Determination and removal of intercluster optical interference

Abstract

The present disclosure describes embodiments of a method, system, and non-transitory computer-readable medium for accurately estimating crosstalk from adjacent clusters of oligonucleotides to a target cluster of oligonucleotides and removing or reducing crosstalk from the target cluster of oligonucleotides emitted by the adjacent clusters of oligonucleotides. For example, the disclosed system can detect intensity values ​​of the target cluster and the adjacent clusters. Based on the intensity values ​​of the adjacent clusters, the disclosed system can determine an inter-cluster interference metric that estimates the crosstalk emitted from the adjacent clusters. The disclosed system can remove the inter-cluster interference metric from the intensity value of the target cluster and generate a corrected intensity value for the target cluster.

Description

[Technical Field]

[0001] Priority application This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 483,428, entitled "DETERMINING AND REMOVING INTER-CLUSTER LIGHT INTERFERENCE," filed February 6, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] In recent years, biotechnology companies and research institutions have improved the hardware and software platforms used to determine the sequence of nucleotide bases (also called "nucleobases") in a sample. For example, some existing sequencing machines and sequencing data analysis software (collectively "existing sequencing systems") use traditional Sanger sequencing or sequencing by synthesis (SBS) methods to determine individual nucleotide bases in a nucleic acid sequence. When using SBS, existing sequencing systems can monitor thousands, tens of thousands, or even more nucleic acid polymers synthesized in parallel to detect more accurate nucleotide base calls. For example, a camera in an SBS platform can capture images of illuminated fluorescent tags from nucleotide bases incorporated into such synthesized nucleic acid sequences (often separated into clusters). After capturing the images, a computing device from the existing system uses sequencing data analysis software to determine the nucleotide bases detected in a given image based on the optical signals captured in the image data. Existing sequencing systems can determine the sequence of nucleotide bases present in a sample by repeatedly incorporating nucleotide bases into oligonucleotides and capturing images of the emitted optical signals at various sequencing cycles.

[0003] To increase sample throughput and efficiency, existing sequencing systems have grouped clusters of oligonucleotides closer together within wells of a flow cell or on other nucleotide sample slides. As cluster density increases, the fluorescent response (e.g., signal) from one cluster is more likely to interfere with the fluorescent response (or lack of response) of adjacent clusters, causing overlapping signals between clusters. Such overlapping signals and optical interference are often referred to as spatial crosstalk. Existing sequencing systems attempt to reduce the interfering signal by reducing optical interference to various components and by implementing computational models to estimate and decompose the interfering response (e.g., DC offset, noise level, and / or point spread function) from the cluster signal. Unfortunately, the increased density and optical interference between clusters makes it more difficult to estimate the point spread function (PSF) for a given cluster or section of a nucleotide sample slide.

[0004] As nucleotide sample slides carry more densely packed clusters, sequencing instruments, along with other intensity detection systems, are more likely to erroneously determine that a cluster is illuminated (instead of not illuminated) due to spatial crosstalk from neighboring clusters. The increased crosstalk, along with variations in amplitude and background noise, reduces the accuracy of nucleobase calls based on signals from a particular cluster. For example, increased crosstalk from multiple neighboring clusters may illuminate a given cluster in an image for a given channel. Such indirect illumination in existing sequencing systems can cause base calling algorithms to determine an incorrect nucleobase call (e.g., adenine) instead of the correct nucleobase call (e.g., guanine) for the nucleobase incorporated by the oligonucleotides of the cluster during a given cycle.

[0005] Because crosstalk has limited the accuracy of nucleic acid base calling by existing sequencing systems, some existing systems maintain a distance between clusters, thereby limiting the sample and cluster throughput of the sequencing device. As described above, when existing sequencing systems increase the cluster density on a flow cell or other nucleotide sample slide, the quality of imaging and the accuracy of nucleic acid base calling decrease, resulting in lower data output. To maintain high-quality imaging and relatively accurate nucleic acid base calling, some existing sequencing systems limit the number and density of clusters on the nucleotide sample slide. By avoiding overclustering (e.g., placing too many clusters on the flow cell) and / or underclustering (e.g., placing fewer clusters on the flow cell), existing systems limit nucleotide sequencing to a narrow range of cluster density on the flow cell, reducing data yield.

[0006] These, along with additional problems and challenges, exist with existing sequencing systems. Summary of the Invention

[0007] The present disclosure describes embodiments of methods, non-transitory computer-readable media, and systems that can estimate crosstalk of adjacent clusters of oligonucleotides on a target cluster of oligonucleotides ("target cluster") and remove or reduce crosstalk from a signal emitted by the target cluster when determining a corrected signal for the target cluster. For example, the disclosed system can detect intensity values ​​of various clusters of oligonucleotides to which labeled nucleotide bases have been added. Based on the intensity values ​​for different sets of clusters, the disclosed system can determine illumination indices for one or more clusters adjacent to the target cluster. From the illumination indices and / or other data for adjacent clusters of oligonucleotides ("adjacent clusters"), the disclosed system determines an inter-cluster interference metric that estimates the optical interference of adjacent clusters on the target cluster. The disclosed system can further remove the inter-cluster interference metric from the intensity value of the target cluster.

[0008] The disclosed system can utilize such inter-cluster interference metrics associated with clusters for various base calling applications, which are further described below. For example, the disclosed system can more accurately determine cluster signals and their corresponding nucleobase calls for a given sequencing cycle by (i) removing the crosstalk of adjacent clusters from the intensity values ​​of the target cluster when determining the intensity values ​​of the target cluster's signal, and (ii) determining the nucleobase calls for the target cluster. To increase the accuracy and efficiency of nucleobase calling, in some cases, the disclosed system iteratively determines and removes or reduces the crosstalk of adjacent subsets of clusters from the target subset of clusters based on the intensity value range for each cluster.

[0009] Additional features and advantages of one or more embodiments of the present disclosure will be set forth in the description that follows, and in part will be obvious from the description, or may be learned by practice of such exemplary embodiments. [Brief explanation of the drawings]

