CCC AAA GCG TTA
10653
8727
59.7
  3



  790


AGG TTT CTT GTC ACA A
 4040





 


AJ_P1
85652
A03
AJ_3
/5AmMC6/CCC AAG TCG TAC
10653
8675
61.8
  4



  791


GAA CTC ACC ACA TGA A
 4041





 


AJ_P1
85652
A04
AJ_4
/5AmMC6/CCC AAA CTT GTT
10653
8672
60.3
  5



  792


CCC TTG AGA CCA GTA A
 4042





 


AJ_P1
85652
A05
AJ_5
/5AmMC6/CCC AAG ACT TCT
10653
8657
60.7
  6



  793


ACC CTA GGT TCC AGA A
 4043





 


AJ_P1
85652
A06
AJ_6
/5AmMC6/CCC AAC CAG ACT
10653
8755
62.3
  7



  794


TGG GTA CGT GAA ACA A
 4044





 


AJ_P1
85652
A07
AJ_7
/5AmMC6/CCC AAC GAC TGG
10653
8786
62.3
  8



  795


TTC TGA AGT GGA ACA A
 4045





 


AJ_P1
85652
A08
AJ_8
/5AmMC6/CCC AAT TTA GCT
10653
8712
60.4
  9



  796


TCG TGA GTC AGA CCA A
 4046





 


AJ_P1
85652
A09
AJ_9
/5AmMC6/CCC AAC TCG AAG
10653
8752
59.8
 10



  797


AGT GGT CAG TCT TTA A
 4047





 


AJ_P1
85652
A10
AJ_10
/5AmMC6/CCC AAT CGC AAG
10653
8745
58.4
 11



  798


GAG ACA TAG TCT TTA A
 4048





 


AJ_P1
85652
A11
AJ_11
/5AmMC6/CCC AAG TCC TAG
10653
8761
60
 12



  799


TGA GAG CAA CGT TTA A
 4049





 


AJ_P1
85652
A12
AJ_12
/5AmMC6/CCC AAG GAA CCT
10653
8697
61.4
 13



  800


ACT GTC CTT GTC AGA A
 4050





 


AJ_P1
85652
B01
AJ_13
/5AmMC6/CCC AAA CTA GAA
10653
8779
59.7
 14



  801


GAC GAG TTC GAG TCA A
 4051





 


AJ_P1
85652
B02
AJ_14
/5AmMC6/CCC AAG GAC ATA
10653
8699
60
 15



  802


CTC AAC GTA GCT CAA A
 4052





 


AJ_P1
85652
B03
AJ_15
/5AmMC6/CCC AAG GCA TTT
10653
8690
61.9
 16



  803


GCA ACC TCA CAT GAA A
 4053





 


AJ_P1
85652
B04
AJ_16
/5AmMC6/CCC AAG TAC CCA
10653
8666
61.3
 17



  804


TCC ACT GTC GAG TAA A
 4054





 


AJ_P1
85652
B05
AJ_17
/5AmMC6/CCC AAA GCG TTT
10653
8745
59.4
 18



  805


GTG TAA CAG ACC ATA A
 4055





 


AJ_P1
85652
B06
AJ_18
/5AmMC6/CCC AAA TGG TCT
10653
8737
62.3
 19



  806


GGT TCG ACA GTC ACA A
 4056





 


AJ_P1
85652
B07
AJ_19
/5AmMC6/CCC AAG AGG TAC
10653
8795
60.6
 20



  807


AAC GAC TCT AGG GTA A
 4057





 


AJ_P1
85652
B08
AJ_20
/5AmMC6/CCC AAG AAC TTC
10653
8687
58.9
 21



  808


TAC TTG CTT CGT GAA A
 4058





 


AJ_P1
85652
B09
AJ_21
/5AmMC6/CCC AAG CAC TTT
10653
8687
58.8
 22



  809


CTG TTA ACT AGC TGA A
 4059





 


AJ_P1
85652
B10
AJ_22
/5AmMC6/CCC AAG AAC CTC
10653
8672
58.6
 23



  810


TCT CTA GTG CTA GTA A
 4060





 


AJ_P1
85652
B11
AJ_23
/5AmMC6/CCC AAG CCT TTA
10653
8681
60.4
 24



  811


AGC CTA AAG TCC TGA A
 4061





 


AJ_P1
85652
B12
AJ_24
/5AmMC6/CCC AAT CTG GTA
10653
8672
60.3
 25



  812


GCT CAA CAT CCT TGA A
 4062





 


AJ_P1
85652
C01
AJ_25
/5AmMC6/CCC AAA GGA CTC
10653
8795
61.8
 26



  813


CAT GGA GAA GTG TCA A
 4063





 


AJ_P1
85652
C02
AJ_26
/5AmMC6/CCC AAG AAC CCT
10653
8657
62.4
 27



  814


TTC TGG AAG CTT CCA A
 4064





 


AJ_P1
85652
C03
AJ_27
/5AmMC6/CCC AAA TTC GCT
10653
8712
60.1
 28



  815


TCC TAG TAG TGG ACA A
 4065





 


AJ_P1
85652
C04
AJ_28
/5AmMC6/CCC AAC CGT ACG
10653
8681
59.4
 29



  816


AAG ACC TAG TTT CTA A
 4066





 


AJ_P1
85652
C05
AJ_29
/5AmMC6/CCC AAT CAC GAA
10653
8745
58.4
 30



  817


GAG AGT CAC TGT TTA A
 4067





 


AJ_P1
85652
C06
AJ_30
/5AmMC6/CCC AAG AAA CAT
10653
8763
59.3
 31



  818


AAA CTC GAG TTG CGA A
 4068





 


AJ_P1
85652
C07
AJ_31
/5AmMC6/CCC AAC CAG TTA
10653
8752
60.7
 32



  819


CGT GAG TGT TGC TAA A
 4069





 


AJ_P1
85652
C08
AJ_32
/5AmMC6/CCC AAA CTC GTG
10653
8672
60.5
 33



  820


ACT CCT GTT TCA GAA A
 4070





 


AJ_P1
85652
C09
AJ_33
/5AmMC6/CCC AAC GGT TGA
10653
8779
60.6
 34



  821


AGA GAC TCC TGA AAA A
 4071





 


AJ_P1
85652
C10
AJ_34
/5AmMC6/CCC AAA TTG CTC
10653
8705
59.8
 35



  822


TGG TCA CAT CGA AAA A
 4072





 


AJ_P1
85652
C11
AJ_35
/5AmMC6/CCC AAC AGG ACT
10653
8752
60.7
 36



  823


TGT GCT ACG TGT TAA A
 4073





 


AJ_P1
85652
C12
AJ_36
/5AmMC6/CCC AAA TTT CGT
10653
8672
61.9
 37



  824


GTG TCA ACC ATG CCA A
 4074





 


AJ_P1
85652
D01
AJ_37
/5AmMC6/CCC AAC GTG AAG
10653
8754
59.7
 38



  825


GCT TAA CAA CAT TGA A
 4075





 


AJ_P1
85652
D02
AJ_38
/5AmMC6/CCC AAT GAA CAC
10653
8723
59
 39



  826


AAC TAC GAA GCT GTA A
 4076





 


AJ_P1
85652
D03
AJ_39
/5AmMC6/CCC AAA CTT CCG
10653
8687
58.7
 40



  827


TTG TTA CTA GTC GAA A
 4077





 


AJ_P1
85652
D04
AJ_40
/5AmMC6/CCC AAG GAG TAC
10653
8786
61
 41



  828


AAG CTT CCT AGG GTA A
 4078





 


AJ_P1
85652
D05
AJ_41
/5AmMC6/CCC AAG TGC TAA
10653
8687
58.8
 42



  829


ACT GCT CTT TAC GTA A
 4079





 


AJ_P1
85652
D06
AJ_42
/5AmMC6/CCC AAA GAA ACT
10653
8705
59.5
 43



  830


GCA TCT CCT TTG GAA A
 4080





 


AJ_P1
85652
D07
AJ_43
/5AmMC6/CCC AAG GAC TAA
10653
8666
61.4
 44



  831


GTT CCA CTC ACC TGA A
 4081





 


AJ_P1
85652
D08
AJ_44
/5AmMC6/CCC AAA GTT GTC
10653
8752
60.5
 45



  832


TGG TTC ACT CGA GAA A
 4082





 


AJ_P1
85652
D09
AJ_45
/5AmMC6/CCC AAC GTT CTA
10653
8727
59.2
 46



  833


AGT TTG CTT CGA AGA A
 4083





 


AJ_P1
85652
D10
AJ_46
/5AmMC6/CCC AAC TAA AGG
10653
8730
61.5
 47



  834


TTG TGC ATC CAA GCA A
 4084





 


AJ_P1
85652
D11
AJ_47
/5AmMC6/CCC AAA GGC TTC
10653
8696
59.4
 48



  835


ACG ACA TGT CAT TTA A
 4085





 


AJ_P1
85652
D12
AJ_48
/5AmMC6/CCC AAC TGC TAG
10653
8681
59.7
 49



  836


GTT CCT ACA CAA GTA A
 4086





 


AJ_P1
85652
E01
AJ_49
/5AmMC6/CCC AAA TCA GTA
10653
8699
59.8
 50



  837


GCT ACA CCA CAG GTA A
 4087





 


AJ_P1
85652
E02
AJ_50
/5AmMC6/CCC AAG ACT GCA
10653
8690
60.6
 51



  838


AGC TCA CTA CAT TGA A
 4088





 


AJ_P1
85652
E03
AJ_51
/5AmMC6/CCC AAG CTA CTC
10653
8690
58.9
 52



  839


CTC TAA GAG CAT AGA A
 4089





 


AJ_P1
85652
E04
AJ_52
/5AmMC6/CCC AAT GGA ACG
10653
8779
60.9
 53



  840


CTA AGG TGT AAA CCA A
 4090





 


AJ_P1
85652
E05
AJ_53
/5AmMC6/CCC AAG AAA CTA
10653
8690
61.5
 54



  841


ACC TTG GCT TGC CAA A
 4091





 


AJ_P1
85652
E06
AJ_54
/5AmMC6/CCC AAC CAT TAG
10653
8672
61.3
 55



  842


ACC TTG TGT TGC CAA A
 4092





 


AJ_P1
85652
E07
AJ_55
/5AmMC6/CCC AAG GTC TGA
10653
8777
61.7
 56



  843


CAG TAG GTG TTC CAA A
 4093





 


AJ_P1
85652
E08
AJ_56
/5AmMC6/CCC AAT TTC GCA
10653
8696
59.7
 57



  844


AGC CTT GGT ACA TAA A
 4094





 


AJ_P1
85652
E09
AJ_57
/5AmMC6/CCC AAG TTT CTA
10653
8657
61.2
 58



  845


GCC TAC CAC TAC GGA A
 4095





 


AJ_P1
85652
E10
AJ_58
/5AmMC6/CCC AAA TAG ACC
10653
8779
60.1
 59



  846


TAA CGG AAG CTG TGA A
 4096





 


AJ_P1
85652
E11
AJ_59
/5AmMC6/CCC AAG GAG TCA
10653
8712
60.7
 60



  847


TCC ATG CAT CTT TGA A
 4097





 


AJ_P1
85652
E12
AJ_60
/5AmMC6/CCC AAC CGT ACT
10653
8721
60.5
 61



  848


AGC TTG GGT TAA ACA A
 4098





 


AJ_P1
85652
F01
AJ_61
/5AmMC6/CCC AAA AAG GCT
10653
8696
59
 62



  849


AGC CTT CTG ACT TTA A
 4099





 


AJ_P1
85652
F02
AJ_62
/5AmMC6/CCC AAA GAG CTC
10653
8705
58.8
 63



  850


TGC ACT ACA AGT TTA A
 4100





 


AJ_P1
85652
F03
AJ_63
/5AmMC6/CCC AAC AGC TAA
10653
8779
60
 64



  851


CGG TAG TAA AGG TCA A
 4101





 


AJ_P1
85652
F04
AJ_64
/5AmMC6/CCC AAA GCT TTC
10653
8696
60
 65



  852


CGT TTC AAA GTG ACA A
 4102





 


AJ_P1
85652
F05
AJ_65
/5AmMC6/CCC AAG TCC ATG
10653
8690
61.3
 66



  853


CTT CCA GTG ACA AAA A
 4103





 


AJ_P1
85652
F06
AJ_66
/5AmMC6/CCC AAG TAG CTT
10653
8687
58.4
 67



  854


TGC TCT ACT CGT AAA A
 4104





 


AJ_P1
85652
F07
AJ_67
/5AmMC6/CCC AAC TTC GAA
10653
8779
59.7
 68



  855


CTA AGG AGT AGA GCA A
 4105





 


AJ_P1
85652
F08
AJ_68
/5AmMC6/CCC AAT TCA GTC
10653
8770
58.5
 69



  856


CTA GAG GAG AGA CTA A
 4106





 


AJ_P1
85652
F09
AJ_69
/5AmMC6/CCC AAT AGG TCT
10653
8672
59.6
 70



  857


GTC TTA CCC AAC GTA A
 4107





 


AJ_P1
85652
F10
AJ_70
/5AmMC6/CCC AAC GTG AGG
10653
8770
60.7
 71



  858


AAA GTT CTG CTA ACA A
 4108





 


AJ_P1
85652
F11
AJ_71
/5AmMC6/CCC AAG TTG GCA
10653
8712
60.7
 72



  859


ACT TGC TCT CTA AGA A
 4109





 


AJ_P1
85652
F12
AJ_72
/5AmMC6/CCC AAG ACA TCT
10653
8690
59
 73



  860


CTC TCA GAG CTA GAA A
 4110





 


AJ_P1
85652
G01
AJ_73
/5AmMC6/CCC AAT TTC GCA
10653
8672
60.9
 74



  861


TGT CTC ATC AGG ACA A
 4111





 


AJ_P1
85652
G02
AJ_74
/5AmMC6/CCC AAA GCC TTC
10653
8721
60.6
 75



  862


CTT GGT ACT GAA AGA A
 4112





 


AJ_P1
85652
G03
AJ_75
/5AmMC6/CCC AAA CTT GCC
10653
8696
59.9
 76



  863


TTG CGT ACT GTA AAA A
 4113





 


AJ_P1
85652
G04
AJ_76
/5AmMC6/CCC AAC ACT TTG
10653
8761
59.7
 77



  864


TAC GGT AGA GAC GTA A
 4114





 


AJ_P1
85652
G05
AJ_77
/5AmMC6/CCC AAG TTT CCA
10653
8681
61.2
 78



  865


TCA ACC GAA GCT TGA A
 4115





 


AJ_P1
85652
G06
AJ_78
/5AmMC6/CCC AAG CAT TAC
10653
8699
61
 79



  866


CAA ACT GGA ACC TGA A
 4116





 


AJ_P1
85652
G07
AJ_79
/5AmMC6/CCC AAC CGT ACA
10653
8687
59.7
 80



  867


ACT TGT TCG TTT GAA A
 4117





 


AJ_P1
85652
G08
AJ_80
/5AmMC6/CCC AAC AGC TAG
10653
8690
60.7
 81



  868


TAG CAC ACC ATT TGA A
 4118





 


AJ_P1
85652
G09
AJ_81
/5AmMC6/CCC AAC CTC ACG
10653
8690
61.2
 82



  869


AAA GCA TCA TTG TGA A
 4119





 


AJ_P1
85652
G10
AJ_82
/5AmMC6/CCC AAA CAA AGT
10653
8739
60.5
 83



  870


GAG GTC ATC TCG ACA A
 4120





 


AJ_P1
85652
G11
AJ_83
/5AmMC6/CCC AAG AAA CCT
10653
8721
59.9
 84



  871


TCT TGT AGG ACT CGA A
 4121





 


AJ_P1
85652
G12
AJ_84
/5AmMC6/CCC AAA AGC CTA
10653
8696
58.9
 85



  872


AGC TCT GTC AGT TTA A
 4122





 


AJ_P1
85652
H01
AJ_85
/5AmMC6/CCC AAA CGT TCC
10653
8681
60.9
 86



  873


CTT CAT GTC GAA AGA A
 4123





 


AJ_P1
85652
H02
AJ_86
/5AmMC6/CCC AAG TAG CAC
10653
8699
60.7
 87



  874


TGA CAC CAA GCA TTA A
 4124





 


AJ_P1
85652
H03
AJ_87
/5AmMC6/CCC AAG TTT GAC
10653
8657
62.2
 88



  875


TCC AAG CCT ACG TCA A
 4125





 


AJ_P1
85652
H04
AJ_88
/5AmMC6/CCC AAA CCG TTG
10653
8770
61.2
 89



  876


GTG AAG CCT TAA AGA A
 4126





 


AJ_P1
85652
H05
AJ_89
/5AmMC6/CCC AAG CCT ACA
10653
8675
61.9
 90



  877


CCT TCA GTG AAC AGA A
 4127





 


AJ_P1
85652
H06
AJ_90
/5AmMC6/CCC AAC AGC TCA
10653
8723
59.2
 91



  878


AGC AGT TAG TAA ACA A
 4128





 


AJ_P1
85652
H07
AJ_91
/5AmMC6/CCC AAT ACG CAA
10653
8745
59.3
 92



  879


GCA TGT AGG TTT ACA A
 4129





 


AJ_P1
85652
H08
AJ_92
/5AmMC6/CCC AAC ACG AGT
10653
8727
59.3
 93



  880


CGT TAG TTG TTT CAA A
 4130





 


AJ_P1
85652
H09
AJ_93
/5AmMC6/CCC AAT TCG GAA
10653
8690
59.6
 94



  881


GAC CTA CTA ACC TGA A
 4131





 


AJ_P1
85652
H10
AJ_94
/5AmMC6/CCC AAA GGT CTC
10653
8763
58.2
 95



  882


TAC GAA AGG AAC ATA A
 4132





 


AJ_P1
85652
H11
AJ_95
/5AmMC6/CCC AAG TGC TAG
10653
8761
60.6
 96



  883


ACG TCT GTG TCA AAA A
 4133





 


AJ_P1
85652
H12
AJ_96
/5AmMC6/CCC AAA CCA GTG
10653
8657
61
 97



  884


GAC TTC TCT CCT AGA A
 4134





 


AJ_P2
85652
A01
AJ_97
/5AmMC6/CCC AAC ATG TAG
10653
8746
61.3
 98



  886


GAG ACG TAG TTC CCA A
 4135





 


AJ_P2
85652
A02
AJ_98
/5AmMC6/CCC AAG AAC TCT
10653
8752
60.2
 99



  887


CTG GTT AGG CTT GAA A
 4136





 


AJ_P2
85652
A03
AJ_99
/5AmMC6/CCC AAG GAC ATC
10653
8675
62.1
100



  888


CAC ATC GTC TGA CAA A
 4137





 


AJ_P2
85652
A04
AJ_100
/5AmMC6/CCC AAA CTT GTT
10653
8736
59.8
101



  889


GGG TTC AGC TAA CAA A
 4138





 


AJ_P2
85652
A05
AJ_101
/5AmMC6/CCC AAC ACG TGT
10653
8681
61.2
102



  890


CCT GTC ATG TCA AAA A
 4139





 


AJ_P2
85652
A06
AJ_102
/5AmMC6/CCC AAT CGG AAA
10653
8705
59.4
103



  891


CCA ACG TTA GCT TTA A
 4140





 


AJ_P2
85652
A07
AJ_103
/5AmMC6/CCC AAG GAC TTA
10653
8777
61.4
104



  892


GGT ACC TGT TCG GAA A
 4141





 


AJ_P2
85652
A08
AJ_104
/5AmMC6/CCC AAG ACT TAA
10653
8739
60.1
105



  893


CAA CCT GTG ACG AGA A
 4142





 


AJ_P2
85652
A09
AJ_105
/5AmMC6/CCC AAG TTA ACA
10653
8779
60.7
106



  894


TGC AGA CGA ACG GTA A
 4143





 


AJ_P2
85652
A10
AJ_106
/5AmMC6/CCC AAG CGT ACA
10653
8687
58.9
107



  895


ACT CTT GTC AGT TTA A
 4144





 


AJ_P2
85652
A11
AJ_107
/5AmMC6/CCC AAG TAA CAC
10653
8746
61.9
108



  896


CTT CTG AGC AGT GGA A
 4145





 


AJ_P2
85652
A12
AJ_108
/5AmMC6/CCC AAG ACC TAC
10653
8675
60.7
109



  897


CTC TCA GGA ACA GTA A
 4146





 


AJ_P2
85652
B01
AJ_109
/5AmMC6/CCC AAA CCT GAC
10653
8739
59.9
110



  898


CTT AGG AAG AGC ATA A
 4147





 


AJ_P2
85652
B02
AJ_110
/5AmMC6/CCC AAC AAA GTT
10653
8736
59.3
111



  899


TGT CTC AGT TAG CGA A
 4148





 


AJ_P2
85652
B03
AJ_111
/5AmMC6/CCC AAC GGT AGC
10653
8752
60.5
112



  900


ATT GTT CCT GTA GAA A
 4149





 


AJ_P2
85652
B04
AJ_112
/5AmMC6/CCC AAC TAG GTT
10653
8727
58
113



  901


TGT TCT AGA CAG CTA A
 4150





 


AJ_P2
85652
B05
AJ_113
/5AmMC6/CCC AAG TCT CTA
10653
8681
59.8
114



  902


CGT TCC ATC GAA AGA A
 4151





 


AJ_P2
85652
B06
AJ_114
/5AmMC6/CCC AAA CCT TCG
10653
8672
60.7
115



  903


TTC TTG AGT ACA GCA A
 4152





 


AJ_P2
85652
B07
AJ_115
/5AmMC6/CCC AAG AAC ACT
10653
8666
62.2
116



  904


CCT CAT GTG ACT GCA A
 4153





 


AJ_P2
85652
B08
AJ_116
/5AmMC6/CCC AAA CGC TTG
10653
8763
59.3
117



  905


GTA ACA AAG ACA GTA A
 4154





 


AJ_P2
85652
B09
AJ_117
/5AmMC6/CCC AAC CCT AGA
10653
8721
58.4
118



  906


GTA GTA CTA CGG TTA A
 4155





 


AJ_P2
85652
B10
AJ_118
/5AmMC6/CCC AAC CTG AGG
10653
8779
60.3
119



  907


TAG TGA CTG AAA CAA A
 4156





 


AJ_P2
85652
B11
AJ_119
/5AmMC6/CCC AAG CTA CGA
10653
8727
59.7
120



  908


ACT TGG TTG TTT CAA A
 4157





 


AJ_P2
85652
B12
AJ_120
/5AmMC6/CCC AAG CAA GTC
10653
8752
60.8
121



  909


CTA GGT TGT GTT CAA A
 4158





 


AJ_P2
85652
C01
AJ_121
/5AmMC6/CCC AAC TCC ATG
10653
8755
61.9
122



  910


TCA AGG AAG GGT ACA A
 4159





 


AJ_P2
85652
C02
AJ_122
/5AmMC6/CCC AAT CCG AAC
10653
8723
58.7
123



  911


ACG AAG TAC AAG TTA A
 4160





 


AJ_P2
85652
C03
AJ_123
/5AmMC6/CCC AAC ACG TTG
10653
8727
60.1
124



  912


ACA TTG TTG GCT TAA A
 4161





 