[0010] The detailed description sets forth various embodiments with additional specificity and detail through the use of the accompanying drawings, which are summarized below. [Figure 1] 1 illustrates an environment in which a crosstalk-aware base calling system can operate in accordance with one or more embodiments of the present disclosure. [Figure 2] FIG. 1 shows a schematic diagram of a crosstalk-aware base calling system that generates corrected intensity values ​​for target clusters by determining and removing inter-cluster interference metrics from the intensity values ​​of the target clusters, in accordance with one or more embodiments of the present disclosure. [Figure 3] 1 shows a diagram demonstrating that optical interference between clusters of oligonucleotides increases as the distance between the clusters of oligonucleotides decreases, in accordance with one or more embodiments of the present disclosure. [Figure 4] 1 illustrates a crosstalk-aware base calling system that determines an illumination index based on fluorescent responses in different channels, according to one or more embodiments of the present disclosure. [Figure 5A] FIG. 1 illustrates a crosstalk-aware base calling system that utilizes a linear equalizer system to generate modified intensity values ​​for a target cluster by determining nucleic acid base calls and illumination indices from neighboring clusters, a signal model for the target cluster, and an inter-cluster interference metric, in accordance with one or more embodiments. [Figure 5B] FIG. 1 illustrates a crosstalk-aware base calling system that utilizes a linear equalizer system to generate modified intensity values ​​for a target cluster by determining nucleic acid base calls and illumination indices from neighboring clusters, a signal model for the target cluster, and an inter-cluster interference metric, in accordance with one or more embodiments. [Figure 6] 1 shows a point spread function estimated for intensity values ​​of a signal from a cluster of oligonucleotides according to one or more embodiments of the present disclosure. [Figure 7A]1 illustrates the effect of optical interference between clusters of oligonucleotides and the elimination of optical interference for specific clusters of oligonucleotides, in accordance with one or more embodiments of the present disclosure. [Figure 7B] 1 illustrates the effect of optical interference between clusters of oligonucleotides and the elimination of optical interference for specific clusters of oligonucleotides, in accordance with one or more embodiments of the present disclosure. [Figure 7C] 1 illustrates the effect of optical interference between clusters of oligonucleotides and the elimination of optical interference for specific clusters of oligonucleotides, in accordance with one or more embodiments of the present disclosure. [Figure 8A] 1 shows histograms of intensity values ​​for a cluster of oligonucleotides both with and without light interference from adjacent clusters of oligonucleotides, in accordance with one or more embodiments of the present disclosure. [Figure 8B] 1 shows histograms of intensity values ​​for a cluster of oligonucleotides both with and without light interference from adjacent clusters of oligonucleotides, in accordance with one or more embodiments of the present disclosure. [Figure 9] 1 illustrates a series of operations for generating a revised set of intensity values ​​for a cluster of oligonucleotides using an inter-cluster interference metric, in accordance with one or more embodiments of the present disclosure. [Figure 10] 1 illustrates a block diagram of an exemplary computing device in accordance with one or more embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure describes one or more embodiments of a crosstalk-aware base calling system that determines an inter-cluster interference metric representing the optical interference of one cluster of oligonucleotides with a target cluster of oligonucleotides and generates a corrected intensity value for the target cluster based on the inter-cluster interference metric. By determining and removing the inter-cluster interference metric, the crosstalk-aware base calling system resolves the optical interference between clusters. To detect and resolve light based on such an inter-cluster interference metric, in some implementations, the crosstalk-aware base calling system determines intensity values ​​of signals from a target cluster and neighboring clusters of oligonucleotides for a given sequencing cycle. Based on the intensity values ​​of the neighboring clusters, the crosstalk-aware base calling system determines an illumination index representing whether the neighboring cluster is illuminated during the given sequencing cycle. Based on the illumination index, the crosstalk-aware base calling system determines an inter-cluster interference metric that estimates the optical interference from the neighboring cluster on the target cluster. The crosstalk-aware base calling system can further subtract (or otherwise remove) the inter-cluster interference metric from the intensity value of the target cluster's signal to create a corrected intensity value for the target cluster.

[0012] As alluded to above, in one or more embodiments, the crosstalk-aware base calling system detects intensity values ​​(e.g., wavelength and / or brightness values) of signals emitted by a target cluster and adjacent clusters in a given sequencing cycle. For example, in some cases, the crosstalk-aware base calling system detects intensity values ​​from signals emitted by each cluster (including the target cluster and adjacent clusters) on the sample nucleotide slide in a given sequencing cycle. In certain embodiments, clusters with higher intensity values ​​are relatively bright, and clusters with lower intensity values ​​are relatively dark. In some cases, the crosstalk-aware base calling system utilizes data from brighter clusters to determine crosstalk of the brighter clusters relative to the darker clusters.

[0013] For example, based on the detected intensity values, the crosstalk-aware base calling system can determine a subset of illumination indices for a subset of clusters, including clusters adjacent to the target cluster. In particular, the crosstalk-aware base calling system determines nucleobase calls for a subset of clusters (e.g., a subset of brighter clusters incorporating adenine) and determines illumination indices for the subset of clusters from the nucleobase calls. Such illumination indices identify whether a given cluster is illuminated or emits a fluorescent response in a given channel (e.g., of two or four channels) during a sequencing cycle, and, together with the fluorescent responses in other given channels, form data for determining the nucleobase call. In some cases, the illumination indices together represent the illumination of the cluster in multiple channels, such as a first illumination indices indicating whether a given cluster is illuminated in a first channel during a given sequencing cycle and a second illumination indices indicating whether a given cluster is illuminated in a second channel during a given sequencing cycle. In contrast, in some cases, the illumination index may be a continuous illumination index, indicating the degree to which a given cluster is illuminated in a given channel.

[0014] Based on such illumination metrics, as previously suggested, the crosstalk-aware base calling system determines an inter-cluster interference metric (e.g., a crosstalk metric). As discussed above, in some instances, crosstalk indicates how the signals (e.g., brightness) of adjacent clusters interfere with, manipulate, and / or modify the signal of the target cluster. In particular, in one or more embodiments, the inter-cluster interference metric estimates the degree or extent to which light from adjacent clusters interferes with or modifies light from the target cluster. In some cases, the crosstalk-aware base calling system can determine multiple inter-cluster interference metrics, each estimating the light interference from a given adjacent cluster on the target cluster.

[0015] Upon determining the inter-cluster interference metric, the crosstalk-aware base calling system can utilize the inter-cluster interference metric to generate a corrected intensity value for the signal emitted by the cluster during a sequencing cycle. By utilizing such a metric, the crosstalk-aware base calling system can determine the amount of crosstalk between clusters and eliminate or reduce crosstalk from the target cluster. By way of example, in one or more embodiments, during a sequencing cycle, the target cluster may have a relatively dimmer (e.g., lower intensity) signal, and an adjacent cluster may have a relatively brighter (e.g., higher intensity) signal. However, the brightness of the adjacent cluster's signal may increase the brightness (e.g., intensity) of the target cluster's signal, making it difficult to determine whether the cluster emits light intensity at a particular frequency (e.g., frequency band or spectral band) in a given channel during a sequencing cycle. In some embodiments, the crosstalk-aware base calling system can determine the inter-cluster interference metric based on the illumination index and other data regarding the adjacent cluster. Based on the inter-cluster interference metric, the crosstalk-aware base calling system can offset (or reduce the effect of) the light emitted from the brighter adjacent cluster's signal from the target cluster's signal. Thus, in some embodiments, the crosstalk-aware base calling system can more accurately determine intensity values ​​of a target cluster in both channels or each relevant channel during a sequencing cycle based on an inter-cluster interference metric, which results in more accurate nucleic acid base calling of the target cluster.

[0016] To increase efficiency and accuracy, in some cases, the crosstalk-aware base calling system determines nucleobase calls and removes crosstalk for clusters according to a specific order. For example, the crosstalk-aware base calling system can (i) identify and determine nucleobase calls for the brightest subset of oligonucleotide clusters that emit signals within a top intensity value range (e.g., the top 10% brightest), and (ii) further determine an inter-cluster interference metric that estimates the optical interference of the brightest subset of oligonucleotide clusters with the next-brightest subset of oligonucleotide clusters that emit signals within a second intensity value range (e.g., the top 20-30% brightest). As described further below, the crosstalk-aware base calling system can similarly perform further iterations to determine crosstalk based on additional intensity value ranges. Instead of ordering nucleobase calls and crosstalk removal according to intensity value ranges, in some embodiments, the crosstalk-aware base calling system can order nucleobase calls and crosstalk removal for clusters using a signal-to-noise ratio (SNR) metric.

[0017] Crosstalk-aware base calling systems offer several advantages over conventional sequencing platforms. In particular, crosstalk-aware base calling systems can resolve light intensity, including the intensity of cluster signals and noise from other sources, improving the accuracy of nucleic acid base calling and increasing the efficiency of flow cells or nucleotide sample slides during sequencing cycles. As described above, crosstalk-aware base calling systems can receive signals from target clusters having uncorrected intensity values, where the uncorrected intensity values ​​of the signals from the target clusters include the signal from the target cluster, crosstalk (e.g., noise) from adjacent clusters, and other noise sources (e.g., background noise or intensity fluctuations). The crosstalk-aware base calling system can resolve light intensity consisting of the target signal and noise. In particular, the crosstalk-aware base calling system can determine an inter-cluster interference metric that estimates light interference from adjacent clusters on the target cluster. The inter-cluster interference metric estimates the crosstalk (e.g., interfering light) of adjacent clusters from composite components (e.g., background noise, the intensity value of the signal of the target cluster, and crosstalk). Once estimated, the crosstalk-aware base calling system can remove or reduce crosstalk by removing the inter-cluster interference metric from the signal of the target cluster. While existing sequencing systems often cannot determine whether a target cluster is emitting a signal based on background noise, crosstalk, and / or amplitude variations, the inter-cluster interference metric allows a crosstalk-aware base calling system to accurately resolve crosstalk from background noise and / or amplitude. Unlike existing sequencing systems, upon determining the extent and source of crosstalk, in one or more embodiments, the crosstalk-aware base calling system can remove the crosstalk from the signal of the affected cluster. By removing the inter-cluster interference metric from the signal of the target cluster, the crosstalk-aware base calling system can generate a corrected signal for the target cluster.Thus, by removing crosstalk from the signal of a target cluster and generating a more accurate signal for the target cluster, the crosstalk-aware base calling system can more accurately and reliably determine nucleic acid base calls for the target cluster.

[0018] In addition to detecting and resolving portions of the signal composed of noise and light, the crosstalk-aware base calling system improves nucleobase calling accuracy. In particular, the crosstalk-aware base calling system can determine an inter-cluster interference metric that estimates light interference from neighboring clusters on the target cluster and remove the inter-cluster interference metric from the intensity value of the signal of the target cluster. The resulting corrected intensity value represents a more accurate and / or purer signal for the target cluster. Based on the more accurate or purer cluster signal, the crosstalk-aware base calling system can similarly determine a more accurate or reliable nucleobase call for the target cluster without or with minimal crosstalk interfering with the signal determining the nucleobase call. For example, the crosstalk-aware base calling system can determine that the corrected intensity value for the target cluster falls within the intensity value boundary of one nucleobase but not another, or improve the reliability score (e.g., QUAL score) of a nucleobase call with a low quality score.

[0019] In addition to improved nucleotide base calling and decomposition of crosstalk from cluster signals, the crosstalk-aware base calling system improves the efficiency with which a sequencing system performs nucleotide sequencing. By determining and removing inter-cluster interference metrics and improving the signal of target clusters, the crosstalk-aware base calling system facilitates more densely grouped clusters on a nucleotide sample slide. Unlike the more limited, less densely grouped clusters of existing sequencing systems, the crosstalk-aware base calling system introduces a model that removes or reduces crosstalk and facilitates more densely grouped clusters and higher throughput on the sequencing device. Thus, by determining and removing inter-cluster interference metrics, the crosstalk-aware base calling system can sequence the nucleotide sequences of more genomic samples with improved accuracy than existing sequencing systems that cannot effectively adjust for crosstalk in densely grouped clusters.

[0020] As indicated by the foregoing discussion, the present disclosure utilizes various terms to describe the features and advantages of the crosstalk-recognition base calling system. Further details regarding the meaning of such terms are now provided. For example, as used herein, the term "nucleotide sample slide" refers to a plate or slide containing oligonucleotides for sequencing nucleotide segments for a sample. In particular, a nucleotide sample slide can refer to a slide containing fluidic channels through which reagents and buffers can travel as part of sequencing. For example, in one or more embodiments, the nucleotide sample slide includes a flow cell (e.g., a patterned or unpatterned flow cell) containing small fluidic channels and short oligonucleotides complementary to adapter sequences.

[0021] Relatedly, as used herein, the term "nucleotide sample slide section" (or "nucleotide sample slide section") refers to an area that is a part of a nucleotide sample slide. In particular, a nucleotide sample slide section can refer to a distinct portion of the nucleotide sample slide that is distinct from other portions of the nucleotide sample slide. For example, a nucleotide sample slide section can include a well (e.g., a nanowell) of a patterned flow cell or a distinct subsection (e.g., a subsection corresponding to a cluster) of an unpatterned flow cell. In some cases, a nucleotide sample slide section includes tiles or subtiles having clusters of the same or similar oligonucleotides growing in parallel.

[0022] Furthermore, as used herein, the term "labeled nucleotide base" refers to a nucleotide base having a fluorescent or light-based indicator or fluorescent dye indicator that indicates the classification of the nucleotide base. In particular, a labeled nucleotide base can refer to a nucleotide base incorporating a fluorescent or light-based indicator or fluorescent dye indicator to identify the base type (e.g., adenine, cytosine, thymine, or guanine). For example, in one or more embodiments, a labeled nucleotide base includes a nucleotide base having a fluorescent tag that emits a signal that identifies the base type, either alone or together with another fluorescent tag. Thus, a nucleotide base can be identified by a mixture of dyes (or a mixture of fluorescent tags) that together indicate the nucleobase type (e.g., an "on" / "on" illumination indicator). Based on the intensity value of the signal emitted by the labeled nucleotide base in a cluster of oligonucleotides, such as a 16-quadrature amplitude modulation (QAM) or pulse amplitude modulation (PAM) format signal, the base type (e.g., adenine, cytosine, thymine, or guanine) can be determined in certain embodiments of the crosstalk recognition base calling system.

[0023] Furthermore, as used herein, the term "cluster of oligonucleotides" refers to a group containing several identical deoxyribonucleic acid (DNA) fragments bound to the surface of a flow cell. For example, in some embodiments, a cluster of oligonucleotides may be composed of a template DNA strand that has been clonally amplified via bridge amplification.

[0024] Furthermore, as used herein, the term "signal" refers to a signal emitted, reflected, or otherwise communicated from a labeled nucleotide base or a group of labeled nucleotide bases (e.g., labeled nucleotide bases added to a cluster of oligonucleotides). In particular, the signal can refer to a signal indicative of the base type. For example, the signal can include an optical signal emitted or reflected from a fluorescent tag of the nucleotide base or from fluorescent tags of multiple nucleotide bases incorporated into the oligonucleotide. As described above, nucleobases incorporated into clusters can similarly emit (in response to a laser) a signal that can be identified as a mixture of dyes (or a mixture of fluorescent tags) that together indicate the nucleobase type (e.g., a cluster with an "on" / "on" illumination indicator). In some implementations, the crosstalk-recognition base calling system triggers a signal via an external stimulus, such as a laser or other light source. In some cases, the crosstalk-recognition base calling system triggers a signal via some internal stimulus. Furthermore, in some embodiments, the crosstalk-recognition base calling system observes the signal using a filter applied when capturing an image of the nucleotide sample slide (e.g., a section of the nucleotide sample slide). As alluded to above, in certain instances, the signal comprises the aggregate of signals provided by each labeled nucleotide base attached to an individual oligonucleotide in a cluster of oligonucleotides.