AJ_P2
85652
C04
AJ_124
/5AmMC6/CCC AAC CTC TAG
10653
8675
60.9
125



  913


GAA CGT AGT ACA CCA A
 4162





 


AJ_P2
85652
C05
AJ_125
/5AmMC6/CCC AAT AGG ACA
10653
8699
60.3
126



  914


CCA CAG TTC ATC GAA A
 4163





 


AJ_P2
85652
C06
AJ_126
/5AmMC6/CCC AAA TGT CGT
10653
8736
59.7
127



  915


TCG GTT AGC TCA AAA A
 4164





 


AJ_P2
85652
C07
AJ_127
/5AmMC6/CCC AAA TCG GTT
10653
8736
59.4
128



  916


GTG TCT AGC TCA AAA A
 4165





 


AJ_P2
85652
C08
AJ_128
/5AmMC6/CCC AAT AAG AAC
10653
8723
59.3
129



  917


GAA ACG TAC CTT GCA A
 4166





 


AJ_P2
85652
C09
AJ_129
/5AmMC6/CCC AAT CGC AAG
10653
8723
59.7
130



  918


AAC CGT TAG TCA AAA A
 4167





 


AJ_P2
85652
C10
AJ_130
/5AmMC6/CCC AAG TCA CAC
10653
8657
61.9
131



  919


GTC TCC ACA GGT TTA A
 4168





 


AJ_P2
85652
C11
AJ_131
/5AmMC6/CCC AAG AGC TTA
10653
8752
59.4
132



  920


CAT CGT TCT AGG GTA A
 4169





 


AJ_P2
85652
C12
AJ_132
/5AmMC6/CCC AAG ACC TTC
10653
8697
61
133



  921


TCC TTG ACA GAG GTA A
 4170





 


AJ_P2
85652
D01
AJ_133
/5AmMC6/CCC AAA AGG CTT
10653
8687
58.6
134



  922


AGC TCT CTT TAC TGA A
 4171





 


AJ_P2
85652
D02
AJ_134
/5AmMC6/CCC AAG TCG TAA
10653
8755
61.9
135



  923


CAG AGG TGT CCA CAA A
 4172





 


AJ_P2
85652
D03
AJ_135
/5AmMC6/CCC AAA CTA CTG
10653
8761
60.5
136



  924


CAA GTG GTA GGT TCA A
 4173





 


AJ_P2
85652
D04
AJ_136
/5AmMC6/CCC AAT TTC GGA
10653
8746
62.3
137



  925


ACC AGT ACC ATG GGA A
 4174





 


AJ_P2
85652
D05
AJ_137
/5AmMC6/CCC AAT CGA GAA
10653
8705
59.1
138



  926


GCA ACT TCC TTG TAA A
 4175





 


AJ_P2
85652
D06
AJ_138
/5AmMC6/CCC AAT GGA GAC
10653
8752
60.3
139



  927


TTC CGT ACT GTT GAA A
 4176





 


AJ_P2
85652
D07
AJ_139
/5AmMC6/CCC AAA CAT GCG
10653
8687
59.6
140



  928


TTT CGT AGT CTT CAA A
 4177





 


AJ_P2
85652
D08
AJ_140
/5AmMC6/CCC AAG AAC CTC
10653
8690
60.4
141



  929


AGC TCT TTC GAA AGA A
 4178





 


AJ_P2
85652
D09
AJ_141
/5AmMC6/CCC AAG TCC TTA
10653
8752
59.7
142



  930


AGC TGT TCG AGA GTA A
 4179





 


AJ_P2
85652
D10
AJ_142
/5AmMC6/CCC AAT CTC GAA
10653
8672
60.5
143



  931


ACT CTT GTG TGA CCA A
 4180





 


AJ_P2
85652
D11
AJ_143
/5AmMC6/CCC AAC CAT TAG
10653
8763
57.7
144



  932


AGG AAC TAA GAG CTA A
 4181





 


AJ_P2
85652
D12
AJ_144
/5AmMC6/CCC AAC CCT AGA
10653
8755
60.7
145



  933


GTG AGT CAG GAA CTA A
 4182





 


AJ_P2
85652
E01
AJ_145
/5AmMC6/CCC AAT GAA CCA
10653
8763
59.1
146



  934


TAA GAG CAA CGG TTA A
 4183





 


AJ_P2
85652
E02
AJ_146
/5AmMC6/CCC AAG AAC CTT
10653
8672
60.3
147



  935


CCC TTA GTC GTT GAA A
 4184





 


AJ_P2
85652
E03
AJ_147
/5AmMC6/CCC AAG TGG TCA
10653
8697
62.1
148



  936


GTA ACC CTT TCC GAA A
 4185





 


AJ_P2
85652
E04
AJ_148
/5AmMC6/CCC AAA GCA TGT
10653
8681
59.3
149



  937


ACG TCT CCT ACT AGA A
 4186





 


AJ_P2
85652
E05
AJ_149
/5AmMC6/CCC AAG GAC TTC
10653
8666
61.9
150



  938


ACC TAC GTT CGA ACA A
 4187





 


AJ_P2
85652
E06
AJ_150
/5AmMC6/CCC AAC GAA CTT
10653
8672
60.7
151



  939


TAC CTT GTC CAT GGA A
 4188





 


AJ_P2
85652
E07
AJ_151
/5AmMC6/CCC AAC AGG TTC
10653
8690
61.1
152



  940


TTA CGC AAC ACA TGA A
 4189





 


AJ_P2
85652
E08
AJ_152
/5AmMC6/CCC AAC TTG TTA
10653
8752
60.1
153



  941


GGG TAG CTG ACT CAA A
 4190





 


AJ_P2
85652
E09
AJ_153
/5AmMC6/CCC AAC TGG AGA
10653
8770
59.2
154



  942


AGA GAC TAC CTG TTA A
 4191





 


AJ_P2
85652
E10
AJ_154
/5AmMC6/CCC AAC TAA GGT
10653
8752
60.3
155



  943


TTG GTC AGT CCT GAA A
 4192





 


AJ_P2
85652
E11
AJ_155
/5AmMC6/CCC AAG CAC ACT
10653
8690
60.7
156



  944


AGC CTT TCT GAA AGA A
 4193





 


AJ_P2
85652
E12
AJ_156
/5AmMC6/CCC AAG TCC TGA
10653
8777
61.6
157



  945


CGA GAG TTT GGT ACA A
 4194





 


AJ_P2
85652
F01
AJ_157
/5AmMC6/CCC AAT CCC AAG
10653
8697
61.8
158



  946


AGT CTC TGG TTG ACA A
 4195





 


AJ_P2
85652
F02
AJ_158
/5AmMC6/CCC AAG GCA TTC
10653
8687
59.8
159



  947


AGC ATT CAT TCT TGA A
 4196





 


AJ_P2
85652
F03
AJ_159
/5AmMC6/CCC AAG TTT GAC
10653
8690
61.3
160



  948


TAC CAA GCA ACT GCA A
 4197





 


AJ_P2
85652
F04
AJ_160
/5AmMC6/CCC AAC CTT AAG
10653
8770
60
161



  949


CTA AGT GTG AGA CGA A
 4198





 


AJ_P2
85652
F05
AJ_161
/5AmMC6/CCC AAC TTA CAG
10653
8736
59.2
162



  950


CTA GTT TGA AGT GCA A
 4199





 


AJ_P2
85652
F06
AJ_162
/5AmMC6/CCC AAC TAG TCT
10653
8727
58.3
163



  951


CTT AGA GTT TGG CAA A
 4200





 


AJ_P2
85652
F07
AJ_163
/5AmMC6/CCC AAT AAA GCT
10653
8763
58
164



  952


CTA GGA GAA CAC GTA A
 4201





 


AJ_P2
85652
F08
AJ_164
/5AmMC6/CCC AAA GCG TAG
10653
8779
59.8
165



  953


TAG TGA CTA ACG ACA A
 4202





 


AJ_P2
85652
F09
AJ_165
/5AmMC6/CCC AAG ACG TAA
10653
8681
59.9
166



  954


ACG CTT CCT TCT AGA A
 4203





 


AJ_P2
85652
F10
AJ_166
/5AmMC6/CCC AAA GCT GTA
10653
8672
59.5
167



  955


GTA CCC TTT CCT AGA A
 4204





 


AJ_P2
85652
F11
AJ_167
/5AmMC6/CCC AAC TCG TAC
10653
8699
59.3
168



  956


AGC ATA CCT AGA AGA A
 4205





 


AJ_P2
85652
F12
AJ_168
/5AmMC6/CCC AAT CGC TAC
10653
8723
58.9
169



  957


ATA GCA ACT GAA AGA A
 4206





 


AJ_P2
85652
G01
AJ_169
/5AmMC6/CCC AAC TTG GCA
10653
8761
61
170



  958


ACG TGT GTA GTA CAA A
 4207





 


AJ_P2
85652
G02
AJ_170
/5AmMC6/CCC AAA CCT GTT
10653
8696
59.9
171



  959


ACG CTT GTG CTA AAA A
 4208





 


AJ_P2
85652
G03
AJ_171
/5AmMC6/CCC AAA GCT TGG
10653
8727
59.4
172



  960


TTG TAA CTT TAC CGA A
 4209





 


AJ_P2
85652
G04
AJ_172
/5AmMC6/CCC AAG AGA CCT
10653
8699
60.5
173



  961


TAG CAA CAA CCT TGA A
 4210





 


AJ_P2
85652
G05
AJ_173
/5AmMC6/CCC AAT ACC GAA
10653
8761
60.1
174



  962


GAG TGC TAG GTT TCA A
 4211





 


AJ_P2
85652
G06
AJ_174
/5AmMC6/CCC AAG ACA TAG
10653
8666
61.6
175



  963


TAC CGT TGC TAC CCA A
 4212





 


AJ_P2
85652
G07
AJ_175
/5AmMC6/CCC AAG GTC TAG
10653
8739
59.7
176



  964


TAA CGA AGC AAC CTA A
 4213





 


AJ_P2
85652
G08
AJ_176
/5AmMC6/CCC AAT AAG CAA
10653
8723
60.1
177



  965


CAA AGG TCA TTG CCA A
 4214





 


AJ_P2
85652
G09
AJ_177
/5AmMC6/CCC AAC TGA GTG
10653
8779
59.5
178



  966


AGA AGT CAG AAC CTA A
 4215





 


AJ_P2
85652
G10
AJ_178
/5AmMC6/CCC AAC TTC GAG
10653
8723
58.8
179



  967


TGA AAC AAG AAC CTA A
 4216





 


AJ_P2
85652
G11
AJ_179
/5AmMC6/CCC AAA GCG TTC
10653
8727
59.4
180



  968


ATG GTT CTG TCA TAA A
 4217





 


AJ_P2
85652
G12
AJ_180
/5AmMC6/CCC AAG AGG TCT
10653
8752
59.7
181



  969


AGG CTT TCG TCT AAA A
 4218





 


AJ_P2
85652
H01
AJ_181
/5AmMC6/CCC AAA GCC ATT
10653
8712
60.7
182



  970


AGT CGT GTC GTT ACA A
 4219





 


AJ_P2
85652
H02
AJ_182
/5AmMC6/CCC AAG GTC TTA
10653
8777
61.9
183



  971


CGT AGG TTG AAG CCA A
 4220





 


AJ_P2
85652
H03
AJ_183
/5AmMC6/CCC AAG AGC TTA
10653
8739
60.2
184



  972


GCG AAC TTA GAA CCA A
 4221





 


AJ_P2
85652
H04
AJ_184
/5AmMC6/CCC AAT GGA ACC
10653
8777
61.9
185



  973


CTA GGG TTG AGT TCA A
 4222





 


AJ_P2
85652
H05
AJ_185
/5AmMC6/CCC AAG AAC ACT
10653
8730
61
186



  974


TGA GCA GAC GTT TCA A
 4223





 


AJ_P2
85652
H06
AJ_186
/5AmMC6/CCC AAT CGA AGG
10653
8763
58.8
187



  975


AAA GCA TGA CTC TAA A
 4224





 


AJ_P2
85652
H07
AJ_187
/5AmMC6/CCC AAC TTA GTG
10653
8761
59.3
188



  976


AGA GTG CTA CTC AGA A
 4225





 


AJ_P2
85652
H08
AJ_188
/5AmMC6/CCC AAA CTT GTT
10653
8736
59.7
189



  977


GAA GTG CTT CAC AGA A
 4226





 


AJ_P2
85652
H09
AJ_189
/5AmMC6/CCC AAG TGC TAA
10653
8672
60.5
190



  978


CAC TGT TCT CCA TGA A
 4227





 


AJ_P2
85652
H10
AJ_190
/5AmMC6/CCC AAC CCT TAG
10653
8666
61.7
191



  979


ACC TGA ACA TCG TGA A
 4228





 


AJ_P2
85652
H11
AJ_191
/5AmMC6/CCC AAC TTA AAG
10653
8770
59.2
192



  980


GGT AGA CCT AGT CGA A
 4229





 


AJ_P2
85652
H12
AJ_192
/5AmMC6/CCC AAG GCA TAG
10653
8712
60.1
193



  981


ACC TGT CGT TCT TAA A
 4230





 


AJ_P3
85652
A01
AJ_193
/5AmMC6/CCC AAA GCG TTT
10653
8761
60.1
194



  983


CTA GGG TAG TAA CCA A
 4231





 


AJ_P3
85652
A02
AJ_194
/5AmMC6/CCC AAG CAA ACT
10653
8705
59.7
195



  984


TTC CAA GAC GTT GTA A
 4232





 


AJ_P3
85652
A03
AJ_195
/5AmMC6/CCC AAT CTG GTA
10653
8672
60.8
196



  985


ACT GCT TTC GAA CCA A
 4233





 


AJ_P3
85652
A04
AJ_196
/5AmMC6/CCC AAT CAG GAG
10653
8779
59.4
197



  986


AGC AAG TAC TAG TCA A
 4234





 


AJ_P3
85652
A05
AJ_197
/5AmMC6/CCC AAA CAT TGT
10653
8687
59.9
198



  987


GTC GTT AAC GCT TCA A
 4235





 


AJ_P3
85652
A06
AJ_198
/5AmMC6/CCC AAG AGG TAC
10653
8770
59.5
199



  988


TTA GGC ATA ACC GTA A
 4236





 


AJ_P3
85652
A07
AJ_199
/5AmMC6/CCC AAA AAC GGT
10653
8739
62.2
200



  989


TTG GCA AAC TGA CCA A
 4237





 


AJ_P3
85652
A08
AJ_200
/5AmMC6/CCC AAC ATA AGG
10653
8755
62.1
201



  990


CAA GGG TAC TGT CCA A
 4238





 


AJ_P3
85652
A09
AJ_201
/5AmMC6/CCC AAA TGA CGA
10653
8795
61.6
202



  991


CAG GAG TAG TGT CCA A
 4239





 


AJ_P3
85652
A10
AJ_202
/5AmMC6/CCC AAG ACC TTT
10653
8712
60.4
203



  992


GCG TTT ACA GGA CTA A
 4240





 


AJ_P3
85652
A11
AJ_203
/5AmMC6/CCC AAG TCT AGA
10653
8699
59.6
204



  993


GTC AAC ACA GCA CTA A
 4241





 


AJ_P3
85652
A12
AJ_204
/5AmMC6/CCC AAG AGA GCT
10653
8715
61.5
205



  994


TAA CCA GAC TGT CCA A
 4242





 


AJ_P3
85652
B01
AJ_205
/5AmMC6/CCC AAG ACC ATA
10653
8690
60.1
206



  995


CTG CAC ATT AGG CTA A
 4243





 


AJ_P3
85652
B02
AJ_206
/5AmMC6/CCC AAG CCA ACT
10653
8706
61.8
207



  996


ACG TCA TAG TGG TCA A
 4244





 


AJ_P3
85652
B03
AJ_207
/5AmMC6/CCC AAT GTC GAA
10653
8699
60.2
208



  997


CGT ACC AAG ACC ATA A
 4245





 


AJ_P3
85652
B04
AJ_208
/5AmMC6/CCC AAC GTG TAG
10653
8761
60
209



  998


GAA GTT CGT ACT CAA A
 4246





 


AJ_P3
85652
B05
AJ_209
/5AmMC6/CCC AAA AAC CGT
10653
8730
61.3
210



  999


AAG CCT TCA TGG TGA A
 4247





 


AJ_P3
85653
B06
AJ_210
/5AmMC6/CCC AAT CGG AAA
10653
8745
59.7
211



  000


CGC AAG TTC ATG TTA A
 4248





 


AJ_P3
85653
B07
AJ_211
/5AmMC6/CCC AAT CGG TAA
10653
8763
58.3
212



  001


CTA GAA AGC ACA GTA A
 4249





 


AJ_P3
85653
B08
AJ_212
/5AmMC6/CCC AAG TCG AAG
10653
8779
60.1
213



  002


TAG GCT AAA GTC CAA A
 4250





 


AJ_P3
85653
B09
AJ_213
/5AmMC6/CCC AAA CGG TAG
10653
8712
59.8
214



  003


TAC CTT GTC GTC ATA A
 4251





 


AJ_P3
85653
B10
AJ_214
/5AmMC6/CCC AAC ATT TGG
10653
8727
59.6
215



  004


AAG TTG CAT CCT GTA A
 4252





 


AJ_P3
85653
B11
AJ_215
/5AmMC6/CCC AAC GAA GTG
10653
8746
62.6
216



  005


TTG GTC AAG TCC ACA A
 4253





 


AJ_P3
85653
B12
AJ_216
/5AmMC6/CCC AAT CAA GGA
10653
8779
60.5
217



  006


AAG GAC TAG TTC GCA A
 4254





 


AJ_P3
85653
C01
AJ_217
/5AmMC6/CCC AAC GAA ACT
10653
8723
58.3
218



  007


TAC AAC GTA GGA CTA A
 4255





 


AJ_P3
85653
C02
AJ_218
/5AmMC6/CCC AAG GCA TGC
10653
8727
59
219



  008


TTA GTC TGA ACT TTA A
 4256





 


AJ_P3
85653
C03
AJ_219
/5AmMC6/CCC AAG AAC CGT
10653
8672
60.5
220



  009


TCC CAT GTA GCT TTA A
 4257





 


AJ_P3
85653
C04
AJ_220
/5AmMC6/CCC AAG GCA TAA
10653
8745
59.1
221



  010


AGT GTT CTC TCG AAA A
 4258





 


AJ_P3
85653
C05
AJ_221
/5AmMC6/CCC AAG GCT ACC
10653
8739
59.6
222



  011


CTT AAA GAG GAC ATA A
 4259





 


AJ_P3
85653
C06
AJ_222
/5AmMC6/CCC AAG TCC TAG
10653
8712
59.8
223



  012


ACT TCG GTT CGT AAA A
 4260





 


AJ_P3
85653
C07
AJ_223
/5AmMC6/CCC AAG GAA CCT
10653
8699
60.2
224



  013


TGT ACA ACA CGA CTA A
 4261





 


AJ_P3
85653
C08
AJ_224
/5AmMC6/CCC AAC ACG TTG
10653
8779
59.3
225



  014


TAG AGA CAG AGA CTA A
 4262





 


AJ_P3
85653
C09
AJ_225
/5AmMC6/CCC AAT CCA AGC
10653
8730
61
226



  015


ACA AGG TAG GTT TCA A
 4263





 


AJ_P3
85653
C10
AJ_226
/5AmMC6/CCC AAA GCC ATA
10653
8736
59
227



  016


CTA GTT GTT GTC GAA A
 4264





 


AJ_P3
85653
C11
AJ_227
/5AmMC6/CCC AAC GAG TAC
10653
8779
59.2
228



  017


CAT AGT GAA GGA CTA A
 4265





 


AJ_P3
85653
C12
AJ_228
/5AmMC6/CCC AAC ATT TGC
10653
8770
60.3
229



  018


CAA GGG TAG AGA CTA A
 4266





 


AJ_P3
85653
D01
AJ_229
/5AmMC6/CCC AAC GAC TGT
10653
8687
59.3
230



  019


TTC CGT AAA GCT TTA A
 4267





 


AJ_P3
85653
D02
AJ_230
/5AmMC6/CCC AAG GAG TAC
10653
8779
59.7
231



  020


GAG ACA TCA AGC TTA A
 4268





 


AJ_P3
85653
D03
AJ_231
/5AmMC6/CCC AAT GGA CTG
10653
8777
61.6
232



  021


TCT GGA GTA ACG TCA A
 4269





 


AJ_P3
85653
D04
AJ_232
/5AmMC6/CCC AAA CCG TTA
10653
8752
60.5
233



  022


CAG GTT TAG TGT CGA A
 4270





 


AJ_P3
85653
D05
AJ_233
/5AmMC6/CCC AAT GAC AAA
10653
8763
58.6
234



  023


GAG TAC GAA CTG CTA A
 4271





 


AJ_P3
85653
D06
AJ_234
/5AmMC6/CCC AAT CAC AAG
10653
8723
59
235



  024


TGA CAA AGT ACG CTA A
 4272





 


AJ_P3
85653
D07
AJ_235
/5AmMC6/CCC AAC TGT AAA
10653
8736
58.1
236



  025


GAG TTG CTA GCT CTA A
 4273





 


AJ_P3
85653
D08
AJ_236
/5AmMC6/CCC AAT GGG AAC
10653
8795
62.3
237



  026


ACT GTG AAG TCG ACA A
 4274





 


AJ_P3
85653
D09
AJ_237
/5AmMC6/CCC AAA TTG CGT
10653
8752
61.9
238



  027


TTG GTC AAC TGG ACA A
 4275





 


AJ_P3
85653
D10
AJ_238
/5AmMC6/CCC AAC GAA GGT
10653
8746
62
239



  028


TCA GGT TAG TCC ACA A
 4276





 


AJ_P3
85653
D11
AJ_239
/5AmMC6/CCC AAA TGC TGT
10653
8687
59.7
240



  029


GTT AAC CTT TAG CCA A
 4277





 


AJ_P3
85653
D12
AJ_240
/5AmMC6/CCC AAC CAC TTG
10653
8712
59.5
241



  030


TAG TAC TAG GTT CGA A
 4278





 


AJ_P3
85653
E01
AJ_241
/5AmMC6/CCC AAC CCA TAG
10653
8712
60.3
242



  031


AGG TTT CAC GTT GTA A
 4279





 


AJ_P3
85653
E02
AJ_242
/5AmMC6/CCC AAC TAG GAA
10653
8763
58.7
243



  032


AGA GTT CAA CGC ATA A
 4280





 


AJ_P3
85653
E03
AJ_243
/5AmMC6/CCC AAT CCG AAG
10653
8779
59.6
244



  033


AAA GGT CTA CAG GTA A
 4281





 


AJ_P3
85653
E04
AJ_244
/5AmMC6/CCC AAT GGA AAC
10653
8714
58.9
245



  034


CCT TAA GAA CTG CTA A
 4282





 


AJ_P3
85653
E05
AJ_245
/5AmMC6/CCC AAG CAA CAT
10653
8699
60.5
246



  035


AAC CTT GAC TCA GGA A
 4283





 


AJ_P3
85653
E06
AJ_246
/5AmMC6/CCC AAT AGA ACC
10653
8723
58.4
247



  036


ACA GAC TTT AGC AGA A
 4284





 


AJ_P3
85653
E07
AJ_247
/5AmMC6/CCC AAT CAC AAG
10653
8763
59.1
248



  037


AGG TTC GTA CGA AAA A
 4285





 


AJ_P3
85653
E08
AJ_248
/5AmMC6/CCC AAA GCT TTG
10653
8705
59.3
249



  038


TCT CCA GTA CGA AAA A
 4286





 


AJ_P3
85653
E09
AJ_249
/5AmMC6/CCC AAT CGG AAG
10653
8770
60.7
250



  039


GTG TTC AGT AAA CCA A
 4287





 


AJ_P3
85653
E10
AJ_250
/5AmMC6/CCC AAA GTG CAT
10653
8723
59.4
251



  040


TCC AAG AAA CGA CTA A
 4288





 


AJ_P3
85653
E11
AJ_251
/5AmMC6/CCC AAG ACG TAA
10653
8690
60.1
252



  041


CCA TCG AAC TCG TTA A
 4289





 


AJ_P3
85653
E12
AJ_252
/5AmMC6/CCC AAC CGT AGA
10653
8687
59.3
253



  042


ACG TTC TTT GCT TAA A
 4290





 


AJ_P3
85653
F01
AJ_253
/5AmMC6/CCC AAG AGC TCA
10653
8755
61.6
254



  043


AGG GTT CTA GAA CCA A
 4291





 


AJ_P3
85653
F02
AJ_254
/5AmMC6/CCC AAT CGG TAG
10653
8770
60
255



  044


TTA CGA GTA AAG CCA A
 4292





 


AJ_P3
85653
F03
AJ_255
/5AmMC6/CCC AAG ACA ACT
10653
8666
61.5
256



  045


AGC TCT TGG ACT CCA A
 4293





 


AJ_P3
85653
F04
AJ_256
/5AmMC6/CCC AAT GAC GAA
10653
8739
59.5
257



  046


GGA CAC TTA GAC CTA A
 4294





 


AJ_P3
85653
F05
AJ_257
/5AmMC6/CCC AAC CGT AGA
10653
8714
59.1
258



  047


ACA TTT GAA GCC ATA A
 4295





 


AJ_P3
85653
F06
AJ_258
/5AmMC6/CCC AAC CAC TCG
10653
8675
62.2
259



  048


AAC ATG GTA ACG TCA A
 4296





 


AJ_P3
85653
F07
AJ_259
/5AmMC6/CCC AAT CGA ACC
10653
8690
60.7
260



  049


GTA ACC ATT TCA GGA A
 4297





 


AJ_P3
85653
F08
AJ_260
/5AmMC6/CCC AAC TAG TGG
10653
8761
60.5
261



  050


TTG GAA CAT GCA CTA A
 4298





 


AJ_P3
85653
F09
AJ_261
/5AmMC6/CCC AAG TGC TTA
10653
8721
60.8
262



  051


CTG TCC ATC GGA AAA A
 4299





 


AJ_P3
85653
F10
AJ_262
/5AmMC6/CCC AAT GAG TCT
10653
8687
58.8
263



  052


GCA TCT CTT TCA AGA A
 4300





 


AJ_P3
85653
F11
AJ_263
/5AmMC6/CCC AAT AGG ACA
10653
8739
59.8
264



  053


AAG ACG TCT TAC CGA A
 4301





 


AJ_P3
85653
F12
AJ_264
/5AmMC6/CCC AAT CAT AGG
10653
8779
59.3
265



  054


CTA AGG GAA GAC CTA A
 4302





 


AJ_P3
85653
G01
AJ_265
/5AmMC6/CCC AAC AGA GGT
10653
8795
61.7
266



  055


AAA GTC CAG TGG TCA A
 4303





 


AJ_P3
85653
G02
AJ_266
/5AmMC6/CCC AAG ACC ACT
10653
8690
60.7
267



  056


ACA ACG TTG CAT GTA A
 4304





 


AJ_P3
85653
G03
AJ_267
/5AmMC6/CCC AAT AGA CCA
10653
8739
60.2
268



  057


CAA GCA TCG TTA GGA A
 4305





 


AJ_P3
85653
G04
AJ_268
/5AmMC6/CCC AAG TCA CTC
10653
8681
59.8
269



  058


ACC TAA GTT CGG TAA A
 4306





 


AJ_P3
85653
G05
AJ_269
/5AmMC6/CCC AAG CTT TCA
10653
8690
60.3
270



  059


AGT ACC ACA CGA GTA A
 4307





 


AJ_P3
85653
G06
AJ_270
/5AmMC6/CCC AAG TCA CAT
10653
8697
61.4
271



  060


CCT CTA GGG TTC GAA A
 4308





 


AJ_P3
85653
G07
AJ_271
/5AmMC6/CCC AAA AAC GTT
10653
8736
59.8
272



  061


CAT TTG GTC TGA CGA A
 4309





 


AJ_P3
85653
G08
AJ_272
/5AmMC6/CCC AAC TGT CCA
10653
8730
61.2
273



  062


TTC GGA ACG TGA AAA A
 4310





 


AJ_P3
85653
G09
AJ_273
/5AmMC6/CCC AAA GTT CTT
10653
8727
59.1
274



  063


TCT TCA GCA AGG GTA A
 4311





 


AJ_P3
85653
G10
AJ_274
/5AmMC6/CCC AAT AGT CCT
10653
8712
59.5
275



  064


GTC GTT AGA ACC GTA A
 4312





 


AJ_P3
85653
G11
AJ_275
/5AmMC6/CCC AAC GTA CAT
10653
8690
60.2
276



  065


CCC TTA GAA ACG TGA A
 4313





 


AJ_P3
85653
G12
AJ_276
/5AmMC6/CCC AAC GGT TCA
10653
8687
59.7
277



  066


GCA CTT TAC ATT TGA A
 4314





 


AJ_P3
85653
H01
AJ_277
/5AmMC6/CCC AAT GCG TAA
10653
8672
60.7
278



  067


ACT CGT TGT CCT ACA A
 4315





 


AJ_P3
85653
H02
AJ_278
/5AmMC6/CCC AAT CGG TAA
10653
8687
59.4
279



  068


ACC TGT TTC GCT TAA A
 4316





 


AJ_P3
85653
H03
AJ_279
/5AmMC6/CCC AAG TGC AAG
10653
8770
61.4
280



  069


CAC AGG TGA CAT TTA A
 4317





 


AJ_P3
85653
H04
AJ_280
/5AmMC6/CCC AAG GGT ACA
10653
8795
60.9
281



  070


GAC GAG TAA CTC TGA A
 4318





 


AJ_P3
85653
H05
AJ_281
/5AmMC6/CCC AAA CCC TAG
10653
8672
59.1
282



  071


TAG TTC TAC TCG TGA A
 4319





 


AJ_P3
85653
H06
AJ_282
/5AmMC6/CCC AAG TAA CCC
10653
8706
61
283



  072


TTC CGT AGG ACA GTA A
 4320





 


AJ_P3
85653
H07
AJ_283
/5AmMC6/CCC AAT TTA GTC
10653
8672
60.3
284



  073


ACT CTG GTC AAC CGA A
 4321





 


AJ_P3
85653
H08
AJ_284
/5AmMC6/CCC AAG TAC ACA
10653
8715
61.6
285



  074


ACC TCT GGT AAC GGA A
 4322





 


AJ_P3
85653
H09
AJ_285
/5AmMC6/CCC AAC ACA AGT
10653
8795
62.2
286



  075


TCA GGT AGG AGT GCA A
 4323





 


AJ_P3
85653
H10
AJ_286
/5AmMC6/CCC AAC TAA AGG
10653
8696
59.4
287



  076


TGT TTA CGC TTC CAA A
 4324





 


AJ_P3
85653
H11
AJ_287
/5AmMC6/CCC AAC TGA AGT
10653
8777
61.4
288



  077


TGG TCT ACC TGA GGA A
 4325





 


AJ_P3
85653
H12
AJ_288
/5AmMC6/CCC AAT GTC GTA
10653
8672
60.8
289



  078


AGT TCC TCA ACT GCA A
 4326





 


AJ_P4
85653
A01
AJ_289
/5AmMC6/CCC AAA CCT GAG
10653
8712
60.5
290



  080


ACC TGT GTT TCG TAA A
 4327





 


AJ_P4
85653
A02
AJ_290
/5AmMC6/CCC AAT AGG CTA
10653
8723
58.4
291



  081


GCT CAA CCA TAA AGA A
 4328





 


AJ_P4
85653
A03
AJ_291
/5AmMC6/CCC AAG TTG ACA
10653
8675
61.8
292



  082


ACG CTA CCC TAG ACA A
 4329





 


AJ_P4
85653
A04
AJ_292
/5AmMC6/CCC AAT CAC GAA
10653
8754
59.7
293



  083


GTG AGC TTG TCA AAA A
 4330





 


AJ_P4
85653
A05
AJ_293
/5AmMC6/CCC AAT GAA ACC
10653
8690
61.2
294



  084


GTA ACT CAC TTG GCA A
 4331





 


AJ_P4
85653
A06
AJ_294
/5AmMC6/CCC AAC TTA GCA
10653
8763
59.2
295



  085


CAA AGT GTA GAA GCA A
 4332





 


AJ_P4
85653
A07
AJ_295
/5AmMC6/CCC AAT GCG TAG
10653
8763
59.1
296



  086


AAC CAT GTA CAA AGA A
 4333





 


AJ_P4
85653
A08
AJ_296
/5AmMC6/CCC AAG AGT TGC
10653
8761
60.4
297



  087


TTC GGT ACT CAA AGA A
 4334





 


AJ_P4
85653
A09
AJ_297
/5AmMC6/CCC AAG CGT AGT
10653
8779
60.3
298



  088


TCG GAA ACA CTA AGA A
 4335





 


AJ_P4
85653
A10
AJ_298
/5AmMC6/CCC AAA AGA GTC
10653
8690
59.4
299



  089


TTA CCG TAC TAC CGA A
 4336





 


AJ_P4
85653
A11
AJ_299
/5AmMC6/CCC AAA AAC GGT
10653
8706
61.7
300



  090


AGG TCT CTG ACT CCA A
 4337





 


AJ_P4
85653
A12
AJ_300
/5AmMC6/CCC AAG GTC AGT
10653
8706
62.4
301



  091


TAA GCC AAC CCT TGA A
 4338





 


AJ_P4
85653
B01
AJ_301
/5AmMC6/CCC AAA CCA GTC
10653
8672
60.1
302



  092


TCT CAG TTT ACG TGA A
 4339





 


AJ_P4
85653
B02
AJ_302
/5AmMC6/CCC AAT AAG ACA
10653
8699
60.7
303



  093


AGG ACT TCC ATG CCA A
 4340





 


AJ_P4
85653
B03
AJ_303
/5AmMC6/CCC AAG TCG AGA
10653
8770
59.9
304



  094


ACA TGG AAG TCC TTA A
 4341





 


AJ_P4
85653
B04
AJ_304
/5AmMC6/CCC AAT GCA GAG
10653
8763
58.4
305



  095


AAA GTA CAT ACC GTA A
 4342





 


AJ_P4
85653
B05
AJ_305
/5AmMC6/CCC AAG TGC ACT
10653
8779
60.5
306



  096


TAA GGA CAA CAG GTA A
 4343





 


AJ_P4
85653
B06
AJ_306
/5AmMC6/CCC AAA CCT GTC
10653
8721
60.2
307



  097


TTA AGG CAT ACG GTA A
 4344





 


AJ_P4
85653
B07
AJ_307
/5AmMC6/CCC AAG TCT CTA
10653
8752
59.6
308



  098


AGT AGG CAT GCT GTA A
 4345





 


AJ_P4
85653
B08
AJ_308
/5AmMC6/CCC AAC GTC TGA
10653
8770
59.8
309



  099


CAT TGG AGA GAA CTA A
 4346





 


AJ_P4
85653
B09
AJ_309
/5AmMC6/CCC AAA AAG CTC
10653
8696
59.3
310



  100


ACG TCT TGG TCT TAA A
 4347





 


AJ_P4
85653
B10
AJ_310
/5AmMC6/CCC AAG GGT AAC
10653
8770
60
311



  101


AGA CAC TTT AGC GTA A
 4348





 


AJ_P4
85653
B11
AJ_311
/5AmMC6/CCC AAT GAC CTA
10653
8795
60.9
312



  102


CGA GTG GAG AGT ACA A
 4349





 


AJ_P4
85653
B12
AJ_312
/5AmMC6/CCC AAA GCT TGC
10653
8723
59.2
313



  103


GAA ACC TAA CTA AGA A
 4350





 


AJ_P4
85653
C01
AJ_313
/5AmMC6/CCC AAT GTC GAC
10653
8699
59.6
314



  104


AGA CCA TAC CTA AGA A
 4351





 


AJ_P4
85653
C02
AJ_314
/5AmMC6/CCC AAG GTC AAC
10653
8675
62.6
315



  105


AAG CCA TAC GTT CCA A
 4352





 


AJ_P4
85653
C03
AJ_315
/5AmMC6/CCC AAC TGG TTA
10653
8770
59.5
316



  106


CTA CGA ACA GGA GTA A
 4353





 


AJ_P4
85653
C04
AJ_316
/5AmMC6/CCC AAT AGA GAC
10653
8690
59.1
317



  107


GTT ACT CCT AAC CGA A
 4354





 


AJ_P4
85653
C05
AJ_317
/5AmMC6/CCC AAA GAC AGT
10653
8690
60.1
318



  108


TGA CAC CTT AGC CTA A
 4355





 


AJ_P4
85653
C06
AJ_318
/5AmMC6/CCC AAA TCG AGA
10653
8690
59.8
319



  109


GTT ACA CCT TAC CGA A
 4356





 


AJ_P4
85653
C07
AJ_319
/5AmMC6/CCC AAA CAG GTT
10653
8779
60.4
320



  110


TCC AAG AAC TAG GGA A
 4357





 


AJ_P4
85653
C08
AJ_320
/5AmMC6/CCC AAG ACA GGT
10653
8777
61.2
321



  111


AGG TCT TGC TAG TCA A
 4358





 


AJ_P4
85653
C09
AJ_321
/5AmMC6/CCC AAG GAG TCT
10653
8715
61.7
322



  112


CAA CCG TTA ACC AGA A
 4359





 


AJ_P4
85653
C10
AJ_322
/5AmMC6/CCC AAG AAA CGT
10653
8681
60.7
323



  113


ACG CTT CTC CAT TGA A
 4360





 


AJ_P4
85653
C11
AJ_323
/5AmMC6/CCC AAC TTA GGA
10653
8675
61.5
324



  114


AGC ACT ACG TAC CCA A
 4361





 


AJ_P4
85653
C12
AJ_324
/5AmMC6/CCC AAG TAA GCT
10658
8657
61.5
325



  115


ACG TTC CTG TAC CCA A
 6457





 


AJ_P4
85653
D01
AJ_325
/5AmMC6/CCC AAC CAA GTA
10653
8795
62.1
326



  116


AGT GGA CAC TGG TGA A
 4363





 


AJ_P4
85653
D02
AJ_326
/5AmMC6/CCC AAC TGT TTA
10653
8761
60
327



  117


CAG AGG TCA GCA GTA A
 4364





 


AJ_P4
85653
D03
AJ_327
/5AmMC6/CCC AAC ACG TCT
10653
8723
58.6
328



  118


TAA AGC AGA GAA CTA A
 4365





 


AJ_P4
85653
D04
AJ_328
/5AmMC6/CCC AAG AGG ACT
10653
8697
61.1
329



  119


GTC CTA CTT CCA TGA A
 4366





 


AJ_P4
85653
D05
AJ_329
/5AmMC6/CCC AAG AAC ATC
10653
8666
61.6
330



  120


TCC ACT GGT CAC GTA A
 4367





 


AJ_P4
85653
D06
AJ_330
/5AmMC6/CCC AAT GAA GCA
10653
8739
60.9
331



  121


ACA AGT GGT ACT CCA A
 4368





 


AJ_P4
85653
D07
AJ_331
/5AmMC6/CCC AAT CCG TAA
10653
8779
59.5
332



  122


CAG TAG GAG AAC GTA A
 4369





 


AJ_P4
85653
D08
AJ_332
/5AmMC6/CCC AAA CCG TAG
10653
8690
60
333



  123


GAA CTA CCA TTC TGA A
 4370





 


AJ_P4
85653
D09
AJ_333
/5AmMC6/CCC AAC CAG TTC
10653
8690
60.8
334



  124


GTT CAA ACA GAC TGA A
 4371





 


AJ_P4
85653
D10
AJ_334
/5AmMC6/CCC AAG TTA AAC
10653
8699
60.4
335



  125


ATC CAG AGC TCA CGA A
 4372





 


AJ_P4
85653
D11
AJ_335
/5AmMC6/CCC AAG TCA CAC
10653
8715
61.9
336



  126


AAC CTA GAG CTT GGA A
 4373





 


AJ_P4
85653
D12
AJ_336
/5AmMC6/CCC AAC ATG TTA
10653
8752
61
337



  127


GGG TTA CCT TGG CAA A
 4374





 


AJ_P4
85653
E01
AJ_337
/5AmMC6/CCC AAG TCA AAG
10653
8666
61.7
338



  128


GTA CTC CAC TTC CGA A
 4375





 


AJ_P4
85653
E02
AJ_338
/5AmMC6/CCC AAG TAG AAC
10653
8699
60
339



  129


GTC AAC CAC TTA CGA A
 4376





 


AJ_P4
85653
E03
AJ_339
/5AmMC6/CCC AAG GAG ACT
10653
8697
60.5
340



  130


TGT CCT ACT CTA CGA A
 4377





 


AJ_P4
85653
E04
AJ_340
/5AmMC6/CCC AAT TTC GTA
10653
8687
58.9
341



  131


GTA CTC ACT TGC GAA A
 4378





 


AJ_P4
85653
E05
AJ_341
/5AmMC6/CCC AAC CTT GTA
10653
8770
59.5
342



  132


CTA GGA AGG AAG CTA A
 4379





 


AJ_P4
85653
E06
AJ_342
/5AmMC6/CCC AAG TCG TAG
10653
8697
62.3
343



  133


TTG TCA CAC TGC ACA A
 4380





 


AJ_P4
85653
E07
AJ_343
/5AmMC6/CCC AAC GAA GTT
10653
8672
61
344



  134


ACG TCT TTC ATG CCA A
 4381





 


AJ_P4
85653
E08
AJ_344
/5AmMC6/CCC AAA AGG CAT
10653
8730
61.3
345



  135


AAG GCT TGT CAT CCA A
 4382





 


AJ_P4
85653
E09
AJ_345
/5AmMC6/CCC AAG TGT CCA
10653
8681
60.8
346



  136


TAC GCT TTA CCG AAA A
 4383





 


AJ_P4
85653
E10
AJ_346
/5AmMC6/CCC AAC GGT TGA
10653
8715
62.3
347



  137


CAC CAG TTA CCA AGA A
 4384





 


AJ_P4
85653
E11
AJ_347
/5AmMC6/CCC AAG TGT GCA
10653
8697
62.2
348



  138


ACC AGT TAC TCC TGA A
 4385





 


AJ_P4
85653
E12
AJ_348
/5AmMC6/CCC AAG CTG ACA
10653
8672
60.1
349



  139


GAC TCT CTT TCA TGA A
 4386





 


AJ_P4
85653
F01
AJ_349
/5AmMC6/CCC AAG AAA GCT
10653
8681
59.4
350



  140


GTA CCC TTC TCT AGA A
 4387





 


AJ_P4
85653
F02
AJ_350
/5AmMC6/CCC AAA TGT TGC
10653
8714
59
351



  141


TAC AAG ACT AAC CGA A
 4388





 


AJ_P4
85653
F03
AJ_351
/5AmMC6/CCC AAG TCT GGA
10653
8777
61.4
352



  142


AGT GCT AGT ACG TCA A
 4389





 


AJ_P4
85653
F04
AJ_352
/5AmMC6/CCC AAT CGC AAC
10653
8687
59.4
353



  143


TTC GGT ACA TTT GTA A
 4390





 


AJ_P4
85653
F05
AJ_353
/5AmMC6/CCC AAC CTG TAA
10653
8754
59
354



  144


CAT TGA AGA AGC GTA A
 4391





 


AJ_P4
85653
F06
AJ_354
/5AmMC6/CCC AAA CTG TTG
10653
8754
59.4
355



 145


GAA AGC TGA ACA CTA A
 4392





 


AJ_P4
85653
F07
AJ_355
/5AmMC6/CCC AAG ACG TAG
10653
8779
58.9
356



  146


CTT AGA GAG AAC CTA A
 4393





 


AJ_P4
85653
F08
AJ_356
/5AmMC6/CCC AAC ATT GTT
10653
8761
60.7
357



  147


GTG GAA CCT CAG AGA A
 4394





 


AJ_P4
85653
F09
AJ_357
/5AmMC6/CCC AAG TGG ACT
10653
8697
61.2
358



  148


AGC TTC CTA CAC TGA A
 4395





 


AJ_P4
85653
F10
AJ_358
/5AmMC6/CCC AAA GGA ACT
10653
8723
59.2
359



  149


GAC ATT CAA CAC GTA A
 4396





 


AJ_P4
85653
F11
AJ_359
/5AmMC6/CCC AAT GTT CGA
10653
8690
60.2
360



  150


GTC CAC AAC TAC AGA A
 4397





 


AJ_P4
85653
F12
AJ_360
/5AmMC6/CCC AAG TAA CTA
10653
8739
59
361



  151


CTC ACA GAG CTA GGA A
 4398





 


AJ_P4
85653
G01
AJ_361
/5AmMC6/CCC AAG AGG ACT
10653
8706
61.2
362



  152


CAC CAG TAC TTT CGA A
 4399





 


AJ_P4
85653
G02
AJ_362
/5AmMC6/CCC AAT AGC GTT
10653
8687
59
363



  153


GTT TCT AAC CAC TGA A
 4400





 


AJ_P4
85653
G03
AJ_363
/5AmMC6/CCC AAC ATT TGT
10653
8736
59
364



  154


TAG TAG CAG TCA CGA A
 4401





 


AJ_P4
85653
G04
AJ_364
/5AmMC6/CCC AAT AAC AGC
10653
8699
60.8
365



  155


AAG ACC TTG TAG CCA A
 4402





 


AJ_P4
85653
G05
AJ_365
/5AmMC6/CCC AAG ACT CTC
10653
8675
61.9
366



  156


CAC ACG TTG AAG ACA A
 4403





 


AJ_P4
85653
G06
AJ_366
/5AmMC6/CCC AAG AAC TCC
10653
8666
61.7
367



  157


ATC CTG TTC GAC AGA A
 4404





 


AJ_P4
85653
G07
AJ_367
/5AmMC6/CCC AAG GTT CTA
10653
8681
60.4
368



  158


GTT CCA ACT AAC GCA A
 4405





 


AJ_P4
85653
G08
AJ_368
/5AmMC6/CCC AAA GTT GCG
10653
8727
59
369



  159


TTT GTC ATA GAC CTA A
 4406





 


AJ_P4
85653
G09
AJ_369
/5AmMC6/CCC AAC GCT TGA
10653
8763
59
370



  160


GGT AAA CTA AAC AGA A
 4407





 


AJ_P4
85653
G10
AJ_370
/5AmMC6/CCC AAT AAC GAG
10653
8739
59
371



  161


TAG AGC TCT AGA CCA A
 4408





 