[0025] As used herein, the term "intensity value" refers to a value that indicates a characteristic or attribute of a signal emitted, reflected, or otherwise communicated from a labeled nucleotide base or a group of labeled nucleotide bases from a cluster of oligonucleotides. In particular, an intensity value can refer to a value associated with color intensity (e.g., wavelength) or light intensity (e.g., brightness). In some cases, a crosstalk-recognition base calling system captures multiple images of a cluster of oligonucleotides bearing labeled nucleotide bases using different filters (or intensity channels). Thus, the intensity value of a signal can correspond to the intensity of the signal observed through a particular filter.

[0026] As used herein, the term "illumination indicator" refers to an indicator of whether a cluster of oligonucleotides is illuminated by light of a specific frequency band or emits light of a specific frequency band intensity during a sequencing cycle. In particular, the illumination indicator indicates whether (i) the cluster of oligonucleotides contains labeled nucleotides that emit light of a specific intensity at a specific frequency (e.g., frequency band) that is illuminated (e.g., turned on or activated), or (ii) does not contain labeled nucleotide bases that are not illuminated by light of a specific intensity at a specific frequency (e.g., frequency band) in an intensity channel during sequencing (e.g., turned off or inactive). In some cases, the illumination indicator can take a couplet format. For example, if a cluster of oligonucleotides incorporates nucleic acid bases with fluorescent tags or other labels that illuminate or emit light intensity at a specific frequency (e.g., frequency band) of light in a channel during a sequencing cycle (in response to light or a laser), the "on" or "illuminated" state of the illumination indicator can be represented by 1. Conversely, if a cluster of oligonucleotides does not incorporate (or incorporates too few) nucleic acid bases bearing fluorescent tags or other labels that illuminate or emit light intensity (in response to light or a laser) at a particular frequency (e.g., a frequency band) within a channel during a sequencing cycle, the "off" or "non-illuminated" state of the illumination indicia can be represented by a 0. To illustrate, [1, 1] can indicate that the illumination indicia for a cluster of oligonucleotides are illuminated in two different channels. While the description and diagrams depict illumination indicia in different channels (e.g., two or four channels), the crosstalk-aware base calling system can simultaneously detect signals from clusters in such different channels.

[0027] In contrast, when a polyclonal cluster of oligonucleotides incorporates nucleobases with different fluorescent tags or other labels that illuminate or emit light (in response to light or laser) within different spectral bands in a given channel during a sequencing cycle, the state of the illumination indicator is not completely "on" or "off" (or completely "illuminated" or "unilluminated"). In some cases, such mixed signals from a polyclonal cluster of oligonucleotides are filtered and discarded based on intensity value boundaries for the different types of nucleobases.

[0028] While the present disclosure frequently uses illumination indicators in the form of "on" or "off" (or corresponding "1" or "0"), the illumination indicators may be channel-specific and are not designed to indicate the presence or absence of background noise or other light. Thus, an "off" or "0" indicator does not indicate the absence of light, but rather indicates an inference that a particular cluster has not incorporated (or has incorporated too few) nucleic acid bases bearing a fluorescent tag or other label that illuminates or emits light intensity (in response to light or a laser) at a particular frequency (e.g., frequency band) in a particular channel during a sequencing cycle. Thus, illumination indicators can take other forms. As an alternative to the couplet format, in some embodiments, the illumination indicator may be continuous and represent the degree to which a given cluster is illuminated during a sequencing cycle. Such a continuous illumination indicator can take the form of, for example, a metric or score (e.g., 0 to 1) indicating the degree to which a cluster is illuminated by light emitted from a particular type of nucleotide incorporated into the cluster during a sequencing cycle.

[0029] Furthermore, as used herein, the term "inter-cluster interference metric" refers to the measurement or quantification of light from one cluster of oligonucleotides interfering with or modifying light from another cluster of oligonucleotides. In particular, an inter-cluster interference metric can refer to the degree, amount, and / or extent to which an optical signal from one cluster of oligonucleotides interferes with another cluster of oligonucleotides.

[0030] As used herein, the term "nucleotide base calling" refers to the determination or prediction of a specific nucleic acid base (or nucleic acid base pair) relative to an oligonucleotide (e.g., a nucleotide read) during a sequencing cycle or to a genomic coordinate of a sample genome. In particular, nucleic acid base calling can refer to the determination or prediction of the type of nucleic acid base incorporated within an oligonucleotide on a nucleotide sample slide (e.g., read-based nucleic acid base calling). In some cases, for a nucleotide read, nucleic acid base calling includes the determination or prediction of a nucleic acid base based on intensity values ​​obtained from fluorescently tagged nucleotides attached to oligonucleotides on a nucleotide sample slide (e.g., in a cluster of a flow cell). As alluded to above, a single nucleic acid base call can be an adenine (A) call, a cytosine (C) call, a guanine (G) call, a thymine (T) call, or a uracil (U) call.

[0031] Additionally, as used herein, the term "sequencing cycle" (or "cycle") refers to repeated addition or incorporation of nucleotide bases into an oligonucleotide, or repeated addition or incorporation of nucleotide bases into an oligonucleotide in parallel. In particular, a cycle can include repeated analysis of one or more images with data indicative of individual nucleotide bases added or incorporated into an oligonucleotide, or into an oligonucleotide in parallel. Thus, a cycle can be repeated as part of sequencing a nucleic acid polymer. For example, in one or more embodiments, each sequencing cycle involves either a single read, in which the DNA or RNA strand is read in only one direction, or a paired-end read, in which the DNA or RNA strand is read from both ends. Furthermore, in certain cases, each sequencing cycle involves a camera capturing images of a nucleotide sample slide or multiple sections of a nucleotide sample slide to generate image data for determining the specific nucleotide bases added or incorporated into a particular oligonucleotide. Following the image capture step, the sequencing system can remove certain fluorescent labels from the incorporated nucleotide bases and perform another sequencing cycle until the nucleic acid polymer is completely sequenced. In one or more embodiments, a sequencing cycle includes a cycle within a sequencing-by-synthesis (SBS) run.

[0032] Additionally, as used herein, the term "nucleotide base call data" refers to digital files, image data, or other digital information representing individual nucleotide bases or a sequence of nucleotide bases in a nucleic acid polymer. In particular, nucleotide base call data can include intensity values ​​(e.g., color or light intensity values ​​of individual clusters) from an image of a nucleotide sample slide taken by a camera, or other data representing individual nucleotide bases or a sequence of nucleotide bases in a nucleic acid polymer. In addition to, or instead of, intensity values, nucleotide base call data can include chromatogram peaks or current changes representing individual nucleic acid bases in a sequence. Additionally, in some embodiments, nucleotide base call data includes individual nucleotide base calls identifying individual nucleotide bases (e.g., A, T, C, or G). For example, nucleotide base call data can include data of nucleotide base calls in a sequence of a nucleic acid polymer, organized in a digital file such as a binary base call (BCL) file, the number of nucleotide base calls corresponding to a particular base (e.g., adenine, cytosine, thymine, or guanine). Furthermore, nucleotide base call data can include error / accuracy information, such as a quality metric, associated with each nucleotide base call. In some embodiments, the nucleotide base call data includes information from a sequencing machine that utilizes sequencing by synthesis (SBS).

[0033] Further details regarding crosstalk-aware base calling systems will now be provided in connection with exemplary diagrams illustrating exemplary embodiments and implementations of crosstalk-aware base calling systems. For example, Figure 1 shows a schematic diagram of a system environment (or "environment") 100 in which a crosstalk-aware base calling system 106 operates according to one or more embodiments. As shown, the environment 100 includes one or more server devices 102 connected to user client devices 108 and sequencing devices 114 via a network 112. While Figure 1 illustrates an embodiment of a crosstalk-aware base calling system 106, alternative embodiments and configurations are possible.

[0034] 1, server device 102, user client device 108, and sequencing device 114 are connected via network 112. Each of the components of environment 100 can communicate via network 112. Network 112 includes any suitable network with which computing devices can communicate. An exemplary network is described in further detail below in connection with FIG. 10.

[0035] 1 , the environment 100 includes a sequencing device 114. The sequencing device 114 includes a device for sequencing a whole genome or other nucleic acid polymer. In some embodiments, the sequencing device 114 analyzes samples and generates data using the computer-implemented methods and systems described herein, either directly or indirectly on the sequencing device 114. In one or more embodiments, the sequencing device 114 sequences the whole genome or other nucleic acid polymer using sequencing-by-synthesis (SBS). As shown, in some embodiments, the sequencing device 114 bypasses the network 112 and communicates directly with the user client device 108.

[0036] As further illustrated by FIG. 1 , the environment 100 includes a server device 102. The server device 102 can generate, receive, analyze, store, receive, and forward electronic data, such as data for sequencing a nucleic acid polymer. The server device 102 can receive data from a sequencing device 114. For example, the server device 102 can collect and / or receive sequencing data, including nucleotide base call data, quality data, and other data related to sequencing a nucleic acid polymer. The server device 102 can also communicate with a user client device 108. In particular, the server device 102 can transmit read data, nucleic acid polymer sequences, error data, and other information to the user client device 108. In some embodiments, the server device 102 comprises a distributed server, where the server device 102 includes several server devices distributed across the network 112 and located in different physical locations. The server device 102 can include a content server, an application server, a communication server, a web hosting server, or another type of server.

[0037] 1 , the server device 102 can include a sequencing system 104. Generally, the sequencing system 104 analyzes sequencing data received from the sequencing device 114 to determine the nucleotide sequence for an entire genome or other nucleic acid polymer. For example, the sequencing system 104 can receive raw data (e.g., base call data of nucleotide reads) from the sequencing device 114 and determine the nucleic acid sequence of the genomic sample. Illustratively, the sequencing system 104 can receive data of nucleotide reads from the sequencing device 114, and the sequencing system 104 generates variant calls (or other nucleic acid base calls) for the genomic sample from the nucleotide reads. In some embodiments, the sequencing system 104 determines the sequence of nucleotide bases in DNA and / or RNA.

[0038] As further illustrated in FIG. 1 , the sequencing device 114 includes a crosstalk-aware base calling system 106. Generally, the crosstalk-aware base calling system 106 determines an inter-cluster interference metric to correct or compensate for a signal for estimated optical interference from adjacent clusters on the target cluster. More specifically, in some embodiments, the crosstalk-aware base calling system 106 detects intensity values ​​for the target cluster and the adjacent clusters in a given sequencing cycle. The crosstalk-aware base calling system 106 determines nucleic acid base calls and illumination indices for the adjacent clusters. The crosstalk-aware base calling system 106 further determines an inter-cluster interface metric for crosstalk of the adjacent clusters on the target cluster. The crosstalk-aware base calling system 106 further generates a corrected intensity value for the target cluster by removing the inter-cluster interference metric from the intensity value of the target cluster.

[0039] 1 further includes a user client device 108. The user client device 108 can generate, store, receive, and transmit digital data. In particular, the user client device 108 can receive sequencing data from the sequencing device 114. Additionally, the user client device 108 can communicate with the server device 102 to receive nucleotide base calls, nucleotide sequences, and variant call files. The user client device 108 can present the sequencing data to a user associated with the user client device 108.

[0040] 1 can include various types of client devices. For example, in some embodiments, user client device 108 includes a non-mobile device, such as a desktop computer or server, or other type of client device. In still other embodiments, user client device 108 includes a mobile device, such as a laptop, tablet, cell phone, smartphone, or the like. Further details of user client device 108 are discussed below with respect to FIG.

[0041] 1 , the user client device 108 includes a sequencing application 110. The sequencing application 110 may be a web application or a native application (e.g., a mobile application, a desktop application, etc.) on the user client device 108. The sequencing application 110 may include instructions that (when executed) cause the user client device 108 to receive or request data from the crosstalk-aware base calling system 106 and present sequencing data. Additionally, the sequencing application 110 may include instructions that (when executed) cause the user client device 108 to provide a graphical visualization of a read pileup or read alignment of nucleotide reads for a genomic sample.

[0042] 1 , the crosstalk-aware base calling system 106 may be located on the user client device 108 as part of the sequencing application 110. As shown, in some embodiments, the crosstalk-aware base calling system 106 is implemented (e.g., located completely or partially) on the user client device 108. In yet other embodiments, the crosstalk-aware base calling system 106 is implemented by one or more other components of the environment 100. In particular, the crosstalk-aware base calling system 106 can be implemented in a variety of different ways across the server device 102, the user client device 108, and the sequencing device 114. In one example, the crosstalk-aware base calling system 106 is located partially on the sequencing device 114 and also on the server device 102. In particular, the crosstalk-aware base calling system 106 can determine an inter-cluster interference metric for crosstalk of neighboring clusters on a target cluster on the sequencing device 114 and modify the intensity value of the target cluster by removing the inter-cluster interference metric as part of the server device 102.

[0043] 1 illustrates components of environment 100 communicating over network 112, in some embodiments, components of environment 100 communicate directly with each other, bypassing the network. For example, as previously described, user client device 108 can communicate directly with sequencing device 114. Furthermore, user client device 108 can communicate directly with crosstalk-aware base calling system 106, bypassing network 112. Furthermore, crosstalk-aware base calling system 106 can access one or more data bases housed on server device 102 or elsewhere in environment 100.

[0044] The following paragraphs provide further details regarding the crosstalk-aware base calling system 106. According to one or more embodiments, Figure 2 shows an overview of the crosstalk-aware base calling system 106 that generates inter-cluster interface metrics and modifies intensity values ​​of a target cluster. As shown in Figure 2, the crosstalk-aware base calling system 106 performs a series of operations including operation 202 of detecting intensity values ​​of a target cluster and adjacent clusters, operation 204 of determining nucleobase calls and illumination indices of the adjacent clusters, operation 206 of determining inter-cluster interference metrics of crosstalk of the adjacent clusters on the target cluster, and operation 208 of generating modified intensity values ​​of the target cluster by removing the inter-cluster interference metrics.