AJ_P4
85653
G11
AJ_371
/5AmMC6/CCC AAG TGA GTC
10653
8739
60.5
372



  162


ATA GCC ATA AGC CAA A
 4409





 


AJ_P4
85653
G12
AJ_372
/5AmMC6/CCC AAC TTA CGT
10653
8672
60.3
373



  163


GAC TTC CAT TCA GGA A
 4410





 


AJ_P4
85653
H01
AJ_373
/5AmMC6/CCC AAA TCA GTG
10653
8672
60.1
374



  164


ACT GTC TCT TCA CGA A
 4411





 


AJ_P4
85653
H02
AJ_374
/5AmMC6/CCC AAA GGT ACT
10653
8657
61.4
375



  165


GAC TTC CAC TCC TGA A
 4412





 


AJ_P4
85653
H03
AJ_375
/5AmMC6/CCC AAT CGA CAT
10653
8779
59.9
376



  166


TAC AGG AAG TAC GGA A
 4413





 


AJ_P4
85653
H04
AJ_376
/5AmMC6/CCC AAC CAC TGG
10653
8739
60.9
377



  167


TTA AAC GTA AAC GGA A
 4414





 


AJ_P4
85653
H05
AJ_377
/5AmMC6/CCC AAG TTC ATT
10653
8672
60.7
378



  168


CCC TAA GCC TTG GAA A
 4415





 


AJ_P4
85653
H06
AJ_378
/5AmMC6/CCC AAG AAA CTA
10653
8730
60.1
379



  169


CTC CAT GGT TAG CGA A
 4416





 


AJ_P4
85653
H07
AJ_379
/5AmMC6/CCC AAC TAA GGG
10653
8745
58.4
380



  170


TTA AAG CTT ACC GTA A
 4417





 


AJ_P4
85653
H08
AJ_380
/5AmMC6/CCC AAG AGA CCT
10653
8690
60.1
381



  171


GTC ACA CTT TAA CGA A
 4418





 


AJ_P4
85653
H09
AJ_381
/5AmMC6/CCC AAT GAA CAA
10653
8723
59.8
382



  172


CAA CAT GCT TAC GGA A
 4419





 


AJ_P4
85653
H10
AJ_382
/5AmMC6/CCC AAT CAG AAA
10653
8763
58.9
383



  173


GCA ACA TTC TAG GGA A
 4420





 


AJ_P4
85653
H11
AJ_383
/5AmMC6/CCC AAT AGG CTT
10653
8705
58.8
384



  174


GAC TCA TTA AAC CGA A
 4421





 


AJ_P4
85653
H12
AJ_384
/5AmMC6/CCC AAA CTG GTT
10653
8712
60.3
385



  175


TGT AGT CCT ACC GAA A
 4422





 


AJ_P5
85653
A01
AJ_385
/5AmMC6/CCC AAA CCT GAC
10653
8687
59
386



  177


AGC TTG TTT CTT AGA A
 4423





 


AJ_P5
85653
A02
AJ_386
/5AmMC6/CCC AAC TTG CTA
10653
8770
59.9
387



  178


CAT AGA GAG AGT GCA A
 4424





 


AJ_P5
85653
A03
AJ_387
/5AmMC6/CCC AAG GTA AAC
10653
8666
61.5
388



  179


CTT CCA GTC TCC AGA A
 4425





 


AJ_P5
85653
A04
AJ_388
/5AmMC6/CCC AAT ACC AAG
10653
8739
60.8
389



  180


TAC GCA AAC TGT GGA A
 4426





 


AJ_P5
85653
A05
AJ_389
/5AmMC6/CCC AAC CGT AAA
10653
8730
60.5
390



  181


CCT TAA GGT GTA GCA A
 4427





 


AJ_P5
85653
A06
AJ_390
/5AmMC6/CCC AAC ATT GTT
10653
8681
61.6
391



  182


TCC CAA GGC ATA GCA A
 4428





 


AJ_P5
85653
A07
AJ_391
/5AmMC6/CCC AAG GTC ATC
10653
8657
61.9
392



  183


CTA CTA GCA TTG CCA A
 4429





 


AJ_P5
85653
A08
AJ_392
/5AmMC6/CCC AAG TTC AAC
10653
8681
60.4
393



  184


ATC ACT GCT ACG GTA A
 4430





 


AJ_P5
85653
A09
AJ_393
/5AmMC6/CCC AAT TCG CAT
10653
8727
60.1
394



  185


GCA TTT AAG GTG TCA A
 4431





 


AJ_P5
85653
A10
AJ_394
/5AmMC6/CCC AAC TTA GCA
10653
8779
59.4
395



  186


CTA GAG AAG GAG TCA A
 4432





 


AJ_P5
85653
A11
AJ_395
/5AmMC6/CCC AAG CTC AGG
10653
8777
62.2
396



  187


ACA GTT GAG TGT TCA A
 4433





 


AJ_P5
85653
A12
AJ_396
/5AmMC6/CCC AAG TCC TAG
10653
8761
59.7
397



  188


CTA AGA GTG TGT CAA A
 4434





 


AJ_P5
85653
B01
AJ_397
/5AmMC6/CCC AAG CTA CAA
10653
8754
59.3
398



  189


GCA TAA GTG GTT CAA A
 4435





 


AJ_P5
85653
B02
AJ_398
/5AmMC6/CCC AAG TCA TAC
10653
8739
60
399



  190


CAA AGC TGA GAC GTA A
 4436





 


AJ_P5
85653
B03
AJ_399
/5AmMC6/CCC AAT TTA GCA
10653
8754
58
400



  191


TAG ACG AGA GAC TCA A
 4437





 


AJ_P5
85653
B04
AJ_400
/5AmMC6/CCC AAT TTC ATG
10653
8745
59.4
401



  192


TAA CGA CAG TGA GCA A
 4438





 


AJ_P5
85653
B05
AJ_401
/5AmMC6/CCC AAT GCA CTT
10653
8754
58.6
402



  193


CGT AGA GTA AGA ACA A
 4439





 


AJ_P5
85653
B06
AJ_402
/5AmMC6/CCC AAA CGT TGT
10653
8752
60.5
403



  194


CTC TGT AGT GGA ACA A
 4440





 


AJ_P5
85653
B07
AJ_403
/5AmMC6/CCC AAC CGA AGT
10653
8699
60.8
404



  195


TAG CAA ACC TCA TGA A
 4441





 


AJ_P5
85653
B08
AJ_404
/5AmMC6/CCC AAC ATT TAG
10653
8754
58.8
405



  196


AAG GAC TTC GAA CGA A
 4442





 


AJ_P5
85653
B09
AJ_405
/5AmMC6/CCC AAG TTC CAA
10653
8675
62
406



  197


CAC TCA GAC AGG TCA A
 4443





 


AJ_P5
85653
B10
AJ_406
/5AmMC6/CCC AAT GAC AAC
10653
8706
61.7
407



  198


CTC TCA GAG TGG TCA A
 4444





 


AJ_P5
85653
B11
AJ_407
/5AmMC6/CCC AAG CCT AGG
10653
8777
61
408



  199


TAG GTT CTG GAA CTA A
 4445





 


AJ_P5
85653
B12
AJ_408
/5AmMC6/CCC AAT CGA ACA
10653
8690
60.6
409



  200


CAC CAT GTT ACT GGA A
 4446





 


AJ_P5
85653
C01
AJ_409
/5AmMC6/CCC AAT AGT CTA
10653
8727
59.3
410



  201


ACT GTT GGC TTG CAA A
 4447





 


AJ_P5
85653
C02
AJ_410
/5AmMC6/CCC AAA AGC TAG
10653
8681
59.8
411



  202


GTA CCT TCT TAC CGA A
 4448





 


AJ_P5
85653
C03
AJ_411
/5AmMC6/CCC AAC TCA GAG
10653
8770
60
412



  203


TAC AGA GAG TTT GCA A
 4449





 


AJ_P5
85653
C04
AJ_412
/5AmMC6/CCC AAG ACA CGT
10653
8795
61.4
413



  204


CAT AGG AGT GTA GCA A
 4450





 


AJ_P5
85653
C05
AJ_413
/5AmMC6/CCC AAT TAA GCA
10653
8763
59.2
414



  205


TAA CGA GAC AGT GCA A
 4451





 


AJ_P5
85653
C06
AJ_414
/5AmMC6/CCC AAG TGT CCA
10653
8795
62.6
415



  206


CAT GAG GTG AAA GCA A
 4452





 


AJ_P5
85653
C07
AJ_415
/5AmMC6/CCC AAC TAA AGG
10653
8770
60.6
416



  207


GTT GAA CGT TCC AGA A
 4453





 


AJ_P5
85653
C08
AJ_416
/5AmMC6/CCC AAA TCG CTT
10653
8727
59.1
417



  208


TCT TTA GTG GAG ACA A
 4454





 


AJ_P5
85653
C09
AJ_417
/5AmMC6/CCC AAA GGT CTT
10653
8696
60.1
418



  209


CAC TTT GTG CAC AAA A
 4455





 


AJ_P5
85653
C10
AJ_418
/5AmMC6/CCC AAG GCT TAA
10653
8755
62.3
419



  210


GGT GAA CCA TCG ACA A
 4456





 


AJ_P5
85653
C11
AJ_419
/5AmMC6/CCC AAC TGT AGA
10653
8699
59.3
420



  211


GCT ACC AAC ACT AGA A
 4457





 


AJ_P5
85653
C12
AJ_420
/5AmMC6/CCC AAC TAA GGG
10653
8752
60.5
421



  212


TTG TTA CGT TAG CCA A
 4458





 


AJ_P5
85653
D01
AJ_421
/5AmMC6/CCC AAG TGG TAC
10653
8697
61.4
422



  213


TCA GCT ACA TCG TCA A
 4459





 


AJ_P5
85653
D02
AJ_422
/5AmMC6/CCC AAG TCC AAA
10653
8675
62.3
423



  214


CAC CTT GAG AGC TCA A
 4460





 


AJ_P5
85653
D03
AJ_423
/5AmMC6/CCC AAT CAC AAG
10653
8779
60.1
424



  215


CTT AGA GTG GAG ACA A
 4461





 


AJ_P5
85653
D04
AJ_424
/5AmMC6/CCC AAC TTT GAC
10653
8752
60.9
425



  216


TTT GGC AAC TAG GGA A
 4462





 


AJ_P5
85653
D05
AJ_425
/5AmMC6/CCC AAC CTC AGT
10653
8737
61
426



  217


CTA AGG GTA GTG TCA A
 4463





 


AJ_P5
85653
D06
AJ_426
/5AmMC6/CCC AAA CAC CTG
10653
8715
61.6
427



  218


TCC AGA GAG TGT ACA A
 4464





 


AJ_P5
85653
D07
AJ_427
/5AmMC6/CCC AAC ATA GTT
10653
8745
59.2
428



  219


GTG AAG CAT CGC TAA A
 4465





 


AJ_P5
85653
D08
AJ_428
/5AmMC6/CCC AAA CGT GTT
10653
8752
60.6
429



  220


GTT GTA CCC TAG GAA A
 4466





 


AJ_P5
85653
D09
AJ_429
/5AmMC6/CCC AAA CTT TGG
10653
8754
59.4
430



  221


TAG AAA CGT AGC CAA A
 4467





 


AJ_P5
85653
D10
AJ_430
/5AmMC6/CCC AAC TCA GTT
10653
8736
59.9
431



  222


GCA TTA AAG TGT GCA A
 4468





 


AJ_P5
85653
D11
AJ_431
/5AmMC6/CCC AAA CTA CTG
10653
8712
60.2
432



  223


TTC TGG ACT TCG GAA A
 4469





 


AJ_P5
85653
D12
AJ_432
/5AmMC6/CCC AAA GAG CAT
10653
8779
60
433



  224


TAG GAC TGT ACG ACA A
 4470





 


AJ_P5
85653
E01
AJ_433
/5AmMC6/CCC AAC ACC ATG
10653
8746
62.3
434



  225


CTG AGT GGT AAG TCA A
 4471





 


AJ_P5
85653
E02
AJ_434
/5AmMC6/CCC AAC TGG AAC
10653
8795
62.2
435



  226


ACG TGT GGT AGA ACA A
 4472





 


AJ_P5
85653
E03
AJ_435
/5AmMC6/CCC AAC CTC AGA
10653
8657
62
436



  227


ACT CGT TGG TTA CCA A
 4473





 


AJ_P5
85653
E04
AJ_436
/5AmMC6/CCC AAT GCC ATA
10653
8687
59.3
437



  228


ACG CTT GTA CTT GTA A
 4474





 


AJ_P5
85653
E05
AJ_437
/5AmMC6/CCC AAA ACC TTG
10653
8763
59
438



  229


TAG ACA AGA AGC GTA A
 4475





 


AJ_P5
85653
E06
AJ_438
/5AmMC6/CCC AAC ACA TGT
10653
8779
59.8
439



  230


TAG AGA CGA CAG GTA A
 4476





 


AJ_P5
85653
E07
AJ_439
/5AmMC6/CCC AAG GTA CTC
10653
8672
59.4
440



  231


TAA CTT GCA GTC CTA A
 4477





 


AJ_P5
85653
E08
AJ_440
/5AmMC6/CCC AAT GCC AAC
10653
8699
60.9
441



  232


CTC AAG AAG TGT ACA A
 4478





 


AJ_P5
85653
E09
AJ_441
/5AmMC6/CCC AAC TAA AGT
10653
8739
61.2
442



  233


TGG GAA CGC ATC ACA A
 4479





 


AJ_P5
85653
E10
AJ_442
/5AmMC6/CCC AAG GAC TAC
10653
8666
61
443



  234


TCC ACT GTC ATC AGA A
 4480





 


AJ_P5
85653
E11
AJ_443
/5AmMC6/CCC AAG AAC CGT
10653
8657
61
444



  235


AGT TCC TTC CCT AGA A
 4481





 


AJ_P5
85653
E12
AJ_444
/5AmMC6/CCC AAC TTT GAG
10653
8752
60.1
445



  236


GTG AGA CTC GTT ACA A
 4482





 


AJ_P5
85653
F01
AJ_445
/5AmMC6/CCC AAT CAG AGA
10653
8739
60.1
446



  237


AGA GTT CGT CAC ACA A
 4483





 


AJ_P5
85653
F02
AJ_446
/5AmMC6/CCC AAG TTT CAT
10653
8672
60.5
447



  238


TCC TCA GAG CTG ACA A
 4484





 


AJ_P5
85653
F03
AJ_447
/5AmMC6/CCC AAG TTG TCA
10653
8657
61.8
448



  239


CTC CTG AGC ACT ACA A
 4485





 


AJ_P5
85653
F04
AJ_448
/5AmMC6/CCC AAA AGG TTC
10653
8681
61.5
449



  240


ATC GCT TTG ACC ACA A
 4486





 


AJ_P5
85653
F05
AJ_449
/5AmMC6/CCC AAT GCC AAG
10653
8752
61.2
450



  241


ACT TGT GGT GTT ACA A
 4487





 


AJ_P5
85653
F06
AJ_450
/5AmMC6/CCC AAA GGC TTC
10653
8739
60.4
451



  242


GGT AAC ACT AAC AGA A
 4488





 


AJ_P5
85653
F07
AJ_451
/5AmMC6/CCC AAC AGC TAG
10653
8752
61.1
452



  243


CAT GGT TTG GTT ACA A
 4489





 


AJ_P5
85653
F08
AJ_452
/5AmMC6/CCC AAG CCA TTA
10653
8657
62.2
453



  244


GCC TAG TTG TCC ACA A
 4490





 


AJ_P5
85653
F09
AJ_453
/5AmMC6/CCC AAC GGT ACA
10653
8777
62.7
454



  245


ACG GTT GGG TTT ACA A
 4491





 


AJ_P5
85653
F10
AJ_454
/5AmMC6/CCC AAC ACC AGT
10653
8715
62.5
455



  246


TGG ACA GGA CAT TCA A
 4492





 


AJ_P5
85653
F11
AJ_455
/5AmMC6/CCC AAT CTC AGA
10653
8786
61.8
456



  247


CTG GAA GGG TTG ACA A
 4493





 


AJ_P5
85653
F12
AJ_456
/5AmMC6/CCC AAG TGT GAC
10653
8739
60.9
457



  248


GAA CCT CAA ACA TGA A
 4494





 


AJ_P5
85653
G01
AJ_457
/5AmMC6/CCC AAT GCG TAC
10653
8779
60.1
458



  249


AGG TAC ATA GGA CAA A
 4495





 


AJ_P5
85653
G02
AJ_458
/5AmMC6/CCC AAC AGT TAA
10653
8763
59.1
459



  250


AGG ACA TGA GCT CAA A
 4496





 


AJ_P5
85653
G03
AJ_459
/5AmMC6/CCC AAT CCG AAA
10653
8770
60.3
460



  251


GGG TTA CAG TTA CGA A
 4497





 


AJ_P5
85653
G04
AJ_460
/5AmMC6/CCC AAC ATT GTG
10653
8721
61.6
461



  252


AAA GTG CAG TTC CCA A
 4498





 


AJ_P5
85653
G05
AJ_461
/5AmMC6/CCC AAA ACC ATG
10653
8675
62.5
462



  253


AGG TCA CGT TAC CCA A
 4499





 


AJ_P5
85653
G06
AJ_462
/5AmMC6/CCC AAT CAA GGA
10653
8739
60.3
463



  254


GAA ACG TGT ACC TCA A
 4500





 


AJ_P5
85653
G07
AJ_463
/5AmMC6/CCC AAT CAG GAG
10653
8795
60.6
464



  255


ACG ACT AGT AGG TCA A
 4501





 


AJ_P5
85653
G08
AJ_464
/5AmMC6/CCC AAG GAC TAG
10653
8706
61
465



  256


GTC ACA CAT CTC TGA A
 4502





 


AJ_P5
85653
G09
AJ_465
/5AmMC6/CCC AAC ATA GAG
10653
8739
59.5
466



  257


AGG ACA TCT TCG ACA A
 4503





 


AJ_P5
85653
G10
AJ_466
/5AmMC6/CCC AAC GAA CTC
10653
8666
62.3
467



  258


ATC CTT GTG GAC ACA A
 4504





 


AJ_P5
85653
G11
AJ_467
/5AmMC6/CCC AAC AGT TGG
10653
8761
61.5
468



  259


TGA GTT CAT GCA CAA A
 4505





 


AJ_P5
85653
G12
AJ_468
/5AmMC6/CCC AAC ATA GGA
10653
8795
62.3
469



  260


CAG GAG TGT TGC ACA A
 4506





 


AJ_P5
85653
H01
AJ_469
/5AmMC6/CCC AAC TAG TAG
10653
8779
59.8
470



  261


AAG ACT GCA TGG ACA A
 4507





 


AJ_P5
85653
H02
AJ_470
/5AmMC6/CCC AAT AGA GCA
10653
8779
60.2
471



  262


AGA ACC TCA GTT GGA A
 4508





 


AJ_P5
85653
H03
AJ_471
/5AmMC6/CCC AAC CAT GTG
10653
8777
62.1
472



  263


GAG TTT CTG AGG ACA A
 4509





 


AJ_P5
85653
H04
AJ_472
/5AmMC6/CCC AAT AGA CAG
10653
8755
61.9
473



  264


GAC AGG TGT TCC CAA A
 4510





 


AJ_P5
85653
H05
AJ_473
/5AmMC6/CCC AAT TCG GAA
10653
8687
58.8
474



  265


GCC ATT TCT CTT AGA A
 4511





 


AJ_P5
85653
H06
AJ_474
/5AmMC6/CCC AAT CGG AAC
10653
8672
60.3
475



  266


AGT TCC TCA TTC TGA A
 4512





 


AJ_P5
85653
H07
AJ_475
/5AmMC6/CCC AAT GAA GCA
10653
8696
59.2
476



  267


GTT CCA TCA TTC TGA A
 4513





 


AJ_P5
85653
H08
AJ_476
/5AmMC6/CCC AAC ATG TGT
10653
8737
61.9
477



  268


CAA GGG TAG CTC TCA A
 4514





 


AJ_P5
85653
H09
AJ_477
/5AmMC6/CCC AAG CCT TTA
10653
8666
62.8
478



  269


CAC CAT GTG GAA CCA A
 4515





 


AJ_P5
85653
H10
AJ_478
/5AmMC6/CCC AAC TAA CTG
10653
8786
61.5
479



  270


CTG AGG TGA GGT ACA A
 4516





 


AJ_P5
85653
H11
AJ_479
/5AmMC6/CCC AAC TCC AAG
10653
8746
61.9
480



  271


TCG AGT GAG TTG ACA A
 4517





 


AJ_P5
85653
H12
AJ_480
/5AmMC6/CCC AAC GAG TTG
10653
8779
60.3
481



  272


AGA AGC TAC ATG ACA A
 4518





 


AJ_P6 
85653
A01
AJ_481
/5AmMC6/CCC AAT TTC TGA
10653
8721
60.2
482



  274


GTG AGC AAC CCT AGA A
 4519





 


AJ_P6 
85653
A02
AJ_482
/5AmMC6/CCC AAG AGT ACA
10653
8675
60.8
483



  275


GCT ACC TCT CCA AGA A
 4520





 


AJ_P6 
85653
A03
AJ_483
/5AmMC6/CCC AAG AGC ACT
10653
8699
60.4
484



  276


CCA CTT GTA CAA AGA A
 4521





 


AJ_P6 
85653
A04
AJ_484
/5AmMC6/CCC AAG CTA CAT
10653
8687
58.3
485



  277


TTC TTG AGT CGA CTA A
 4522





 


AJ_P6 
85653
A05
AJ_485
/5AmMC6/CCC AAA CCG TAG
10653
8699
60.2
486



  278


GAC TAC AAC ACT TGA A
 4523





 


AJ_P6 
85653
A06
AJ_486
/5AmMC6/CCC AAA TTC CTG
10653
8752
60.8
487



  279


TTG TGA CGA AGT CGA A
 4524





 


AJ_P6 
85653
A07
AJ_487
/5AmMC6/CCC AAA GTT CTG
10653
8752
60.8
488



  280


TGG TTC ACA AGT CGA A
 4525





 


AJ_P6 
85653
A08
AJ_488
/5AmMC6/CCC AAG TAC TCG
10653
8672
59.8
489



  281


AGT TCC CTT TAA CGA A
 4526





 


AJ_P6 
85653
A09
AJ_489
/5AmMC6/CCC AAG CTG AAG
10653
8763
59.3
490



  282


GTT AAC AAC AAG CTA A
 4527





 


AJ_P6 
85653
A10
AJ_490
/5AmMC6/CCC AAT CGC ATG
10653
8723
59.8
491



  283


GTA AAC AAA CAC TGA A
 4528





 


AJ_P6 
85653
A11
AJ_491
/5AmMC6/CCC AAC TGG TAC
10653
8699
61
492



  284


TAA AGC CAA ACT GCA A
 4529





 


AJ_P6 
85653
A12
AJ_492
/5AmMC6/CCC AAC GTT AAG
10653
8730
59.4
493



  285


AAG GTA CCT AGC CTA A
 4530





 


AJ_P6 
85653
B01
AJ_493
/5AmMC6/CCC AAC AGT GAA
10653
8721
60.6
494



  286


AGT TGT CCT TCC AGA A
 4531





 