[0045] As described above, FIG. 2 illustrates operation 202 of detecting intensity values ​​for a target cluster and adjacent clusters. In some embodiments, the crosstalk-aware base calling system 106 may detect a set of intensity values ​​for the target cluster and a set of intensity values ​​for adjacent clusters through laser (e.g., light) excitation and imaging. During a sequencing cycle, the crosstalk-aware base calling system 106 can direct a light source having a specific wavelength toward the nucleotide sample slide (or a portion of the nucleotide sample slide) and capture images of clusters within the nucleotide sample slide that emit signals. In some embodiments, the crosstalk-aware base calling system 106 captures multiple images of clusters that emit signals. For example, the crosstalk-aware base calling system 106 can capture multiple images using different filters or intensity channels. Illustratively, in some embodiments, the crosstalk-aware base calling system 106 utilizes a two-channel implementation by capturing two images of a section of the nucleotide sample slide per sequencing cycle. In particular, the crosstalk-aware base calling system 106 captures a first image using a first filter and a second image using a second filter. The first and second images can capture the intensity of signals emitted from the target cluster and neighboring clusters corresponding to the filters.

[0046] However, the crosstalk-aware base calling system 106 can implement a sequencing run using alternative channel-based approaches. In some implementations, the crosstalk-aware base calling system 106 utilizes a four-channel implementation and captures four different images of a section of the flow cell. Similar to a two-channel implementation, the crosstalk-aware base calling system 106 can capture each image of the four-channel implementation using a different filter. Each image can capture the intensity of the emitted signal based on the image filter used for that image. Thus, in some cases, each of the four images shows an emitted signal with a different intensity. Additionally, the crosstalk-aware base calling system 106 can utilize a single-channel implementation and capture one image of a section of the nucleotide sample slide and capture the intensity of the emitted signal using a specific filter. In other embodiments, the crosstalk-aware base calling system 106 can utilize a one-channel implementation and capture one image of a section of the nucleotide sample slide (or a three-channel implementation and capture three images) and capture the intensity values ​​of the emitted signal by using a specific filter.

[0047] Based on the captured images of the intensities (e.g., color intensity and / or light intensity) of the signals emitted by the target cluster and adjacent clusters, the crosstalk-aware base calling system 106 can measure the intensities of the signals of the target cluster and adjacent clusters and provide intensity values ​​(e.g., wavelength and / or brightness) of the signals of the target cluster and adjacent clusters. For example, while utilizing two intensity channels, the crosstalk-aware base calling system 106 can measure the wavelengths of the signals emitted by the target cluster and adjacent clusters in a first channel and a second channel.

[0048] As further shown in FIG. 2 , the crosstalk-aware base calling system 106 can perform operation 204, which determines nucleobase calls and illumination indices for adjacent clusters. As previously described, the emitted signals of a cluster can indicate the type of nucleotide base. For example, in some embodiments, the crosstalk-aware base calling system 106 analyzes the intensity values ​​of signals from a given cluster in both channels or in each of multiple channels to determine a nucleobase call. In some embodiments, based on the intensity values ​​of the signals of the cluster in each channel, the crosstalk-aware base calling system 106 can utilize expectation maximization and Gaussian probability distribution to calculate the probability that the signal falls within the intensity value boundary of a particular base (A, C, G, or T). The crosstalk-aware base calling system 106 can then call the nucleobase incorporated in the cluster by selecting the intensity value boundary of the nucleobase with the highest probability. For example, based on the intensity values ​​emitted by the signals of the cluster, the crosstalk-aware base calling system 106 can determine that the intensity value boundary of the nucleobase with the highest probability for the cluster is adenine (A).

[0049] After determining the nucleobase call of the cluster, in some embodiments, the crosstalk-aware base calling system 106 determines an illumination index for the cluster. Based on the nucleobase call, for example, the crosstalk-aware base calling system 106 can determine whether the cluster was "on" (e.g., illuminated or actively emitting light intensity at a particular frequency) or off (e.g., not illuminated or not emitting light intensity at a particular frequency) in a given intensity channel during a sequencing cycle. For example, if the nucleobase call for the cluster is adenine (A), the crosstalk-aware base calling system 106 can determine that the first channel signal and the second channel signal of the cluster were "on" (or the cluster emitted light in both the first and second channels) during a sequencing cycle.

[0050] Although the previous embodiment describes the crosstalk-aware base calling system 106 determining the nucleobase call before determining the illumination index, in some embodiments, the crosstalk-aware base calling system 106 can perform these operations in the reverse order. For example, in some embodiments, the crosstalk-aware base calling system 106 can determine whether the illumination index was "on" or "off" for a given channel during a sequencing cycle and then determine the nucleobase call for the cluster based on the illumination index.

[0051] In some cases, the crosstalk-aware base calling system 106 can represent the states of illumination indices in the intensity channels of adjacent clusters as a set of illumination indices as a couplet. For example, in some embodiments, the crosstalk-aware base calling system 106 determines the adenine (A) nucleobase calls of adjacent clusters and, consequently, determines the corresponding illumination indices of adjacent clusters in two different channels as on / on or [1, 1]. As shown by FIG. 2 , in some embodiments, the illumination indices of cytosine (C), thymine (T), and guanine (G) nucleobase calls can be represented as on / off or [1, 0], off / on or [0, 1], and off / off or [0, 0], respectively.

[0052] As further shown in FIG. 2 , after determining the sets of nucleic acid base calls and illumination indices for the adjacent clusters, the crosstalk-aware base calling system 106 performs operation 206 of determining an inter-cluster interface metric for crosstalk of the adjacent clusters on the target cluster. For example, the crosstalk-aware base calling system 106 determines an inter-cluster interference metric based on the sets of illumination indices for the adjacent clusters. As described further below, in some embodiments, the crosstalk-aware base calling system 106 determines the inter-cluster interference metric based on the amplitude of the adjacent clusters, the set of illumination indices encoded for the adjacent clusters, and an estimated point spread function response from the position of the adjacent cluster to the position of the target cluster. Based on the estimated amplitude, illumination indices, and point spread function of the adjacent clusters, the crosstalk-aware base calling system 106 can measure the amount of crosstalk (e.g., optical interference) from the adjacent clusters on the target cluster.

[0053] In some cases, the crosstalk-aware base calling system 106 utilizes an inter-cluster interference metric as part of a function for subtracting crosstalk from a target cluster. Figure 5B and the corresponding paragraph below illustrate how the crosstalk-aware base calling system 106, in accordance with one or more embodiments, utilizes an inter-cluster interference metric to subtract the amplitudes of neighboring clusters.

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[0057] After determining the inter-cluster interference metric, the crosstalk-aware base calling system 106 performs operation 208, which generates a corrected intensity value for the target cluster by removing the inter-cluster interference metric. Based on the corrected intensity value of the target cluster, the crosstalk-aware base calling system 106 can make a more accurate nucleobase call for the target cluster. For example, the crosstalk-aware base calling system 106 can determine that the corrected intensity value of the target cluster resulted in a guanine (G) nucleobase call, while the uncorrected intensity value for the target cluster initially resulted in a cytosine (C) nucleobase call.

[0058] As previously described, the crosstalk-aware base calling system 106 can determine illumination indices and crosstalk in a given sequencing cycle according to a specific cluster order. For example, the crosstalk-aware base calling system 106 can identify a first subset of oligonucleotide clusters that emit the brightest signals within a top intensity value range (e.g., the top 10% brightest). The crosstalk-aware base calling system 106 then determines (i) nucleic acid base calls for the first subset of oligonucleotide clusters and (ii) an inter-cluster interference metric that estimates the interference of clusters from the first subset of oligonucleotide clusters on a second subset of oligonucleotide clusters that emits signals within a second intensity value range (e.g., the top 20-30% brightest). The order of the remaining clusters can follow the same or similar pattern based on additional intensity value ranges. In some cases, for example, the crosstalk-aware base calling system 106 determines (i) nucleic acid base calls for the second subset of oligonucleotide clusters, and (ii) an inter-cluster interference metric that estimates the interference of clusters from the second subset of oligonucleotide clusters on the third subset of oligonucleotide clusters that emit signals within a third intensity value range (e.g., top 30-40% brightest).