AJ_P6 
85653
B02
AJ_494
/5AmMC6/CCC AAC AGG AGT
10653
8786
61.4
495



  287


TGG GTA CCA GTC TAA A
 4532





 


AJ_P6 
85653
B03
AJ_495
/5AmMC6/CCC AAG AAA CTG
10653
8699
61.1
496



  288


TGC AAA CAC TCC TGA A
 4533





 


AJ_P6 
85653
B04
AJ_496
/5AmMC6/CCC AAT CGT AGT
10653
8690
60.1
497



  289


TCG ACA AAC TCC AGA A
 4534





 


AJ_P6 
85653
B05
AJ_497
/5AmMC6/CCC AAC AGG TTA
10653
8666
62.1
498



  290


GTT CAC ACC ATC CGA A
 4535





 


AJ_P6 
85653
B06
AJ_498
/5AmMC6/CCC AAG GTT TAC
10653
8657
61.7
499



  291


GTC ACT CCA TCC AGA A
 4536





 


AJ_P6 
85653
B07
AJ_499
/5AmMC6/CCC AAG TTT AAC
10653
8687
59.3
500



  292


CTC ATG CTT TAG CGA A
 4537





 


AJ_P6 
85653
B08
AJ_500
/5AmMC6/CCC AAT TTG TAC
10653
8672
60.9
501



  293


GTT CCA ACC TAG GCA A
 4538





 


AJ_P6 
85653
B09
AJ_501
/5AmMC6/CCC AAA TCG TTT
10653
8727
59.8
502



  294


GTT TCC AGT AGG CAA A
 4539





 


AJ_P6 
85653
B10
AJ_502
/5AmMC6/CCC AAG CAT CCT
10658
8672
60.7
503



  295


TGT CTT AAC TGC AGA A
 6458





 


AJ_P6 
85653
B11
AJ_503
/5AmMC6/CCC AAA CTG GTA
10653
8697
62
504



  296


AGT CTT GGC TAC CCA A
 4541





 


AJ_P6 
85653
B12
AJ_504
/5AmMC6/CCC AAG TCC ATG
10653
8675
63
505



  297


TGC AAC ACC AAC TGA A
 4542





 


AJ_P6
85653
C01
AJ_505
/5AmMC6/CCC AAG TCA CAG
10653
8675
61.7
506



  298


GAC TCC TCA ACA TGA A
 4543





 


AJ_P6
85653
C02
AJ_506
/5AmMC6/CCC AAG TAC TCT
10657
8672
60.1
507



  299


CAT TCT GTG CAG ACA A
 7185





 


AJ_P6
85653
C03
AJ_507
/5AmMC6/CCC AAG GTT CCA
10657
8657
62.6
508



  300


CAC TTT GTC ACG ACA A
 7186





 


AJ_P6
85653
C04
AJ_508
/5AmMC6/CCC AAA CTC GTC
10658
8681
60.1
509



  301


TGT CCA TAA AGT CGA A
 6459





 


AJ_P6
85653
C05
AJ_509
/5AmMC6/CCC AAC AAG GTG
10657
8712
60.6
510



  302


TGT TCT ACC ATT CGA A
 7187





 


AJ_P6
85653
C06
AJ_510
/5AmMC6/CCC AAA CTC GTG
10657
8736
58.6
511



  303


TTG TAC TTA GAA CGA A
 7188





 


AJ_P6
85653
C07
AJ_511
/5AmMC6/CCC AAA GGC ATT
10653
8723
59.9
512



  304


GTC AAC AAA CCA GTA A
 4549





 


AJ_P6
85653
C08
AJ_512
/5AmMC6/CCC AAC AGT AGT
10657
8736
58.5
513



  305


TGT TAA CGA CTG CTA A
 7189





 


AJ_P6
85653
C09
AJ_513
/5AmMC6/CCC AAT GCT CAG
10653
8723
59.1
514



  306


GTC AAA CAA ACT AGA A
 4551





 


AJ_P6
85653
C10
AJ_514
/5AmMC6/CCC AAT GTC GTA
10658
8727
58.3
515



  307


CTT TGA GTA AGC CTA A
 6460





 


AJ_P6
85653
C11
AJ_515
/5AmMC6/CCC AAG GCT AGA
10653
8739
60.2
516



  308


CGA ACA TTA CCA TGA A
 4553





 


AJ_P6
85653
C12
AJ_516
/5AmMC6/CCC AAC GAG TGT
10658
8752
60
517



  309


TCT AGT GTT ACA CGA A
 6461





 


AJ_P6
85653
D01
AJ_517
/5AmMC6/CCC AAC AGG TTT
10653
8752
60.3
518



  310


ACG TGT GTA CAG CTA A
 4555





 


AJ_P6
85653
D02
AJ_518
/5AmMC6/CCC AAA GGT TCC
10653
8672
60.6
519



  311


TTC CAT GTA AGC TCA A
 4556





 


AJ_P6
85653
D03
AJ_519
/5AmMC6/CCC AAA GGC TTT
10653
8727
59.3
520



  312


GCT GTT ACT TAG ACA A
 4557





 


AJ_P6
85653
D04
AJ_520
/5AmMC6/CCC AAC AAA GTA
10653
8745
59.9
521



  313


ACT GTT CGT TGC GAA A
 4558





 


AJ_P6
85653
D05
AJ_521
/5AmMC6/CCC AAA TGC TTG
10653
8696
59.1
522



  314


GAA CTT CTA ACT CGA A
 4559





 


AJ_P6
85653
D06
AJ_522
/5AmMC6/CCC AAC CTG AGT
10653
8721
60.4
523



  315


ACT GTG CTC TGA AAA A
 4560





 


AJ_P6
85653
D07
AJ_523
/5AmMC6/CCC AAG GAC TCA
10653
8657
61.6
524



  316


AGT CTT CCT TCA CGA A
 4561





 


AJ_P6
85653
D08
AJ_524
/5AmMC6/CCC AAA GGG TTC
10653
8721
60.7
525



  317


CGT TCA CTA ACA TGA A
 4562





 


AJ_P6
85653
D09
AJ_525
/5AmMC6/CCC AAC CAG TAC
10653
8712
60.5
526



  318


TGC ATT TCT TGG AGA A
 4563





 


AJ_P6
85653
D10
AJ_526
/5AmMC6/CCC AAC AAG CCT
10653
8712
60.5
527



  319


AGT TCT GGT TGT ACA A
 4564





 


AJ_P6
85653
D11
AJ_527
/5AmMC6/CCC AAC AGA CCT
10653
8672
60.5
528



  320


ACC TTT GTT GTA GCA A
 4565





 


AJ_P6
85653
D12
AJ_528
/5AmMC6/CCC AAG AAC CCT
10653
8681
60.9
529



  321


TCT TTG ACT GCA AGA A
 4566





 


AJ_P6
85653
E01
AJ_529
/5AmMC6/CCC AAA GTC GTT
10653
8672
60.2
530



  322


TAG TCC TCT GAC CAA A
 4567





 


AJ_P6
85653
E02
AJ_530
/5AmMC6/CCC AAA GTC TCT
10653
8712
60.2
531



  323


TCG TTC AAC TGG AGA A
 4568





 


AJ_P6
85653
E03
AJ_531
/5AmMC6/CCC AAC GCA TTC
10653
8714
58.6
532



  324


TTA ACA GAG ACA GTA A
 4569





 


AJ_P6
85653
E04
AJ_532
/5AmMC6/CCC AAC GAG TCT
10653
8770
59.4
533



  325


CTT GAG AGG AAA CTA A
 4570





 


AJ_P6
85653
E05
AJ_533
/5AmMC6/CCC AAC GTA GTG
10653
8755
61
534



  326


AGT AGA CGT ACA CCA A
 4571





 


AJ_P6
85653
E06
AJ_534
/5AmMC6/CCC AAA AAG CTT
10653
8696
59.6
535



  327


GTT ACC TTC TGC AGA A
 4572





 


AJ_P6
85653
E07
AJ_535
/5AmMC6/CCC AAA CTT TGT
10653
8745
59.1
536



  328


ACT GGA GTA GCC AAA A
 4573





 


AJ_P6
85653
E08
AJ_536
/5AmMC6/CCC AAG CTT ACC
10653
8705
58.9
537



  329


TCT TAA GTG CAA GAA A
 4574





 


AJ_P6
85653
E09
AJ_537
/5AmMC6/CCC AAG AAC CTC
10653
8723
58.9
538



  330


TTA AAG CTA AGC GAA A
 4575





 


AJ_P6
85653
E10
AJ_538
/5AmMC6/CCC AAG ACC TAA
10653
8739
60.4
539



  331


ACA AGC TTG AGT CGA A
 4576





 


AJ_P6
85653
E11
AJ_539
/5AmMC6/CCC AAT TTG CAT
10653
8687
59.5
540



  332


AGG TTC TTC CAA CGA A
 4577





 


AJ_P6
85653
E12
AJ_540
/5AmMC6/CCC AAG CAA GTT
10653
8672
61.1
541



  333


GCA TTC CTC TCA TGA A
 4578





 


AJ_P6
85653
F01
AJ_541
/5AmMC6/CCC AAT CGG TAC
10653
8739
59.9
542



  334


ACG ACA TAC ATG AGA A
 4579





 


AJ_P6
85653
F02
AJ_542
/5AmMC6/CCC AAA CCT CTG
10653
8712
60.2
543



  335


TTT CTG AGT CGA AGA A
 4580





 


AJ_P6
85653
F03
AJ_543
/5AmMC6/CCC AAA CAC GTG
10653
8745
59.3
544



  336


TTG GCT AGT CTA AAA A
 4581





 


AJ_P6
85653
F04
AJ_544
/5AmMC6/CCC AAC GGT TTA
10653
8672
61.3
545



  337


AGC CTT TCA CCA TGA A
 4582





 


AJ_P6
85653
F05
AJ_545
/5AmMC6/CCC AAC GGT TCA
10653
8786
61.7
546



  338


TGG ACT AAC TGA GGA A
 4583





 


AJ_P6
85653
F06
AJ_546
/5AmMC6/CCC AAA CCG TTC
10653
8721
61.4
547



  339


AGT TTC ACA TGG GAA A
 4584





 


AJ_P6
85653
F07
AJ_547
/5AmMC6/CCC AAG ACC TCT
10653
8657
61
548



  340


CCA CTT GAC TGT AGA A
 4585





 


AJ_P6
85653
F08
AJ_548
/5AmMC6/CCC AAG TCT TTA
10653
8712
59.7
549



  341


CCT CAG TGT AGC AGA A
 4586





 


AJ_P6
85653
F09
AJ_549
/5AmMC6/CCC AAA CAG CTG
10653
8690
60.2
550



  342


AGT CCT TCC ATA AGA A
 4587





 


AJ_P6
85653
F10
AJ_550
/5AmMC6/CCC AAA ACT GTC
10653
8672
60.6
551



  343


ATT GCC TTC CTA GGA A
 4588





 


AJ_P6
85653
F11
AJ_551
/5AmMC6/CCC AAG TCC ATT
10653
8712
60.6
552



  344


CAT TCG TTC GAA GGA A
 4589





 


AJ_P6
85653
F12
AJ_552
/5AmMC6/CCC AAG TCA CCT
10653
8737
61.6
553



  345


CTT GGT AGT AAG GCA A
 4590





 


AJ_P6
85653
G01
AJ_553
/5AmMC6/CCC AAC CAT CAG
10653
8712
60.9
554



  346


CTT TAG TTG GTG ACA A
 4591





 


AJ_P6
85653
G02
AJ_554
/5AmMC6/CCC AAG TTA CCT
10653
8666
61.7
555



  347


GAC TCC ACT GGA CAA A
 4592





 


AJ_P6
85653
G03
AJ_555
/5AmMC6/CCC AAA GTT GGC
10653
8727
60.1
556



  348


ATC TTT GTC GTC AAA A
 4593





 


AJ_P6
85653
G04
AJ_556
/5AmMC6/CCC AAA CGT TGT
10653
8687
59.4
557



  349


GTC TTT AAC ATC CGA A
 4594





 


AJ_P6
85653
G05
AJ_557
/5AmMC6/CCC AAC AGT TTG
10653
8712
61.5
558



  350


GCT TTG ACA TCA CGA A
 4595





 


AJ_P6
85653
G06
AJ_558
/5AmMC6/CCC AAA CGG TTT
10653
8687
60.1
559



  351


GCA ACT CAT TCT TGA A
 4596





 


AJ_P6
85653
G07
AJ_559
/5AmMC6/CCC AAG ACG ACT
10653
8672
59.8
560



  352


GTT TAC TTC CTC AGA A
 4597





 


AJ_P6
85653
G08
AJ_560
/5AmMC6/CCC AAG GAC TCC
10653
8657
61.9
561



  353


ATT TCG ACT TCG ACA A
 4598





 


AJ_P6
85653
G09
AJ_561
/5AmMC6/CCC AAA TCA AGT
10653
8763
58
562



  354


CTA GAC AGA AGG CTA A
 4599





 


AJ_P6
85653
G10
AJ_562
/5AmMC6/CCC AAG TCG TCA
10653
8699
60.4
563



  355


TCA GCA AGA AAC CTA A
 4600





 


AJ_P6
85653
G11
AJ_563
/5AmMC6/CCC AAT CGT GTA
10653
8754
59.1
564



  356


CAT GGA AAG CAC ATA A
 4601





 


AJ_P6
85653
G12
AJ_564
/5AmMC6/CCC AAC TTT GAA
10653
8754
59.1
565



  357


GCA TGG AGA ACA CTA A
 4602





 


AJ_P6
85653
H01
AJ_565
/5AmMC6/CCC AAA AGT CCT
10653
8727
58.6
566



  358


CTG TTT AGT TAG CGA A
 4603





 


AJ_P6
85653
H02
AJ_566
/5AmMC6/CCC AAG TAA CCA
10653
8699
60.5
567



  359


AAC CAT GCT AGT CGA A
 4604





 


AJ_P6
85653
H03
AJ_567
/5AmMC6/CCC AAG GAC ATT
10653
8675
62.4
568



  360


GAC TCA CCA TCA GCA A
 4605





 


AJ_P6
85653
H04
AJ_568
/5AmMC6/CCC AAT GGG TAC
10653
8730
60
569



  361


TGC ATA CAC CAT AGA A
 4606





 


AJ_P6
85653
H05
AJ_569
/5AmMC6/CCC AAA GAA CTC
10653
8696
58.8
570



  362


GTC TTC ATT TAC GGA A
 4607





 


AJ_P6
85653
H06
AJ_570
/5AmMC6/CCC AAA GGT CTT
10653
8752
59.5
571



  363


TGT CCT AGT ACG AGA A
 4608





 


AJ_P6
85653
H07
AJ_571
/5AmMC6/CCC AAC ATG GTT
10653
8770
60.3
572



  364


AAG GTC AAC TCG AGA A
 4609





 


AJ_P6
85653
H08
AJ_572
/5AmMC6/CCC AAG CTT GTA
10653
8672
59.9
573



  365


ACG ACT TAC TCT CGA A
 4610





 


AJ_P6
85653
H09
AJ_573
/5AmMC6/CCC AAG ACC ACT
10653
8657
61.7
574



  366


CTC CTA GCA TTT GGA A
 4611





 


AJ_P6
85653
H10
AJ_574
/5AmMC6/CCC AAG TCC ATT
10653
8697
62.2
575



  367


CCC ATT GGT AGC AGA A
 4612





 


AJ_P6
85653
H11
AJ_575
/5AmMC6/CCC AAC ACT CTG
10653
8737
61.3
576



  368


TGT CGT ACA TAG GGA A
 4613





 


AJ_P6
85653
H12
AJ_576
/5AmMC6/CCC AAA CTT GTG
10653
8706
62.8
577



  369


TGG AAA CCG TAC CCA A
 4614





 


AJ_P7
85653
A01
AJ_577
/5AmMC6/CCC AAA TGC CTT
10653
8761
61
578



  371


GGT GTC ATA CAG GAA A
 4711





 


AJ_P7
85653
A02
AJ_578
/5AmMC6/CCC AAT CGG AAG
10653
8763
57.8
579



  372


TCA GAC TAG AAA CTA A
 4712





 


AJ_P7
85653
A03
AJ_579
/5AmMC6/CCC AAC CAG TAC
10653
8755
61
580



  373


CAG AGG TGA AGT CTA A
 4713





 


AJ_P7
85653
A04
AJ_580
/5AmMC6/CCC AAC ATA AAG
10653
8763
58.4
581



  374


GGA AAC TGA GCT CTA A
 4714





 


AJ_P7
85653
A05
AJ_581
/5AmMC6/CCC AAC TAA GAG
10653
8779
59.6
582



  375


GAG AAC TCC AGT TGA A
 4715





 


AJ_P7
85653
A06
AJ_582
/5AmMC6/CCC AAC TAG GAA
10653
8681
59.5
583



  376


GTT TAC TCC ACT CGA A
 4716





 


AJ_P7
85653
A07
AJ_583
/5AmMC6/CCC AAC AAC GTC
10653
8730
60.3
584



  377


TGC TAA AGT AGG TCA A
 4717





 


AJ_P7
85653
A08
AJ_584
/5AmMC6/CCC AAC GTC ATC
10653
8714
58.4
585



  378


AAC ATA GTA GGC TAA A
 4718





 


AJ_P7
85653
A09
AJ_585
/5AmMC6/CCC AAA TCG TCA
10653
8763
57.3
586



  379


CTA GAG AGA GAA CTA A
 4719





 


AJ_P7
85653
A10
AJ_586
/5AmMC6/CCC AAC TTG TCA
10653
8730
60
587



  380


CAT GAA GGA GAC CTA A
 4720





 


AJ_P7
85653
A11
AJ_587
/5AmMC6/CCC AAG GAG ACT
10653
8739
59.5
588



  381


CTA GAA ACT TCC GAA A
 4721





 


AJ_P7
85653
A12
AJ_588
/5AmMC6/CCC AAG AGT TAC
10653
8672
59.7
589



  382


GCT TCT ACT TCC AGA A
 4722





 


AJ_P7
85653
B01
AJ_589
/5AmMC6/CCC AAA CCA GTC
10653
8746
61.5
590



  383


CTT AAG GGT AGG TCA A
 4723





 


AJ_P7
85653
B02
AJ_590
/5AmMC6/CCC AAA AGC CTA
10653
8723
58.8
591



  384


GAA CAT TAC ATC GGA A
 4724





 


AJ_P7
85653
B03
AJ_591
/5AmMC6/CCC AAG CTG AAA
10653
8690
61.1
592



  385


GCA CTC CAT CAT TGA A
 4725





 


AJ_P7
85653
B04
AJ_592
/5AmMC6/CCC AAT CAG TGT
10653
8657
61.1
593



  386


GAC TCC ATC CCT AGA A
 4726





 


AJ_P7
85653
B05
AJ_593
/5AmMC6/CCC AAG CTA CTT
10653
8687
58.6
594



  387


AAC TCT GTT TCG GAA A
 4727





 


AJ_P7
85653
B06
AJ_594
/5AmMC6/CCC AAA TGC TTT
10653
8752
60.6
595



  388


CAC TGG TCT AGG GAA A
 4728





 


AJ_P7
85653
B07
AJ_595
/5AmMC6/CCC AAC AGT TGT
10653
8712
60.9
596



  389


TCG TTC ATG ACC AGA A
 4729





 


AJ_P7
85653
B08
AJ_596
/5AmMC6/CCC AAT CAC GAA
10653
8739
59.8
597



  390


ACG ACT ACT TAG GGA A
 4730





 


AJ_P7
85653
B09
AJ_597
/5AmMC6/CCC AAC ATT GTT
10653
8727
60.1
598



  391


TGG TTC ATC AAG CGA A
 4731





 


AJ_P7
85653
B10
AJ_598
/5AmMC6/CCC AAA TTC TTG
10653
8736
59.8
599



  392


TGG TAC AAC ATG CGA A
 4732





 


AJ_P7
85653
B11
AJ_599
/5AmMC6/CCC AAC CTG ACC
10653
8672
61
600



  393


AAC GGT TCA TTT GTA A
 4733





 


AJ_P7
85653
B12
AJ_600
/5AmMC6/CCC AAG ACC ATT
10653
8672
60.8
601



  394


ACG TCT TGC CTT GAA A
 4734





 


AJ_P7
85653
C01
AJ_601
/5AmMC6/CCC AAG CCA TAC
10653
8672
60.8
602



  395


CTC ATT GAG CTT TGA A
 4735





 


AJ_P7
85653
C02
AJ_602
/5AmMC6/CCC AAA GGA CTC
10653
8657
61.7
603



  396


TTC CGT AAC CTG TCA A
 4736





 


AJ_P7
85653
C03
AJ_603
/5AmMC6/CCC AAG GAG TGC
10653
8761
60.8
604



  397


ATT TCG TAA CCT GAA A
 4737





 


AJ_P7
85653
C04
AJ_604
/5AmMC6/CCC AAT CAC AAG
10653
8754
58.6
605



  398


CGA AAG TAG TGT CTA A
 4738





 


AJ_P7
85653
C05
AJ_605
/5AmMC6/CCC AAT CGA AGA
10653
8770
60.1
606



  399


GAC GAC TTG AGT TCA A
 4739





 


AJ_P7
85653
C06
AJ_606
/5AmMC6/CCC AAA TGG CTT
10653
8761
61.2
607



  400


TGG TAC AAC TGA CGA A
 4740





 


AJ_P7
85653
C07
AJ_607
/5AmMC6/CCC AAG AGA CGT
10653
8755
61.6
608



  401


TGG AAC ACC TAC TGA A
 4741





 


AJ_P7
85653
C08
AJ_608
/5AmMC6/CCC AAG AAA GCT
10653
8699
61.1
609



  402


GTT CAA ACC TCA CGA A
 4742





 


AJ_P7
85653
C09
AJ_609
/5AmMC6/CCC AAG TGA GTC
10653
8761
60.4
610



 403


TTC GAA ACT TCG GAA A
 4743





 


AJ_P7
85653
C10
AJ_610
/5AmMC6/CCC AAC CAG TGT
10653
8786
62.4
611



  404


TAA CGG AAC TTG GGA A
 4744





 


AJ_P7
85653
C11
AJ_611
/5AmMC6/CCC AAC AGG TGT
10653
8777
61.3
612



  405


ACT TGG TAC TAC GGA A
 4745





 


AJ_P7
85653
C12
AJ_612
/5AmMC6/CCC AAG TAC CAT
10653
8672
59.8
613



  406


CCT TAC GTA GCT TGA A
 4746





 


AJ_P7
85653
D01
AJ_613
/5AmMC6/CCC AAG CTA CTT
10653
8706
60.9
614



  407


CCA CTA GGT ACA GGA A
 4747





 


AJ_P7
85653
D02
AJ_614
/5AmMC6/CCC AAG TAC CTC
10653
8699
60.1
615



  408


AAC AAG TCA AGG CTA A
 4748





 


AJ_P7
85653
D03
AJ_615
/5AmMC6/CCC AAG TAC CCA
10653
8739
59.8
616



  409


AGA GAC TAA GCT TGA A
 4749





 


AJ_P7
85653
D04
AJ_616
/5AmMC6/CCC AAT GAA CCA
10653
8699
61
617



  410


AAC ACT GAC CTG TGA A
 4750





 