[0059] As just described, the crosstalk-aware base calling system 106 can determine nucleic acid base calls, illumination indices, and crosstalk in a given sequencing cycle in an order based on intensity value ranges. As an alternative to using intensity value ranges, the crosstalk-aware base calling system 106 can (i) identify a first subset of oligonucleotide clusters based on the DC offset and amplitude of the signals emitted by the clusters, and (ii) determine an inter-cluster interference metric that estimates the interference of clusters from the first subset of oligonucleotide clusters on a second subset of oligonucleotide clusters emitting signals. For example, the crosstalk-aware base calling system 106 can (i) identify a first subset of oligonucleotide clusters that exhibit a combination of DC offset and amplitude within a first threshold difference in received intensity values ​​for a given cluster, and (ii) identify a second subset of oligonucleotide clusters that exhibit a combination of DC offset and amplitude within a second threshold difference in received intensity values ​​for the given cluster.

[0060] Figure 2 provides an overview of operations performed by the crosstalk-aware base calling system 106 as part of generating a corrected intensity value for a target cluster by utilizing an inter-cluster interface metric to remove or reduce crosstalk from neighboring clusters on the target cluster. According to one or more embodiments, Figure 3 shows an example of crosstalk (e.g., optical interference) increasing between clusters as the distance between the clusters decreases. In particular, Figure 3 shows a one-dimensional cross-section of a two-dimensional nucleotide sample slide containing three clusters of oligonucleotides to illustrate how the distance between clusters affects crosstalk between clusters.

[0061] As previously discussed, some existing sequencing systems limit the number and density of oligonucleotide clusters on a flow cell to maintain accurate nucleic acid base calling. As shown in Figure 3, if there is sufficient distance between adjacent clusters of oligonucleotides 302, a central cluster of oligonucleotides 304, and an adjacent cluster of oligonucleotides 306, the signal of the central cluster of oligonucleotides 304, which has a relatively high intensity value, does not overlap (or only minimally overlaps) with the signals of adjacent clusters of oligonucleotides 302 and adjacent clusters of oligonucleotides 306, which have relatively low intensity values.

[0062] Because there is relatively little overlap between the signals of adjacent clusters of oligonucleotides 302, the central cluster of oligonucleotides 304, and the adjacent clusters of oligonucleotides 306, existing sequencing systems can more easily detect interference from the signal of the central cluster of oligonucleotides 304 on the intensity values ​​of the signals emitted by adjacent clusters of oligonucleotides 302 and adjacent clusters of oligonucleotides 306. Using more accurate or purer intensity values ​​for adjacent clusters of oligonucleotides 302 and adjacent clusters of oligonucleotides 306, crosstalk-aware base calling system 106 can more accurately make nucleic acid base calls and determine whether adjacent clusters of oligonucleotides 302 and adjacent clusters of oligonucleotides 306 are "on" (e.g., illuminating or emitting light intensity at a particular frequency) or "off" (e.g., not illuminating or emitting light intensity at a particular frequency) in a particular intensity channel during a sequencing cycle.

[0063] As further shown in Figure 3, as the density between clusters of oligonucleotides increases, it becomes more difficult to accurately determine intensity values; nucleic acid bases require clusters of oligonucleotides that emit relatively lower (e.g., relatively dimmer) intensity values, because light interference from clusters of oligonucleotides that emit relatively higher (e.g., relatively brighter) intensity values ​​affects the intensity values ​​of dimmer clusters of oligonucleotides.

[0064] 3 , for example, a decrease in the distance between adjacent clusters of oligonucleotides 308, the central cluster of oligonucleotides 310, and the adjacent clusters of oligonucleotides 312 causes increased crosstalk that interferes with the relatively low intensity values ​​of the adjacent clusters of oligonucleotides 308 and 312. More specifically, the optical signal emitted from the central cluster of oligonucleotides 310 interferes with or makes more difficult to detect the intensity values ​​of the adjacent clusters of oligonucleotides 308 and 312. As a result of optical interference and an increased likelihood of detecting the intensity value of the central cluster of oligonucleotides 310 being inaccurately attributed to the adjacent clusters of oligonucleotides 308 and 312, existing sequencing systems often cannot accurately make nucleobase calls for the adjacent clusters of oligonucleotides 308 and 312.

[0065] As described above, the crosstalk-aware base calling system 106 can determine nucleobase calls and corresponding illumination indices. According to one or more embodiments, Figure 4 shows the crosstalk-aware base calling system 106 determining nucleobase calls and corresponding sets of illumination indices for clusters of oligonucleotides in different channels for a given sequencing cycle. As described above, the illumination indices indicate whether and / or to what extent a cluster provides a fluorescent response in a particular intensity channel during sequencing.

[0066] In particular, Figure 4 shows the on / off states of sets of illumination indices in two different intensity channels for a cluster of oligonucleotides corresponding to a particular type of nucleotide base. To illustrate such on / off states, Figure 4 shows the light intensity at particular frequencies (e.g., frequency bands) that emit or do not emit from a cluster of oligonucleotides 402 in a cropped image shown in rows aligned with the nucleobase calls of adenine (A) 408, cytosine (C) 410, thymine (T) 412, and guanine (G) 414.

[0067] 4, when making a nucleobase call of adenine (A) 408 for cluster of oligonucleotides 402, crosstalk-aware base calling system 106 determines a first set of illumination indicia indicating that cluster of oligonucleotides 402 is "on" (e.g., illuminated or emitting a light intensity of a particular frequency) in both a first channel captured by first channel image 404 and a second channel captured by second channel image 406. In contrast, when making a nucleobase call of cytosine (C) 410 for cluster of oligonucleotides 402, crosstalk-aware base calling system 106 determines a second set of illumination indicia indicating that cluster of oligonucleotides 402 is "on" in the first channel captured by first channel image 404 and "off" (e.g., not illuminated or not emitting a light intensity of a particular frequency) in the second channel captured by second channel image 406. When making a nucleobase call of thymine (T) 412 for cluster of oligonucleotides 402, crosstalk-aware base calling system 106 determines a third set of illumination indices indicating that cluster of oligonucleotides 402 is "off" in the first channel captured by first channel image 404 and "on" in the second channel captured by second channel image 406. Finally, when making a nucleobase call of guanine (G) 414 for cluster of oligonucleotides 402, crosstalk-aware base calling system 106 determines a fourth set of illumination indices indicating that cluster of oligonucleotides 402 is "off" in both the first channel captured by first channel image and the second channel captured by second channel image 406.

[0068] As previously mentioned, the illumination state (e.g., on / active or off / inactive state) of an illumination indicium can take a couplet or a continuum form. For example, if an illumination indicium is "on" (and emits a light intensity of a particular frequency) in an intensity channel during sequencing, the "on" state can be represented by a 1. Conversely, if an illumination indicium is "off" (and does not emit a light intensity of a particular frequency) in an intensity channel during sequencing, the "off" state can be represented by a 0.