AJ_P7
85653
D05
AJ_617
/5AmMC6/CCC AAG TGC ACA
10653
8699
60.8
618



  411


TCG AAC CAA CTT AGA A
 4751





 


AJ_P7
85653
D06
AJ_618
/5AmMC6/CCC AAT GCT TAG
10653
8696
58.2
619



  412


CGT ACT ACC ATT AGA A
 4752





 


AJ_P7
85653
D07
AJ_619
/5AmMC6/CCC AAG TTT GAC
10653
8681
61.2
620



  413


GTT CAA CCA TCA CGA A
 4753





 


AJ_P7
85653
D08
AJ_620
/5AmMC6/CCC AAT TTA GCT
10653
8712
60.2
621



  414


TGT CCA CTC AGA GGA A
 4754





 


AJ_P7
85653
D09
AJ_621
/5AmMC6/CCC AAC GCT ACT
10653
8687
58.4
622



  415


TTC TTA GTT AGA GCA A
 4755





 


AJ_P7
85653
D10
AJ_622
/5AmMC6/CCC AAA AGC CTT
10653
8672
60.9
623



  416


TCC ACT GTT ACT GGA A
 4756





 


AJ_P7
85653
D11
AJ_623
/5AmMC6/CCC AAC CTG TTA
10653
8697
61.9
624



  417


CCT CAG ACA TTG GGA A
 4757





 


AJ_P7
85653
D12
AJ_624
/5AmMC6/CCC AAC GTC ATT
10653
8752
59.6
625



  418


TAG GTC TCT AAG GGA A
 4758





 


AJ_P7
85653
E01
AJ_625
/5AmMC6/CCC AAA CGT CTT
10653
8712
60.2
626



  419


GGG TTA CAC TAC TGA A
 4759





 


AJ_P7
85653
E02
AJ_626
/5AmMC6/CCC AAT CAC AGA
10653
8690
60.8
627



  420


ACC AGT CAG CTT TGA A
 4760





 


AJ_P7
85653
E03
AJ_627
/5AmMC6/CCC AAG TGG TAC
10653
8697
60.9
628



  421


TCT CGT AAC TCC AGA A
 4761





 


AJ_P7
85653
E04
AJ_628
/5AmMC6/CCC AAG AAC TCC
10653
8675
61.2
629



  422


TAC CAA GAC TCG TGA A
 4762





 


AJ_P7
85653
E05
AJ_629
/5AmMC6/CCC AAT TTG ACT
10653
8705
59.7
630



  423


TGA ACG CAT AAC CGA A
 4763





 


AJ_P7
85653
E06
AJ_630
/5AmMC6/CCC AAT TGA GAC
10653
8699
59.8
631



  424


CTC ACG AGA ACA CTA A
 4764





 


AJ_P7
85653
E07
AJ_631
/5AmMC6/CCC AAA CAA AGT
10653
8745
59.8
632



  425


CAT TGG GTT CGC TAA A
 4765





 


AJ_P7
85653
E08
AJ_632
/5AmMC6/CCC AAT CGA ACA
10653
8699
60.4
633



  426


AAC CTA GAG TGC TCA A
 4766





 


AJ_P7
85653
E09
AJ_633
/5AmMC6/CCC AAG GTC TTA
10653
8681
59.8
634



  427


GCT ACA ACC TCA TGA A
 4767





 


AJ_P7
85653
E10
AJ_634
/5AmMC6/CCC AAG CTT TGA
10653
8681
60.7
635



  428


AGC CTT CCA ACT AGA A
 4768





 


AJ_P7
85653
E11
AJ_635
/5AmMC6/CCC AAT ACA GGT
10653
8699
60.5
636



  429


GTC ACA AAC TCA CGA A
 4769





 


AJ_P7
85653
E12
AJ_636
/5AmMC6/CCC AAC CGT TCA
10653
8699
60.4
637



  430


TAA CAA GGG AAC CTA A
 4770





 


AJ_P7
85653
F01
AJ_637
/5AmMC6/CCC AAA GTA CCC
10653
8739
61
638



  431


AAA GCA TGT CTG GAA A
 4771





 


AJ_P7
85653
F02
AJ_638
/5AmMC6/CCC AAA TGT TCT
10653
8727
58.8
639



  432


CTT TAC GCT AGG GAA A
 4772





 


AJ_P7
85653
F03
AJ_639
/5AmMC6/CCC AAT TTG ACT
10653
8745
59.3
640



  433


TCA GAC GAA AGC TGA A
 4773





 


AJ_P7
85653
F04
AJ_640
/5AmMC6/CCC AAT ACA GAA
10653
8723
59
641



  434


ACG ACA TAC GCT TGA A
 4774





 


AJ_P7
85653
F05
AJ_641
/5AmMC6/CCC AAT CAC CAG
10653
8699
60
642



  435


AAG AAC TAC CTG TGA A
 4775





 


AJ_P7
85653
F06
AJ_642
/5AmMC6/CCC AAT ACG AAC
10653
8770
60.2
643



  436


GAC AGG TCA TGG TTA A
 4776





 


AJ_P7
85653
F07
AJ_643
/5AmMC6/CCC AAG AAC TCC
10653
8690
59.9
644



  437


AAC CAT GTA GTC GTA A
 4777





 


AJ_P7
85653
F08
AJ_644
/5AmMC6/CCC AAA TTG CGT
10653
8696
59.6
645



  438


TCT TCA GTA CAC GAA A
 4778





 


AJ_P7
85653
F09
AJ_645
/5AmMC6/CCC AAA TCT GCT
10653
8672
60
646



  439


TCC TGT AGT ACA CGA A
 4779





 


AJ_P7
85653
F10
AJ_646
/5AmMC6/CCC AAG GTC ACT
10653
8675
62.3
647



  440


TGC AAC CTA GAA CCA A
 4780





 


AJ_P7
85653
F11
AJ_647
/5AmMC6/CCC AAG GCT TAG
10653
8715
61.5
648



  441


TAC GAC AGT AAC CCA A
 4781





 


AJ_P7
85653
F12
AJ_648
/5AmMC6/CCC AAC AAG TGA
10653
8795
62
649



  442


AGT GGT CTG ACC AGA A
 4782





 


AJ_P7
85653
G01
AJ_649
/5AmMC6/CCC AAC AGA GTA
10653
8770
59.3
650



  443


GTG TGA CTA GCC TAA A
 4783





 


AJ_P7
85653
G02
AJ_650
/5AmMC6/CCC AAT CAC AAG
10653
8754
59.1
651



  444


GAG TAG CAA CTT TGA A
 4784





 


AJ_P7
85653
G03
AJ_651
/5AmMC6/CCC AAC CTG TAA
10653
8779
60.5
652



  445


GTG AAA CGA CTG GAA A
 4785





 


AJ_P7
85653
G04
AJ_652
/5AmMC6/CCC AAC CCT AGT
10653
8755
61.8
653



  446


TGA GGA CAA ACT GGA A
 4786





 


AJ_P7
85653
G05
AJ_653
/5AmMC6/CCC AAG GCA TCA
10653
8690
60.8
654



  447


CAC CTA GCA AGT TTA A
 4787





 


AJ_P7
85653
G06
AJ_654
/5AmMC6/CCC AAG ACC TAC
10653
8666
60.9
655



  448


CCT ACA GAG CTT GTA A
 4788





 


AJ_P7
85653
G07
AJ_655
/5AmMC6/CCC AAT TTC GTA
10653
8736
59.1
656



  449


ACA AGT TGG ACT CGA A
 4789





 


AJ_P7
85653
G08
AJ_656
/5AmMC6/CCC AAT CAA AGA
10653
8763
59.8
657



  450


AAC AGG TTG CAC TGA A
 4790





 


AJ_P7
85653
G09
AJ_657
/5AmMC6/CCC AAC GTC TTA
10653
8657
61.7
658



  451


GAG TCC TTG AAC CCA A
 4791





 


AJ_P7
85653
G10
AJ_658
/5AmMC6/CCC AAT GCT GAA
10653
8687
59.8
659



  452


ACG TTT CCC TTG TAA A
 4792





 


AJ_P7
85653
G11
AJ_659
/5AmMC6/CCC AAC AGG TTT
10653
8752
60.8
660



  453


GTT TGA CTC AGA CGA A
 4793





 


AJ_P7
85653
G12
AJ_660
/5AmMC6/CCC AAC CTT CGA
10653
8723
59
661



  454


CAT AAA GAA AGC GTA A
 4794





 


AJ_P7
85653
H01
AJ_661
/5AmMC6/CCC AAT GAA CCA
10653
8763
59.4
662



  455


TTA GCA AGC AAG GTA A
 4795





 


AJ_P7
85653
H02
AJ_662
/5AmMC6/CCC AAT GAA CCT
10653
8739
61.3
663



  456


TGA GCA CAA ACT GGA A
 4796





 


AJ_P7
85653
H03
AJ_663
/5AmMC6/CCC AAA GGG TTC
10653
8737
61.8
664



  457


TTG GAC AGT ACC TCA A
 4797





 


AJ_P7
85653
H04
AJ_664
/5AmMC6/CCC AAC TGT AAA
10653
8721
59.5
665



  458


GGA GTT CGT ACC CTA A
 4798





 


AJ_P7
85653
H05
AJ_665
/5AmMC6/CCC AAT CGA GAA
10653
8779
59.8
666



  459


GGA AGT CAC ACT GTA A
 4799





 


AJ_P7
85653
H06
AJ_666
/5AmMC6/CCC AAC TAA AGG
10653
8770
60.4
667



  460


AAG TGT CAG CTG TCA A
 4800





 


AJ_P7
85653
H07
AJ_667
/5AmMC6/CCC AAG CAC ATA
10653
8779
61.1
668



  461


AGG TCA AAC GTG TGA A
 4801





 


AJ_P7
85653
H08
AJ_668
/5AmMC6/CCC AAC GTT GAA
10653
8779
61
669



  462


GGA ACA TTC ACA GGA A
 4802





 


AJ_P7
85653
H09
AJ_669
/5AmMC6/CCC AAT GTG AGC
10653
8763
59.8
670



  463


TGA CAA ACA ACA TGA A
 4803





 


AJ_P7
85653
H10
AJ_670
/5AmMC6/CCC AAG CTA CTC
10653
8675
61.4
671



  464


TAA CAC GAC TGG ACA A
 4804





 


AJ_P7
85653
H11
AJ_671
/5AmMC6/CCC AAG CCT AAC
10653
8681
60.8
672



  465


CTT CAA GTG CAT GTA A
 4805





 


AJ_P7
85653
H12
AJ_672
/5AmMC6/CCC AAG TAA ACA
10653
8730
59.9
673



  466


CCT CTA GGT TCG GAA A
 4806





 


AJ_P8
85653
A01
AJ_673
/5AmMC6/CCC AAG TCT TGA
10653
8681
60
674



  468


CTC TCG ACT CGA AAA A
 4807





 


AJ_P8
85653
A02
AJ_674
/5AmMC6/CCC AAC TGC AGA
10653
8779
61.1
675



  469


GTG GAC TTG ACA AAA A
 4808





 


AJ_P8
85653
A03
AJ_675
/5AmMC6/CCC AAC AGC TCT
10653
8721
60
676



  470


GGT GTA CTT AAG ACA A
 4809





 


AJ_P8
85653
A04
AJ_676
/5AmMC6/CCC AAT ACG AGA
10653
8779
59.6
677



  471


GAG ACG TTT ACG ACA A
 4810





 


AJ_P8
85653
A05
AJ_677
/5AmMC6/CCC AAG TAC CCT
10653
8666
60.9
678



  472


ACT CTC GTC AAG GAA A
 4811





 


AJ_P8
85653
A06
AJ_678
/5AmMC6/CCC AAT AAC GAC
10653
8699
60.5
679



  473


ACA ACT GGT TAC CGA A
 4812





 


AJ_P8
85653
A07
AJ_679
/5AmMC6/CCC AAC ACG TCA
10653
8675
61.5
680



  474


TAA CGG TAG ACC TCA A
 4813





 


AJ_P8
85653
A08
AJ_680
/5AmMC6/CCC AAT CCC AAG
10653
8699
60.2
681



  475


CAA CAG TCA GTA GTA A
 4814





 


AJ_P8
85653
A09
AJ_681
/5AmMC6/CCC AAT AAA CGA
10653
8699
60.8
682



  476


ACA CCT GTG AGC TCA A
 4815





 


AJ_P8
85653
A10
AJ_682
/5AmMC6/CCC AAG TTA CCA
10653
8699
60.2
683



  477


GAC TCA ACA ACG GTA A
 4816





 


AJ_P8
85653
A11
AJ_683
/5AmMC6/CCC AAG TTA GCT
10653
8690
60.8
684



  478


TGA CCA ACC AAC GTA A
 4817





 


AJ_P8
85653
A12
AJ_684
/5AmMC6/CCC AAG ACC ATC
10653
8675
60.7
685



  479


ACT ACA GGA GTC CTA A
 4818





 


AJ_P8
85653
B01
AJ_685
/5AmMC6/CCC AAG TAC TCT
10653
8712
59.3
686



  480


TCT TAC GGT AGC AGA A
 4819





 


AJ_P8
85653
B02
AJ_686
/5AmMC6/CCC AAT TTG CCA
10653
8714
60.4
687



  481


TCG ACA ACG TGA AAA A
 4820





 


AJ_P8
85653
B03
AJ_687
/5AmMC6/CCC AAA GTC TCT
10653
8752
60.2
688



  482


TGG GTA CAA CGT GTA A
 4821





 


AJ_P8
85653
B04
AJ_688
/5AmMC6/CCC AAT GAC CTT
10653
8672
60.2
689



  483


CTC GTT ACA ACG GTA A
 4822





 


AJ_P8
85653
B05
AJ_689
/5AmMC6/CCC AAT ACC GTT
10653
8736
58.6
690



  484


CTG TTA AGA AGC GTA A
 4823





 


AJ_P8
85653
B06
AJ_690
/5AmMC6/CCC AAA GTC CTT
10653
8672
59.6
691



  485


CCT CTA GTT ACG GAA A
 4824





 


AJ_P8
85653
B07
AJ_691
/5AmMC6/CCC AAG CCA TAC
10653
8730
60.6
692



  486


AAC ATT GGA CTG GTA A
 4825





 


AJ_P8
85653
B08
AJ_692
/5AmMC6/CCC AAC CTG AGA
10653
8761
59.8
693



  487


GGT AAG CTT GAC TTA A
 4826





 


AJ_P8
85653
B09
AJ_693
/5AmMC6/CCC AAC ACC TAG
10653
8746
61.2
694



  488


TAG TCG TTG GAC AGA A
 4827





 


AJ_P8
85653
B10
AJ_694
/5AmMC6/CCC AAG TAC ACT
10653
8723
59.6
695



  489


AAA CCG TTG CGA AAA A
 4828





 


AJ_P8
85653
B11
AJ_695
/5AmMC6/CCC AAC CAC TGG
10653
8730
60.8
696



  490


TAC GGA AAG CTT TAA A
 4829





 


AJ_P8
85653
B12
AJ_696
/5AmMC6/CCC AAG ACC ACT
10653
8746
61.2
697



  491


CTT TGA GGA GTA CGA A
 4830





 


AJ_P8
85653
C01
AJ_697
/5AmMC6/CCC AAG ACT GAC
10653
8795
62
698



  492


CTT GGA AAG TAG GCA A
 4831





 


AJ_P8
85653
C02
AJ_698
/5AmMC6/CCC AAC TCA CGT
10653
8739
60
699



  493


TAC GAA ACA GAG GTA A
 4832





 


AJ_P8
85653
C03
AJ_699
/5AmMC6/CCC AAG CGT AAC
10653
8687
59.2
700



  494


GTC ATT TAC TTT CGA A
 4833





 


AJ_P8
85653
C04
AJ_700
/5AmMC6/CCC AAC GAA CGT
10653
8687
59.7
701



  495


GTC ATT TCA CTT TGA A
 4834





 


AJ_P8
85653
C05
AJ_701
/5AmMC6/CCC AAA TCT CTG
10653
8657
62.1
702



  496


GTG TCC ATC CGA ACA A
 4835





 


AJ_P8
85653
C06
AJ_702
/5AmMC6/CCC AAA GCT TTG
10653
8761
61.1
703



  497


GAG TCT GTG ACA ACA A
 4836





 


AJ_P8
85653
C07
AJ_703
/5AmMC6/CCC AAG GGT ACT
10653
8786
61.9
704



  498


AGG CTT GTG ACA ACA A
 4837





 


AJ_P8
85653
C08
AJ_704
/5AmMC6/CCC AAT AGC GAA
10653
8699
60
705



  499


CAC CTA GTT ACG ACA A
 4838





 


AJ_P8
85653
C09
AJ_705
/5AmMC6/CCC AAT CAC GAG
10653
8675
61.7
706



  500


TCC AAG AGT TAC CCA A
 4839





 


AJ_P8
85653
C10
AJ_706
/5AmMC6/CCC AAT GAG AAC
10653
8763
59.1
707



  501


AAA GGC TAA CCG TTA A
 4840





 


AJ_P8
85653
C11
AJ_707
/5AmMC6/CCC AAC TCG TCA
10653
8699
59.9
708



  502


TAG AAC ACC AAG GTA A
 4841





 


AJ_P8
85653
C12
AJ_708
/5AmMC6/CCC AAC TCC ATG
10653
8723
59
709



  503


CAA GTA AAG AAC GTA A
 4842





 


AJ_P8
85653
D01
AJ_709
/5AmMC6/CCC AAA TGT GAC
10653
8705
59.3
710



  504


TAC CGA AAC GCT TTA A
 4843





 


AJ_P8
85653
D02
AJ_710
/5AmMC6/CCC AAG ACA AGT
10653
8699
60.8
711



  505


TGA CCA ACG CAT CTA A
 4844





 


AJ_P8
85653
D03
AJ_711
/5AmMC6/CCC AAC TGC ACA
10653
8690
60.5
712



  506


GTT TAC AAC CTA GGA A
 4845





 


AJ_P8
85653
D04
AJ_712
/5AmMC6/CCC AAG CTG ACT
10653
8672
59.8
713



  507


GTC TTA ACC CTT AGA A
 4846





 


AJ_P8
85653
D05
AJ_713
/5AmMC6/CCC AAG GTC AAG
10653
8739
60
714



  508


TCG ACA AGC TAA CTA A
 4847





 


AJ_P8
85653
D06
AJ_714
/5AmMC6/CCC AAC ACG TGA
10653
8675
62
715



  509


GTT CCA ACC CTA AGA A
 4848





 


AJ_P8
85653
D07
AJ_715
/5AmMC6/CCC AAG CCA TAA
10653
8690
59.9
716



  510


CCA TCA GTC TGA GTA A
 4849





 


AJ_P8
85653
D08
AJ_716
/5AmMC6/CCC AAG TCA ACA
10653
8699
60
717



  511


CAC TCA GCA GTA GTA A
 4850





 


AJ_P8
85653
D09
AJ_717
/5AmMC6/CCC AAG TAC CTA
10653
8672
60
718



  512


CTC ATG CTT GCA GTA A
 4851





 


AJ_P8
85653
D10
AJ_718
/5AmMC6/CCC AAA TGT ACG
10653
8723
59.2
719



  513


TAA AGC ACA AGC CTA A
 4852





 


AJ_P8
85653
D11
AJ_719
/5AmMC6/CCC AAC GTG TAA
10653
8779
60
720



  514


AGG AAC TAG GCT ACA A
 4853





 


AJ_P8
85653
D12
AJ_720
/5AmMC6/CCC AAG GTC ACT
10653
8675
61.4
721



  515


AAC TCA GGA ACT CCA A
 4854





 


AJ_P8
85653
E01
AJ_721
/5AmMC6/CCC AAT TCG AAG
10653
8723
59.4
722



  516


TAA GCA ACA CCA TGA A
 4855





 


AJ_P8
85653
E02
AJ_722
/5AmMC6/CCC AAT CGG AAG
10653
8779
60.6
723



  517


TGT AAA CTG GAC ACA A
 4856





 


AJ_P8
85653
E03
AJ_723
/5AmMC6/CCC AAG ACT CAC
10653
8690
60.2
724



  518


AAA CCG TAC TTG GTA A
 4857





 


AJ_P8
85653
E04
AJ_724
/5AmMC6/CCC AAC ATT CTG
10653
8770
60.2
725



  519


CAT AGG AGA CAG TGA A
 4858





 


AJ_P8
85653
E05
AJ_725
/5AmMC6/CCC AAA CCC ATG
10653
8699
61.3
726



  520


CAC ATT GAG AAC TGA A
 4859





 


AJ_P8
85653
E06
AJ_726
/5AmMC6/CCC AAT GGT CAG
10653
8739
59.7
727



  521


GAC TAA ACT ACC AGA A
 4860





 


AJ_P8
85653
E07
AJ_727
/5AmMC6/CCC AAG CTT CCA
10653
8712
60.6
728



  522


GAA CTT TAC TTG GGA A
 4861





 


AJ_P8
85653
E08
AJ_728
/5AmMC6/CCC AAG TTC AAC
10653
8675
62.3
729



  523


TCC AAC GTC AGG ACA A
 4862





 


AJ_P8
85653
E09
AJ_729
/5AmMC6/CCC AAG TTA CTA
10653
8681
59.4
730



  524


CCA TAC GAC TCG TGA A
 4863





 


AJ_P8
85653
E10
AJ_730
/5AmMC6/CCC AAC AGA CAT
10653
8699
60.5
731



  525


GCA CTT AAC TCA GGA A
 4864





 


AJ_P8
85653
E11
AJ_731
/5AmMC6/CCC AAC TTG AAC
10653
8779
60.2
732



  526


CTA GAA AGG GTA GCA A
 4865





 


AJ_P8
85653
E12
AJ_732
/5AmMC6/CCC AAG TCC TAC
10653
8730
58.8
733



  527


CTT AAG AGA CGA GTA A
 4866





 


AJ_P8
85653
F01
AJ_733
/5AmMC6/CCC AAC AGT TAG
10653
8761
61.1
734



  528


GGA AGC TTT GCA TCA A
 4867





 


AJ_P8
85653
F02
AJ_734
/5AmMC6/CCC AAC GTC TAG
10653
8745
58.2
735



  529


CTA GAA GAA GTT TCA A
 4868





 


AJ_P8
85653
F03
AJ_735
/5AmMC6/CCC AAT TTA GTC
10653
8672
60
736



  530


ACC TCT GGA ACC GTA A
 4869





 


AJ_P8
85653
F04
AJ_736
/5AmMC6/CCC AAC AGT GAA
10653
8730
60.9
737



  531


GGA ACC TTT CGT CAA A
 4870





 


AJ_P8
85653
F05
AJ_737
/5AmMC6/CCC AAA GGC TTC
10653
8687
59.1
738



  532


CTT TCA GAC AGT TTA A
 4871





 


AJ_P8
85653
F06
AJ_738
/5AmMC6/CCC AAA CGG TTG
10653
8761
60.9
739



  533


TTG AGT CGA ACC ATA A
 4872





 


AJ_P8
85653
F07
AJ_739
/5AmMC6/CCC AAA CCT CTG
10653
8730
60.5
740



  534


AGT TGG CTA AAC AGA A
 4873





 


AJ_P8
85653
F08
AJ_740
/5AmMC6/CCC AAG CAG TTG
10653
8779
60.3
741



  535


TAA GAC CAA GAC GTA A
 4874





 


AJ_P8
85653
F09
AJ_741
/5AmMC6/CCC AAG AGA GCT
10653
8727
58.6
742



  536


ACC GTT TCT TTG TAA A
 4875





 


AJ_P8
85653
F10
AJ_742
/5AmMC6/CCC AAA GGG TTC
10653
8761
60.4
743



  537


TCC AAG TTT ACA GGA A
 4876





 


AJ_P8
85653
F11
AJ_743
/5AmMC6/CCC AAC GTT AGT
10653
8727
59.6
744



  538


GTG TTC AAG CTT CAA A
 4877





 


AJ_P8
85653
F12
AJ_744
/5AmMC6/CCC AAC TCA CTG
10653
8739
60.8
745



  539


CAA AGG TAA AGG TCA A
 4878





 


AJ_P8
85653
G01
AJ_745
/5AmMC6/CCC AAG AGC TCA
10653
8786
61.9
746



  540


CAA GGT GTT AGG TCA A
 4879





 


AJ_P8
85653
G02
AJ_746
/5AmMC6/CCC AAC TGT CTA
10653
8752
60.4
747



  541


CTG AAG GAG TTT GCA A
 4880





 


AJ_P8
85653
G03
AJ_747
/5AmMC6/CCC AAA GCT TCC
10653
8687
58.3
748



  542


TTT ACT GAC TAG TGA A
 4881





 


AJ_P8
85653
G04
AJ_748
/5AmMC6/CCC AAC TGC TAC
10653
8681
60.1
749



  543


CCT TGA GTA AAG TCA A
 4882





 


AJ_P8
85653
G05
AJ_749
/5AmMC6/CCC AAG CTC ATT
10653
8681
60.2
750



  544


CCC TTG AAC AGA GTA A
 4883





 


AJ_P8
85653
G06
AJ_750
/5AmMC6/CCC AAG AGA CTG
10653
8715
61.9
751



  545


TGC ACA ACC CTT AGA A
 4884





 


AJ_P8
85653
G07
AJ_751
/5AmMC6/CCC AAC GGT TAA
10653
8714
59.4
752



  546


CCT CAA GTG CTA AAA A
 4885





 


AJ_P8
85653
G08
AJ_752
/5AmMC6/CCC AAA CCC TTG
10653
8755
61.7
753



  547


GGT AAG CTA GAG ACA A
 4886





 


AJ_P8
85653
G09
AJ_753
/5AmMC6/CCC AAA TTG CTC
10653
8672
61.2
754



  548


ACG TTC TCA TGG ACA A
 4887





 


AJ_P8
85653
G10
AJ_754
/5AmMC6/CCC AAC CCT AGG
10653
8690
60.3
755



  549


AAG CCA TCA GTT TAA A
 4888





 


AJ_P8
85653
G11
AJ_755
/5AmMC6/CCC AAA CCG TTT
10653
8672
61.3
756



  550


GAA CCT TCT GGT CAA A
 4889





 


AJ_P8
85653
G12
AJ_756
/5AmMC6/CCC AAT CCG AAG
10653
8739
60.7
757



  551


GAG AAC TTT GAC CAA A
 4890





 


AJ_P8
85653
H01
AJ_757
/5AmMC6/CCC AAT TGA GTC
10653
8754
59.1
758



  552


TGA AGC AAC CAA GTA A
 4891





 


AJ_P8
85653
H02
AJ_758
/5AmMC6/CCC AAC TGT TTA
10653
8727
58.7
759



  553


GAG TGA CAT TGC CTA A
 4892





 


AJ_P8
85653
H03
AJ_759
/5AmMC6/CCC AAT ACT GTT
10653
8705
58.5
760



  554


AAG GCT ACA ACG CTA A
 4893





 


AJ_P8
85653
H04
AJ_760
/5AmMC6/CCC AAA TCG GTT
10653
8672
60.1
761



  555


CGT TCA CTA CTC AGA A
 4894





 


AJ_P8
85653
H05
AJ_761
/5AmMC6/CCC AAC CAA GGT
10653
8737
62
762



  556


TGG CTT AGT AGT CCA A
 4895





 


AJ_P8
85653
H06
AJ_762
/5AmMC6/CCC AAG GCT ACA
10653
8672
60.6
763



  557


GAC TTT CCC ATT TGA A
 4896





 


AJ_P8
85653
H07
AJ_763
/5AmMC6/CCC AAG AAC CTC
10653
8712
61
764



  558


ACG TGT GCT TGT TAA A
 4897





 


AJ_P8
85653
H08
AJ_764
/5AmMC6/CCC AAG ACA TCC
10653
8672
60.5
765



  559


ACT CTT GTT TGA CGA A
 4898





 


AJ_P8
85653
H09
AJ_765
/5AmMC6/CCC AAG GTA CAC
10653
8657
62.2
766



  560


ACC TTT GCC TTA CGA A
 4899





 


AJ_P8
85653
H10
AJ_766
/5AmMC6/CCC AAC GAG TTG
10653
8779
60.1
767



  561


GAG TAA CAT ACG ACA A
 4900





 


AJ_P8
85653
H11
AJ_767
/5AmMC6/CCC AAA CGG TTG
10653
8761
60.3
768



  562


TGG TAA CAT CCT AGA A
 4901





 


AJ_P8
85653
H12
AJ_768
/5AmMC6/CCC AAG ACC TTG
10658
8795
61.7
769



  563


ACT GGA GAA ACG GTA A
 6462





 


AJ_P9
85653
A01
AJ_769
/5AmMC6/CCC AAG CTC ACT
10653
8672
60.6
770



  565


ACC ATT GTC ATT GGA A
 4903





 


AJ_P9
85653
A02
AJ_770
/5AmMC6/CCC AAT CCG TTA
10653
8770
60.6
771



  566


CGT GAA GGG TAA ACA A
 4904





 


AJ_P9
85653
A03
AJ_771
/5AmMC6/CCC AAT ACA GAC
10653
8699
60.1
772



  567


TGC ACA CTC AGG TAA A
 4905





 


AJ_P9
85653
A04
AJ_772
/5AmMC6/CCC AAT TTA CGT
10653
8696
59.3
773



  568


AGT CCA ACT TGC GAA A
 4906





 


AJ_P9
85653
A05
AJ_773
/5AmMC6/CCC AAG ACC TTA
10653
8690
59.4
774



  569


CTA CCT GAA GCA GTA A
 4907





 


AJ_P9
85653
A06
AJ_774
/5AmMC6/CCC AAC ATT GTT
10653
8687
59.4
775



  570


TCT CTG ACA AGC TGA A
 4908





 


AJ_P9
85653
A07
AJ_775
/5AmMC6/CCC AAC AGC AGT
10653
8739
61
776



  571


TTA GCC AAG AAG TCA A
 4909





 


AJ_P9
85653
A08
AJ_776
/5AmMC6/CCC AAG ACC TTG
10653
8657
60.9
777



  572


GAC TCT CTC TAA CGA A
 4910





 


AJ_P9
85653
A09
AJ_777
/5AmMC6/CCC AAG TAC TTT
10653
8672
60.1
778



  573


CTT CCA GTC AGA GCA A
 4911





 


AJ_P9
85653
A10
AJ_778
/5AmMC6/CCC AAT CAG ACA
10653
8681
60.7
779



  574


ACC TTG TTC ATC GGA A
 4912





 


AJ_P9
85653
A11
AJ_779
/5AmMC6/CCC AAT CAC CTG
10653
8712
60.7
780



  575


TTG CAT TCA TAG GGA A
 4913





 


AJ_P9
85653
A12
AJ_780
/5AmMC6/CCC AAT TTG CAG
10653
8714
59.8
781



  576


TGA ACA CCA ACA GTA A
 4914





 


AJ_P9
85653
B01
AJ_781
/5AmMC6/CCC AAG TCT GCA
10653
8699
60.4
782



  577


GTA ACA CAC CAA GTA A
 4915





 


AJ_P9
85653
B02
AJ_782
/5AmMC6/CCC AAT GTC TCA
10653
8672
59.7
783



  578


GTC TCC ACA TTA GGA A
 4916





 


AJ_P9
85653
B03
AJ_783
/5AmMC6/CCC AAG TAC ACC
10653
8672
61.2
784



  579


ATT TCG CAT TTC GGA A
 4917





 


AJ_P9
85653
B04
AJ_784
/5AmMC6/CCC AAG CTA CCA
10653
8730
60
785



  580


CTT TAG AAG TAG GCA A
 4918





 


AJ_P9
85653
B05
AJ_785
/5AmMC6/CCC AAT CAC AAG
10653
8739
60
786



  581


GTT ACC ACA GGA GTA A
 4919





 


AJ_P9
85653
B06
AJ_786
/5AmMC6/CCC AAC ACC ATG
10653
8715
61.9
787



  582


GAC ACT TCT AAG GGA A
 4920





 


AJ_P9
85653
B07
AJ_787
/5AmMC6/CCC AAC CTG AAA
10653
8736
59.3
788



  583


GAG TTT CTT GCG TAA A
 4921





 


AJ_P9
85653
B08
AJ_788
/5AmMC6/CCC AAG AGA CGT
10653
8706
61.3
789



  584


GTC ATC TCA TCC AGA A
 4922





 


AJ_P9
85653
B09
AJ_789
/5AmMC6/CCC AAT AGC GTA
10653
8723
59.2
790



  585


GAC AAC TTC AAA GCA A
 4923





 


AJ_P9
85653
B10
AJ_790
/5AmMC6/CCC AAA GTT CTC
10653
8687
58.6
791



  586


TCG TTC ATA GCT GAA A
 4924





 


AJ_P9
85653
B11
AJ_791
/5AmMC6/CCC AAA TTG GTC
10653
8736
59.7
792



  587


TTC TGC ATA AAG CGA A
 4925





 


AJ_P9
85653
B12
AJ_792
/5AmMC6/CCC AAT CGA AGG
10653
8761
58.8
793



  588


AGT AGT CTA CCT GTA A
 4926





 


AJ_P9
85653
C01
AJ_793
/5AmMC6/CCC AAT CAG GAC
10653
8715
61.8
794



  589


TAC GGA AAG TTC CCA A
 4927





 


AJ_P9
85653
C02
AJ_794
/5AmMC6/CCC AAC CGT AAC
10653
8675
62
795



  590


ATC CAT GAG ACG TCA A
 4928





 


AJ_P9
85653
C03
AJ_795
/5AmMC6/CCC AAT GCG AAA
10653
8770
60.8
796



  591


GAG GTA CCG TTT ACA A
 4929





 


AJ_P9
85653
C04
AJ_796
/5AmMC6/CCC AAG ACA CAT
10653
8675
62.1
797



  592


CCA ACT GGT GAC TCA A
 4930





 


AJ_P9
85653
C05
AJ_797
/5AmMC6/CCC AAG ACC ATC
10653
8675
61.7
798



  593


CTT CAA GAG ACG TCA A
 4931





 


AJ_P9
85653
C06
AJ_798
/5AmMC6/CCC AAG CTC TCA
10653
8690
60.6
799



  594


AGT CTA AAC AGT GCA A
 4932





 


AJ_P9
85653
C07
AJ_799
/5AmMC6/CCC AAC AAA GTA
10653
8763
58.6
800



  595


GAA ACT CGT AGC TGA A
 4933





 


AJ_P9
85653
C08
AJ_800
/5AmMC6/CCC AAC CAG AGT
10653
8795
61.3
801



  596


GTG AAC ACT AGG GTA A
 4934





 


AJ_P9
85653
C09
AJ_801
/5AmMC6/CCC AAC CTC ATG
10653
8715
61.5
802



  597


AAG ACT CCA AGG GTA A
 4935





 


AJ_P9
85653
C10
AJ_802
/5AmMC6/CCC AAA CCT GTG
10653
8666
62.1
803



  598


GAC ACT ACA CCT TGA A
 4936





 


AJ_P9
85653
C11
AJ_803
/5AmMC6/CCC AAA GTT CAG
10653
8672
59.9
804



  599


AGT TCT CTC CAC TGA A
 4937





 


AJ_P9
85653
C12
AJ_804
/5AmMC6/CCC AAG CTA CTT
10653
8721
60.4
805



  600


TCA ACT GAC AGT GGA A
 4938





 


AJ_P9
85653
D01
AJ_805
/5AmMC6/CCC AAG CCA TCT
10653
8681
60.1
806



  601


TCT ACT GAA CGG TAA A
 4939





 


AJ_P9
85653
D02
AJ_806
/5AmMC6/CCC AAT GTT TCA
10653
8687
59.4
807



  602


GTC CAT TGA ACG CTA A
 4940





 


AJ_P9
85653
D03
AJ_807
/5AmMC6/CCC AAA TTG CTT
10653
8687
59.1
808



  603


CTC ACG TCA TTA GGA A
 4941





 


AJ_P9
85653
D04
AJ_808
/5AmMC6/CCC AAT GGG AAC
10653
8739
60.8
809



  604


TCT GAA ACA TCC GAA A
 4942





 


AJ_P9
85653
D05
AJ_809
/5AmMC6/CCC AAT CGT AGA
10653
8723
58.4
810



  605


GTC AAA CCA CAA GTA A
 4943





 


AJ_P9
85653
D06
AJ_810
/5AmMC6/CCC AAC AGG TGT
10653
8786
62.5
811



  606


CGT GTG AAA CAG TCA A
 4944





 


AJ_P9
85653
D07
AJ_811
/5AmMC6/CCC AAG GTC ATT
10653
8657
62
812



  607


AAG CCT TCG ACT CCA A
 4945





 


AJ_P9
85653
D08
AJ_812
/5AmMC6/CCC AAC TTG AAG
10653
8779
61.3
813



  608


TGA AGG CAA CCA TGA A
 4946





 


AJ_P9
85653
D09
AJ_813
/5AmMC6/CCC AAC AAC TAG
10653
8761
60.1
814



  609


GAG TGC TCT GGT TAA A
 4947





 


AJ_P9
85653
D10
AJ_814
/5AmMC6/CCC AAG ACC ATA
10653
8675
61.9
815



  610


GCA TCC AAG TCG TCA A
 4948





 


AJ_P9
85653
D11
AJ_815
/5AmMC6/CCC AAT CGA GAA
10653
8723
58.4
816



  611


ACA CCT GTA CAA GTA A
 4949





 


AJ_P9
85653
D12
AJ_816
/5AmMC6/CCC AAC AGT CTT
10653
8754
58.3
817



  612


TAA GCA GAA GGA CTA A
 4950





 


AJ_P9
85653
E01
AJ_817
/5AmMC6/CCC AAC GTC AAC
10653
8699
59.8
818



  613


TAC ACA GAA GGT CTA A
 4951





 


AJ_P9
85653
E02
AJ_818
/5AmMC6/CCC AAG TCG ACA
10653
8730
60
819



  614


ACA GCA TTA GGT CTA A
 4952





 


AJ_P9
85653
E03
AJ_819
/5AmMC6/CCC AAT TGG TCA
10653
8687
59.9
820



  615


GAA CTT TCC TTG CAA A
 4953





 


AJ_P9
85653
E04
AJ_820
/5AmMC6/CCC AAC CTA GGT
10653
8752
60.5
821



  616


CAA GTT TAG GTT GCA A
 4954





 


AJ_P9
85653
E05
AJ_821
/5AmMC6/CCC AAG TCA TCT
10653
8666
61.6
822



  617


GCA TCC ACA CTA GGA A
 4955





 


AJ_P9
85653
E06
AJ_822
/5AmMC6/CCC AAA TCG CTT
10653
8690
61
823



  618


GAA CCA TAC CAT GGA A
 4956





 


AJ_P9
85653
E07
AJ_823
/5AmMC6/CCC AAA TCT GAA
10653
8763
58.8
824



  619


CTG AGG AAC AAG CTA A
 4957





 


AJ_P9
85653
E08
AJ_824
/5AmMC6/CCC AAC GTG AGC
10653
8770
61.2
825



  620


ATC AGG AAC ATT TGA A
 4958





 


AJ_P9
85653
E09
AJ_825
/5AmMC6/CCC AAT CCC TAG
10653
8697
61.2
826



  621


TTC CAG TCA TGA GGA A
 4959





 


AJ_P9
85653
E10
AJ_826
/5AmMC6/CCC AAC TCC TAG
10653
8657
60.7
827



  622


TCC TGT AGT CCA GAA A
 4960





 


AJ_P9
85653
E11
AJ_827
/5AmMC6/CCC AAG AGT CAA
10653
8699
60.2
828



  623


CTC CAT GAA AGC CTA A
 4961





 


AJ_P9
85653
E12
AJ_828
/5AmMC6/CCC AAG GTA GTC
10653
8755
61
829



  624


TCA GAG AAC ACC TGA A
 4962





 


AJ_P9
85653
F01
AJ_829
/5AmMC6/CCC AAG CTG TAG
10653
8779
59.8
830



  625


GAC ATA AGA ACC GTA A
 4963





 


AJ_P9
85653
F02
AJ_830
/5AmMC6/CCC AAG TCC AAC
10653
8739
60.4
831



  626


TGA AAC AGA GCT GTA A
 4964





 


AJ_P9
85653
F03
AJ_831
/5AmMC6/CCC AAG TGC AAC
10653
8795
62.1
832



  627


TAC AGG ACA GTG TGA A
 4965





 


AJ_P9
85653
F04
AJ_832
/5AmMC6/CCC AAT GAA ACA
10653
8763
59.3
833



  628


GAC AAG TAG CGT TCA A
 4966





 


AJ_P9
85653
F05
AJ_833
/5AmMC6/CCC AAA AAC TGT
10653
8696
59.5
834



  629


AGC TTT CCC TTG GAA A
 4967





 


AJ_P9
85653
F06
AJ_834
/5AmMC6/CCC AAT CCG TAG
10653
8761
60
835



  630


AGC AGT GAG TTT ACA A
 4968





 


AJ_P9
85653
F07
AJ_835
/5AmMC6/CCC AAG GTT CAT
10653
8672
60.6
836



  631


GCA TCC TCT TCA AGA A
 4969





 


AJ_P9
85653
F08
AJ_836
/5AmMC6/CCC AAA CCT TTG
10653
8761
61.3
837



  632


TGG AGT CAA GCA TGA A
 4970





 


AJ_P9
85653
F09
AJ_837
/5AmMC6/CCC AAA CCT TTG
10653
8736
59.7
838



  633


TGA GCA GAG CAT TTA A
 4971





 


AJ_P9
85653
F10
AJ_838
/5AmMC6/CCC AAA CTG TTT
10653
8672
60.5
839



  634


CCC TTA GAG CAG TCA A
 4972





 


AJ_P9
85653
F11
AJ_839
/5AmMC6/CCC AAG CTG TAG
10653
8752
59.4
840



  635


GAG TTA CAT CTC TGA A
 4973





 


AJ_P9
85653
F12
AJ_840
/5AmMC6/CCC AAG TGG ACA
10653
8706
61.3
841



  636


CTC CAG AAC TCT GTA A
 4974





 


AJ_P9
85653
G01
AJ_841
/5AmMC6/CCC AAC GTC ATC
10653
8699
59.8
842



  637


TGA CAG AAC AGA CTA A
 4975





 


AJ_P9
85653
G02
AJ_842
/5AmMC6/CCC AAG TCC AAC
10653
8699
60.8
843



  638


GAA GCA TGA CAC TTA A
 4976





 


AJ_P9
85653
G03
AJ_843
/5AmMC6/CCC AAA GCC TAA
10653
8721
61.2
844



  639


AGC CTT TGG GTT ACA A
 4977





 


AJ_P9
85653
G04
AJ_844
/5AmMC6/CCC AAC CGT TCA
10653
8699
60.4
845



  640


AAC GAC TAA GAG TCA A
 4978





 


AJ_P9
85653
G05
AJ_845
/5AmMC6/CCC AAT CGG AAC
10653
8672
61.5
846



  641


ACC TTT GGT TTC CAA A
 4979





 


AJ_P9
85653
G06
AJ_846
/5AmMC6/CCC AAT GAC CAT
10653
8687
60
847



  642


CAT GTT TGG CTT CAA A
 4980





 


AJ_P9
85653
G07
AJ_847
/5AmMC6/CCC AAG ACC ATG
10653
8672
60.5
848



  643


AGC TCT CTT GTT CAA A
 4981





 


AJ_P9
85653
G08
AJ_848
/5AmMC6/CCC AAC TAG GTG
10653
8755
61.9
849



  644


AAG TGA CAG CAT CCA A
 4982





 


AJ_P9
85653
G09
AJ_849
/5AmMC6/CCC AAC AAG TTA
10653
8779
60.4
850



  645


GGA GAC TGA CTG CAA A
 4983





 


AJ_P9
85653
G10
AJ_850
/5AmMC6/CCC AAT CAG CAC
10653
8714
58.6
851



  646


ACG AGT TCT AGT AAA A
 4984





 


AJ_P9
85653
G11
AJ_851
/5AmMC6/CCC AAA CGT CAC
10653
8737
62.1
852



  647


CTA GGT TGG GTT ACA A
 4985





 


AJ_P9
85653
G12
AJ_852
/5AmMC6/CCC AAA CCT TGT
10653
8672
60.6
853



  648


CTC TTA GCC ATG GAA A
 4986





 


AJ_P9
85653
H01
AJ_853
/5AmMC6/CCC AAA CCT TGT
10653
8712
60.6
854



  649


TAC TGT GCT AGA GCA A
 4987





 


AJ_P9
85653
H02
AJ_854
/5AmMC6/CCC AAA CAG AGT
10653
8681
60.8
855



  650


GCT TCC AAC TTC TGA A
 4988





 


AJ_P9
85653
H03
AJ_855
/5AmMC6/CCC AAT CGT TCA
10653
8761
60.2
856



  651


CGA AGT AGG GTT ACA A
 4989





 


AJ_P9
85653
H04
AJ_856
/5AmMC6/CCC AAA AAC ATG
10653
8705
60.1
857



  652


TTC CGT AGT TGC CAA A
 4990





 


AJ_P9
85653
H05
AJ_857
/5AmMC6/CCC AAT GAC CAC
10653
8699
60.9
858



  653


AAC ATA GCA TGT CGA A
 4991





 


AJ_P9
85653
H06
AJ_858
/5AmMC6/CCC AAG CAT AAA
10653
8739
60.2
859



  654


CAC TCT GGA CAG GTA A
 4992





 


AJ_P9
85653
H07
AJ_859
/5AmMC6/CCC AAG CTA ACA
10653
8699
59.7
860



  655


ACC ATC GAG AGT CTA A
 4993





 


AJ_P9
85653
H08
AJ_860
/5AmMC6/CCC AAG TGA AAC
10653
8699
59.7
861



  656


TCA CAC GAG ACT CTA A
 4994





 


AJ_P9
85653
H09
AJ_861
/5AmMC6/CCC AAG TAA CAA
10653
8739
60.9
862



  657


ACC CAT GAG CTG TGA A
 4995





 


AJ_P9
85653
H10
AJ_862
/5AmMC6/CCC AAG TCG ACA
10653
8755
61.9
863



  658


TCA CAG TCA AGG TGA A
 4996





 


AJ_P9
85653
H11
AJ_863
/5AmMC6/CCC AAG AAC TCT
10653
8672
60.4
864



  659


CTC TGC ACA TTG TGA A
 4997





 


AJ_P9
85653
H12
AJ_864
/5AmMC6/CCC AAC TGC ACA
10653
8687
60.1
865



  660


CAT GGT TTC TTT GAA A
 4998





 


AJ_P10
85653
A01
AJ_865
/5AmMC6/CCC AAT AAA GCA
10653
8754
58.7
866



  662


CTT TGA GAG TAC CGA A
 4999





 


AJ_P10
85653
A02
AJ_866
/5AmMC6/CCC AAA TCG CTT
10653
8687
59.1
867



  663


GTT TAA CCT ACT GGA A
 5000





 


AJ_P10
85653
A03
AJ_867
/5AmMC6/CCC AAC GTT GAG
10653
8745
59
868



  664


TTT AAG CTA CCA GAA A
 5001





 


AJ_P10
85653
A04
AJ_868
/5AmMC6/CCC AAG TTT CAC
10653
8681
60
869



  665


TAC ACG ACT TCG AGA A
 5002





 


AJ_P10
85653
A05
AJ_869
/5AmMC6/CCC AAT GGA GAC
10653
8712
60.4
870



  666


AGT CTT CCC TTT GAA A
 5003





 


AJ_P10
85653
A06
AJ_870
/5AmMC6/CCC AAG TTT CAC
10653
8712
61.1
871



  667


TGC ACT TCA AGG TGA A
 5004





 


AJ_P10
85653
A07
AJ_871
/5AmMC6/CCC AAC CAG TCT
10653
8657
61.2
872



  668


GGT TCT ACT ACA CGA A
 5005





 


AJ_P10
85653
A08
AJ_872
/5AmMC6/CCC AAA TTC TCG
10653
8712
59.9
873



  669


TTC TCA GAG TCA GGA A
 5006





 


AJ_P10
85653
A09
AJ_873
/5AmMC6/CCC AAG TTA CCA
10653
8699
60.1
874



  670


ACA CCT GAG AAG CTA A
 5007





 


AJ_P10
85653
A10
AJ_874
/5AmMC6/CCC AAA CTA CTG
10653
8779
60.1
875



  671


TCA AAG GAG TAG GCA A
 5008





 


AJ_P10
85653
A11
AJ_875
/5AmMC6/CCC AAG TTC CCA
10653
8675
62
876



  672


AGA CCT ACA AGC TGA A
 5009





 


AJ_P10
85653
A12
AJ_876
/5AmMC6/CCC AAT TTA GCC
10653
8714
59
877



  673


TAA CAG CAA CAG GTA A
 5010





 


AJ_P10
85653
B01
AJ_877
/5AmMC6/CCC AAA TCT GTT
10653
8687
59
878



  674


CTC TGC AAA GTC GTA A
 5011





 


AJ_P10
85653
B02
AJ_878
/5AmMC6/CCC AAA GTC CTT
10653
8681
60.3
879



  675


GTC TCA AAC TCA GGA A
 5012





 


AJ_P10
85653
B03
AJ_879
/5AmMC6/CCC AAA TCT TGT
10653
8736
59.3
880



  676


GTG TCG AAG CAA CTA A
 5013





 


AJ_P10
85653
B04
AJ_880
/5AmMC6/CCC AAG TGC AAC
10653
8770
60.4
881



  677


TGG AGA CAG ACT TTA A
 5014





 


AJ_P10
85653
B05
AJ_881
/5AmMC6/CCC AAA CTG TCT
10653
8696
59.3
882



  678


TGT TCG AAC AGC ATA A
 5015





 


AJ_P10
85653
B06
AJ_882
/5AmMC6/CCC AAT TTG TAC
10653
8687
59.4
883



  679


ATC GCT TCA TCG GAA A
 5016





 


AJ_P10
85653
B07
AJ_883
/5AmMC6/CCC AAT ACA GAA
10653
8739
58.9
884



  680


GGA GTA CCT GAC CTA A
 5017





 


AJ_P10
85653
B08
AJ_884
/5AmMC6/CCC AAT CGC AAA
10653
8714
59.4
885



  681


GAA GTA CCA GTT TCA A
 5018





 


AJ_P10
85653
B09
AJ_885
/5AmMC6/CCC AAC TGG TAG
10653
8779
60.4
886



  682


ACA TGC ATA GAA GCA A
 5019





 


AJ_P10
85653
B10
AJ_886
/5AmMC6/CCC AAG AGA ACT
10653
8795
62.2
887



  683


ACC GTT GTG AAG GCA A
 5020





 


AJ_P10
85653
B11
AJ_887
/5AmMC6/CCC AAT TTC GAG
10653
8714
58.8
888



  684


AGT CAC ATC AAC AGA A
 5021





 


AJ_P10
85653
B12
AJ_888
/5AmMC6/CCC AAC GGT AAG
10653
8712
60.1
889



  685


GCT ACC TCT TTG TAA A
 5022





 


AJ_P10
85653
C01
AJ_889
/5AmMC6/CCC AAC TAC GCT
10653
8723
58.5
890



  686


ACT AAA GTA AAG GCA A
 5023





 


AJ_P10
85653
C02
AJ_890
/5AmMC6/CCC AAC GTG AGT
10653
8721
60
891



  687


TCG TTA ACT ACC AGA A
 5024





 


AJ_P10
85653
C03
AJ_891
/5AmMC6/CCC AAT GGT CTA
10653
8681
60.2
892



  688


GCA TTC AAC TAC CGA A
 5025





 


AJ_P10
85653
C04
AJ_892
/5AmMC6/CCC AAT GTT TCA
10653
8672
60
893



  689


GAC CTG ACT ACC TGA A
 5026





 


AJ_P10
85653
C05
AJ_893
/5AmMC6/CCC AAT AAC AGA
10657
8699
60.3
894



  690


ACC CAT GCT CAG GTA A
 7190





 


AJ_P10
85653
C06
AJ_894
/5AmMC6/CCC AAA CAC GTT
10653
8687
60
895



  691


GCA CTT TAC TTT GGA A
 5028





 


AJ_P10
85653
C07
AJ_895
/5AmMC6/CCC AAT GCT GAC
10653
8723
59.3
896



  692


GTA CAC AAA CAA GTA A
 5029





 


AJ_P10
85653
C08
AJ_896
/5AmMC6/CCC AAA GCT GTT
10653
8736
59.9
897



  693


GCT GTT AAA CCG TAA A
 5030





 


AJ_P10
85653
C09
AJ_897
/5AmMC6/CCC AAC ATG TTG
10653
8761
60.8
898



  694


TGG TAG CTA CCG AAA A
 5031





 


AJ_P10
85653
C10
AJ_898
/5AmMC6/CCC AAA TCT CTG
10653
8761
60.2
899



  695


TGG TAG CAT AAC GGA A
 5032





 


AJ_P10
85653
C11
AJ_899
/5AmMC6/CCC AAG AGC TCT
10653
8736
57.8
900



  696


CGT GTT ACT AAA GTA A
 5033





 


AJ_P10
85653
C12
AJ_900
/5AmMC6/CCC AAA GCC TTG
10653
8727
59.5
901



  697


GTT GTC AGT CTT AAA A
 5034





 


AJ_P10
85653
D01
AJ_901
/5AmMC6/CCC AAG TAC CTC
10653
8657
60.2
902



  698


TAC TCT GAC TCA GGA A
 5035





 


AJ_P10
85653
D02
AJ_902
/5AmMC6/CCC AAG GCA TAC
10653
8666
61.9
903



  699


AAC TCT GAC CTG TCA A
 5036





 


AJ_P10
85653
D03
AJ_903
/5AmMC6/CCC AAC CAG TAA
10653
8675
62.6
904



  700


ACC AGT GAC TTG CCA A
 5037





 


AJ_P10
85653
D04
AJ_904
/5AmMC6/CCC AAG ACT CCT
10653
8721
60.6
905



  701


TGG TTC AAC GGT AAA A
 5038





 


AJ_P10
85653
D05
AJ_905
/5AmMC6/CCC AAC TTA GGT
10653
8770
59.7
906



  702


AGG TAG CAC ACT GAA A
 5039





 


AJ_P10
85653
D06
AJ_906
/5AmMC6/CCC AAA GTC CAG
10653
8714
58.8
907



  703


AGC ACA TTT CAT AGA A
 5040





 


AJ_P10
85653
D07
AJ_907
/5AmMC6/CCC AAG GCT ACA
10653
8675
61.6
908



  704


TGT CAC CTA ACC AGA A
 5041





 


AJ_P10
85653
D08
AJ_908
/5AmMC6/CCC AAT GTC CAT
10653
8672
60.4
909



  705


GAC TTT CCT AAC GGA A
 5042





 


AJ_P10
85653
D09
AJ_909
/5AmMC6/CCC AAG CAC ATG
10653
8699
61.2
910



  706


GTT CCA CAT AAA CGA A
 5043





 


AJ_P10
85653
D10
AJ_910
/5AmMC6/CCC AAG CCA TGT
10653
8699
61.4
911



  707


TGC ACA CTA CAA AGA A
 5044





 


AJ_P10
85653
D11
AJ_911
/5AmMC6/CCC AAA CGC ATC
10653
8739
60.9
912



  708


CAA AGT TAG GGT ACA A
 5045





 


AJ_P10
85653
D12
AJ_912
/5AmMC6/CCC AAC CAC TCG
10653
8706
60.1
913



  709


TAG TCT ACT AGG AGA A
 5046





 


AJ_P10
85653
E01
AJ_913
/5AmMC6/CCC AAA CTG TGT
10653
8752
59.8
914



  710


TGT CTC ACT AGA GGA A
 5047





 


AJ_P10
85653
E02
AJ_914
/5AmMC6/CCC AAG TAC TCC
10653
8657
61.1
915



  711


TAC TCG TAC ATG GCA A
 5048





 


AJ_P10
85653
E03
AJ_915
/5AmMC6/CCC AAT AAC ACG
10653
8714
59.9
916



  712


AAA GCT TGT GCA TCA A
 5049





 


AJ_P10
85653
E04
AJ_916
/5AmMC6/CCC AAT TTC TAG
10653
8687
59.6
917



  713


AAC TGT GCT TGC ACA A
 5050





 


AJ_P10
85653
E05
AJ_917
/5AmMC6/CCC AAG GTG TAC
10653
8777
61.7
918



 714


CTT TGA CCA GTG AGA A
 5051





 


AJ_P10
85653
E06
AJ_918
/5AmMC6/CCC AAG TTA CCT
10653
8681
60.1
919



  715


CTT GCC ATA CGA GAA A
 5052





 


AJ_P10
85653
E07
AJ_919
/5AmMC6/CCC AAG AAC GTT
10653
8714
59.6
920



  716


CTG CTC ATA GCA AAA A
 5053





 


AJ_P10
85653
E08
AJ_920
/5AmMC6/CCC AAA CGC TTC
10653
8696
59.4
921



  717


TTC ATT GTA ACA GGA A
 5054





 


AJ_P10
85653
E09
AJ_921
/5AmMC6/CCC AAG AGT CTC
10653
8657
59.8
922



  718


GAC TCC TCT ACT AGA A
 5055





 


AJ_P10
85653
E10
AJ_922
/5AmMC6/CCC AAG AGT ACA
10653
8690
59.8
923



  719


GAA CCT CAC TTT CGA A
 5056





 


AJ_P10
85653
E11
AJ_923
/5AmMC6/CCC AAG TAC TGC
10653
8699
60.3
924



  720


TGA CAC AAC TAA CGA A
 5057





 


AJ_P10
85653
E12
AJ_924
/5AmMC6/CCC AAA GCC TTT
10653
8761
60.1
925



  721


GGT AGT CAG ACA GTA A
 5058





 


AJ_P10
85653
F01
AJ_925
/5AmMC6/CCC AAA GGC TAC
10653
8714
59
926



  722


TCA GAA CAA CTT TGA A
 5059





 


AJ_P10
85653
F02
AJ_926
/5AmMC6/CCC AAG TAC CTC
10653
8675
61.6
927



  723


ACT CAA GCA TCA GGA A
 5060





 


AJ_P10
85653
F03
AJ_927
/5AmMC6/CCC AAA TAG TCT
10653
8752
60.3
928



  724


CAG TGT GCT AGT GCA A
 5061





 


AJ_P10
85653
F04
AJ_928
/5AmMC6/CCC AAG TCC ACT
10653
8697
62
929



  725


TTC TGC ACT AAG GGA A
 5062





 


AJ_P10
85653
F05
AJ_929
/5AmMC6/CCC AAC AGT GCT
10653
8723
60.3
930



  726


TGC AAA CAT CAA AGA A
 5063





 


AJ_P10
85653
F06
AJ_930
/5AmMC6/CCC AAA CTT GTC
10653
8712
59.5
931



  727


TCT CTG AGT ACA GGA A
 5064





 


AJ_P10
85653
F07
AJ_931
/5AmMC6/CCC AAA GTT CTC
10653
8730
60.2
932



  728


CAC AAG TGT CAG AGA A
 5065





 


AJ_P10
85653
F08
AJ_932
/5AmMC6/CCC AAG TCT TCA
10653
8690
60.4
933



  729


CAC TCA GAA CGT GAA A
 5066





 


AJ_P10
85653
F09
AJ_933
/5AmMC6/CCC AAT CCG AAG
10653
8754
59
934



  730


TTG CGT AGA CTA AAA A
 5067





 


AJ_P10
85653
F10
AJ_934
/5AmMC6/CCC AAG AAA CAT
10653
8699
60.1
935



  731


CGT ACA CAG TCT CGA A
 5068





 


AJ_P10
85653
F11
AJ_935
/5AmMC6/CCC AAG AAC CAT
10653
8675
62.4
936



  732


CAC CTG TCA GCA TGA A
 5069





 


AJ_P10
85653
F12
AJ_936
/5AmMC6/CCC AAC GAC ATA
10653
8739
60.5
937



  733


CCT AAA GCA TGG TGA A
 5070





 


AJ_P10
85653
G01
AJ_937
/5AmMC6/CCC AAG GTC ACA
10653
8666
61.9
938



  734


GCA CTT TCC ACT AGA A
 5071





 


AJ_P10
85653
G02
AJ_938
/5AmMC6/CCC AAC GAG TTA
10653
8705
59.6
939



  735


CAC GTT TGC CTA AAA A
 5072





 


AJ_P10
85653
G03
AJ_939
/5AmMC6/CCC AAG TAC GCT
10653
8681
58.9
940



  736


AGT CTC TCA CAT AGA A
 5073





 


AJ_P10
85653
G04
AJ_940
/5AmMC6/CCC AAA GTG TCT
10653
8681
59.9
941



  737


GAC CAT ACT TAC CGA A
 5074





 


AJ_P10
85653
G05
AJ_941
/5AmMC6/CCC AAC GTT CCA
10653
8699
60
942



  738


TAC CAA GGA CAT AGA A
 5075





 


AJ_P10
85653
G06
AJ_942
/5AmMC6/CCC AAG GAC TTC
10653
8681
59.1
943



  739


GAC TTC CTA CTA AGA A
 5076





 


AJ_P10
85653
G07
AJ_943
/5AmMC6/CCC AAT GAC GTT
10653
8681
60.5
944



  740


GTA AAC CTC TCA CGA A
 5077





 


AJ_P10
85653
G08
AJ_944
/5AmMC6/CCC AAG CAC TGT
10653
8690
61
945



  741


GTA AAC AAC CTT CGA A
 5078





 


AJ_P10
85653
G09
AJ_945
/5AmMC6/CCC AAC ATG TAG
10653
8763
58.1
946



  742


AGA AAC TCT CGA GAA A
 5079





 


AJ_P10
85653
G10
AJ_946
/5AmMC6/CCC AAC AGC TTC
10653
8672
59.5
947



  743


CTC ATA GTC TTA GGA A
 5080





 


AJ_P10
85653
G11
AJ_947
/5AmMC6/CCC AAG TCC TAC
10653
8675
61.3
948



  744


ACA CAG TCA TAC GGA A
 5081





 


AJ_P10
85653
G12
AJ_948
/5AmMC6/CCC AAG TCC ATA
10653
8666
62.3
949



  745


CAT CCG AAC TGT GCA A
 5082





 


AJ_P10
85653
H01
AJ_949
/5AmMC6/CCC AAG AAC TTC
10653
8681
61
950



  746


CAC TTA GCA TGT GCA A
 5083





 


AJ_P10
85653
H02
AJ_950
/5AmMC6/CCC AAG GTT CTA
10653
8690
60.7
951



  747


CAT CAC GTA CGC AAA A
 5084





 


AJ_P10
85653
H03
AJ_951
/5AmMC6/CCC AAG AGT GCT
10653
8730
60
952



  748


ACC TTC GTA CAG AAA A
 5085





 


AJ_P10
85653
H04
AJ_952
/5AmMC6/CCC AAA TAA GTC
10653
8763
58.8
953



  749


CTG AAG GAA CGC ATA A
 5086





 


AJ_P10
85653
H05
AJ_953
/5AmMC6/CCC AAG TGC AAC
10653
8739
61.4
954



  750


GAG ACC TTT GAC AAA A
 5087





 


AJ_P10
85653
H06
AJ_954
/5AmMC6/CCC AAG CAC TGT
10653
8681
61.5
955



  751


TGA AAC CCT TTC GAA A
 5088





 


AJ_P10
85653
H07
AJ_955
/5AmMC6/CCC AAA CTC GTC
10653
8681
61
956



  752


ACC TTT GGG TAA ACA A
 5089





 


AJ_P10
85653
H08
AJ_956
/5AmMC6/CCC AAA GCC TTC
10653
8721
60.2
957



  753


TTG GTC ATA GAC AGA A
 5090





 


AJ_P10
85653
H09
AJ_957
/5AmMC6/CCC AAC AAC GGT
10653
8752
61.1
958



  754


ACT TTG TTG GTA GCA A
 5091





 


AJ_P10
85653
H10
AJ_958
/5AmMC6/CCC AAC ATT CTG
10653
8752
60.6
959



  755


GTG TTA CGA ACT GGA A
 5092





 


AJ_P10
85653
H11
AJ_959
/5AmMC6/CCC AAT GAA ACC
10653
8699
61
960



  756


ATC CAT GTC AGA GCA A
 5093





 


AJ_P10
85653
H12
AJ_960
/5AmMC6/CCC AAA ACT GAC
10653
8761
61.9
961



  757


CAT TGT GGT GTG CAA A
 5094









Example 11—Titration or Dilution Series
The ability to perform multiple experiments in parallel enables straightforward exploration of the counting results from samples with a range of starting concentrations or amounts of target, sometimes known as a titration experiment or a dilution series.
An example of dilution series data for a labeled RPLPO gene sequence is shown in FIG. 26. Plotted in the figure is the counting result N for each array where the nominal starting concentration of mRNA in the reaction is shown on the X axis.
Some of the graphical output from the analysis software for the same experiment is shown in FIG. 27. For each array, there is a compound display with the following elements: (i) an intensity histogram (green) for the index spots, (ii) a blue line registered with the histogram, showing the dynamic threshold for spot counting, (iii) a 32×32 grid which is a digital representation of each array. A white site in the grid denotes a spot whose intensity was above the dynamic threshold, and a black site denotes that the intensity was below the dynamic threshold, and (iv) the result N and the quality score Q is reported for each array as text.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments may be provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

## Claims

1. A method for determining the number of a target molecule on an array, comprising:
(a) acquiring image data of a region of an array, wherein the array comprises a plurality of stochastically labeled and non-labeled features, and wherein the features comprise oligonucleotide probes;
(b) measuring a signal intensity and a local background intensity for one of the plurality of stochastically labeled and non-labeled features based on the acquired image data;
(c) calculating a local background corrected signal intensity for the one of the plurality of stochastically labeled and non-labeled features using the signal intensity and the local background intensity measured in (b);
(d) transforming the local background corrected signal intensity calculated in (c) for the one of the plurality of stochastically labeled and non-labeled features to determine the number of stochastically labeled and non-labeled features in the of the array; and
(e) calculating the number of a target molecule based on the number of stochastically labeled and non-labeled features determined in (d) within the region of the array.

2. The method of claim 1, wherein acquiring the image data of the region of the array comprises acquiring the image data of the region of the array from an optical imaging system, wherein the optical imaging system is a fluorescence imaging system or a phosphorescence imaging system, and wherein the optical imaging system operates in a transmitted light, reflected light, or scattered light imaging mode, or any combination thereof.

3. The method of claim 1, wherein the oligonucleotide probes comprise a fluorescein dye, a Cy3 dye, a Cy5 dye, or a phycoerythrin fluorophore.

4. The method of claim 1, further comprising illuminating the region of the array using an illumination system prior to (a) acquiring the image data of the region of the array.

5. The method of claim 1, wherein acquiring the image data of the region of the array comprises automatically locating one or more of the plurality of stochastically labeled and non-labeled features of the array.

6. The method of claim 1, further comprising analyzing the local background corrected signal intensities for the plurality of stochastically labeled and non-labeled features to determine a dynamic signal intensity threshold for distinguishing the stochastically labeled and non-labeled features of the array.

7. The method of claim 6, wherein determining the dynamic signal intensity threshold for distinguishing the stochastically labeled and non-labeled features of the array comprises performing, a k-means clustering analysis, a k-medoids clustering analysis, a mixture model statistical analysis, an empirical analysis based on sorting of the background corrected signal intensity values for the plurality of stochastically labeled and non-labeled features, or an empirical analysis based on sorting of pairwise differences in background corrected signal intensity values for the plurality of stochastically labeled and non-labeled features to determine a dynamic signal intensity threshold for distinguishing the plurality of stochastically labeled and non-labeled features of the array.

8. The method of claim 6, wherein determining the dynamic signal intensity threshold for distinguishing the stochastically labeled and non-labeled features of the array comprises maximizing a quality metric relating to a statistical difference between feature intensities above the dynamic signal intensity threshold and feature intensities below the dynamic signal intensity threshold.

9. The method of claim 6, wherein calculating the local background corrected signal intensity for the one of the plurality of stochastically labeled and non-labeled features using the signal intensity and the local background intensity measured in (b) comprises:
(i) centering a predefined analysis window on an array feature within an image;
(ii) calculating an intensity value statistic for signal and background pixels according to a predefined pattern of pixels within the array feature; and
(iii) utilizing the intensity value statistic for signal and background pixels to calculate the local background corrected signal intensity for the array feature.

10. The method of claim 1, wherein the intensity value statistic used for calculating the background corrected signal intensity for each of the plurality of stochastically labeled and non-labeled features is the mean, the median, or the ratio of signal to background intensities.

11. The method of claim 1, wherein calculating the number of the target molecule based on the number of stochastically labeled and non-labeled features determined in (d) within the region of the array comprises calculating the number of the target molecule based on the number of stochastically labeled features, the number of non-labeled features within the region of the array, and the predictions of the Poisson distribution.

12. The method of claim 1, further comprising calculating a confidence interval for the calculated number of the target molecule.

13. The method of claim 5, wherein automatically locating the one or more of plurality of stochastically labeled and non-labeled features of the array comprises automatically locating the one or more of the plurality of stochastically labeled and non-labeled features of the array using a predefined set of fiducial features on the array.

14. The method of claim 9, wherein calculating the intensity value statistic for signal and background pixels comprises subtracting the mean or median background intensity from the mean or median signal intensity to determine a background corrected signal intensity value for each array feature.