[0069] Thus, the illumination state of a cluster of oligonucleotides in two or more channels can be represented by a set of illumination indices. For example, a set of illumination indices represented by [1, 1] can indicate that the illumination indices for the cluster of oligonucleotides are "on" in the first intensity channel and the second intensity channel. Additionally, the crosstalk-aware base calling system 106 can decode the set of illumination indices based on the nucleic acid base call. For example, a set of illumination indices for a cluster of oligonucleotides having an adenine (A) nucleotide base can be represented by [1, 1]. A cytosine (C) nucleotide base can be represented by [1, 0], a thymine (T) nucleotide base can be represented by [0, 1], and a guanine (G) nucleotide base can be represented by [0, 0].

[0070] As described above, the illumination state of the illumination indicia can be continuous. More specifically, a given illumination indicia can indicate the degree to which a cluster of oligonucleotides is illuminated by light intensity at a particular frequency (e.g., a frequency band). For example, based on the likelihood that the cluster of oligonucleotides falls within an intensity value boundary defined by a Gaussian mixture model, the crosstalk-aware base calling system 106 can determine the degree to which the illumination indicia is illuminated in a given intensity channel. Furthermore, the crosstalk-aware base calling system 106 can determine the degree to which the continuous illumination indicia is illuminated based on the intensity value of the cluster of oligonucleotides.

[0071] Considering the relationship between the illumination indices and the nucleobase calls, in some embodiments, the crosstalk-aware base calling system 106 can update or adjust the set of illumination indices based on the corrected signal of the target cluster. For example, the crosstalk-aware base calling system 106 can generate a corrected (and more accurate) intensity value of the target cluster by removing an inter-cluster interference metric from the initial intensity value of the target cluster. Based on the corrected intensity value of the target cluster, the crosstalk-aware base calling system 106 can make a different nucleobase call for the target cluster. Based on the different, more accurate nucleobase call, the crosstalk-aware base calling system 106 can adjust the set of illumination indices to more accurately represent the "on" or "off" state of the illumination indices of the target cluster in the intensity channel. For example, in one or more embodiments, based on the initial intensity value of the target cluster, the crosstalk-aware base calling system 106 determines the nucleobase call of A and the set of illumination indices for the target cluster as [1, 1]. However, based on the corrected intensity values ​​of the target cluster and the corresponding nucleic acid base calls of T, the crosstalk-aware base calling system 106 determines that the adjusted set of illumination indices is [0, 1].

[0072] As described above, in one or more embodiments, the crosstalk-aware base calling system 106 can utilize an inter-cluster interference metric to remove crosstalk from adjacent clusters on a target cluster. According to one or more embodiments, Figures 5A and 5B show a crosstalk-aware base calling system 106 that utilizes an equalizer system to determine an inter-cluster interference metric that represents optical interference of adjacent clusters on a target cluster and generate a modified intensity value for the target cluster based on the inter-cluster interference metric.

[0073] As previously mentioned, the crosstalk-aware base calling system 106 can utilize an equalizer to estimate the modified signal. In some embodiments, the crosstalk-aware base calling system 106 can utilize a linear equalizer to determine intensity values ​​of target clusters by processing the received image. Generally, a linear equalizer is a linear filter that can be designed or optimized to remove noise. In certain embodiments, the equalizer can convert the intensity energy distributed over the received pixels into received intensity values ​​of the target cluster and neighboring clusters by linearly weighting the pixel intensities. In some embodiments, the linear filter can be applied to each cluster individually or across the entire image. In particular, FIG. 5A illustrates a model of the equalizer system.

[0074] When implemented on a sequencing device, in some embodiments, the crosstalk-aware base calling system 106 can utilize a linear equalizer to calculate a weighted sum of intensity values ​​of pixels depicting intensity emissions from a target cluster and one or more adjacent clusters. The equalizer may be trained, for example, to generate equalizer coefficients configured to mix / combine the intensity values ​​of pixels depicting intensity emissions from the target cluster and adjacent clusters in a manner that maximizes the signal-to-noise ratio.

[0075] As shown in FIG. 5A, the crosstalk-aware base calling system 106 can receive an input image 503 of a section of a nucleotide sample slide. The input image can include pixels that indicate intensity values ​​of a target cluster and nearby neighboring clusters. Based on the received input image, an equalizer collects light energy from the pixels and quantizes the energy into an intensity value (y i,c,j ) The system model of the equalizer 505 can be transformed into y i,c,j =a i,c,j v i,c,j +d i,c,j +n i,c,jIt can be modeled as the amplification factor a i,c,j accounts for the scale variation between clusters on the nucleotide sample slide for cycle (c), channel (j), and cluster (i). i,c,j ) accounts for the unsealed and unshifted signal for cycle (c), channel (j), and cluster (i). DC offset (d i,c,j ) accounts for random noise caused by different cluster sizes, different background intensities, varying stimulus responses, varying focus, varying sensor sensitivity, and varying lens aberrations for cycle (c), channel (j), and cluster (i). i,c,j ) represents the additive noise of cycle (c), channel (j), and cluster (i).

[0076] Using the system model for equalizer 505 to process the input, crosstalk-aware base calling system 106 can determine the intensity value 507 P[x,y,c,j] of the pixel at cycle (c), position (x,y), and channel (j). As discussed below in FIG. 5B, crosstalk-aware base calling system 106 can use the pixel intensities to determine modified intensity values ​​for the target cluster. While the described embodiment uses a linear equalizer to determine the intensities of the pixels that describe the target cluster, other embodiments may use the methods described in conjunction with an intensity detection system and / or intensity extraction system. In some embodiments, the crosstalk-aware base calling system 106 utilizes an equalizer such as that described by U.S. Pat. No. 11,188,778, entitled "Equalization-Based Image Processing and Spatial Crosstalk Attenuator," by Eric Ojard et al., and U.S. patent application Ser. No. 18 / 059,326, entitled "Generating Cluster-Specific-Signal Corrections for Determining Nucleotide-Base Calls," by Eric Ojard et al., each of which is incorporated by reference in its entirety.

[0077] 5B and as discussed above, the crosstalk-aware base calling system 106 can perform operations to determine nucleobase calls and illumination indices for adjacent clusters 502. As previously described, the crosstalk-aware base calling system 106 can detect and / or measure light emitted by adjacent clusters in a given channel during a sequencing cycle and determine intensity values ​​of the emitted light. In some cases, based on the intensity values ​​for the adjacent clusters, the crosstalk-aware base calling system 106 determines nucleobase calls for the adjacent clusters.

[0078] To illustrate such intensity-value-based base calling, in some embodiments, the crosstalk-aware base calling system 106 can apply the expected maximum to a 2D Gaussian mixture model to define intensity value boundaries corresponding to each type of nucleobase (A, C, T, or G). Based on the intensity values ​​of light emitted by labeled nucleotides incorporated into a cluster of oligonucleotides for a given sequencing cycle, the crosstalk-aware base calling system 106 can determine the probability that the intensity value of the cluster of oligonucleotides falls within one of four intensity value boundaries corresponding to each type of nucleobase. The crosstalk-aware base calling system 106 can then call the nucleobase for the cluster of oligonucleotides by selecting the nucleobase with the highest probability according to the intensity value boundaries. As discussed above, in some embodiments, based on the nucleic acid base calls, the crosstalk-aware base calling system 106 can determine a set of illumination indices for the clusters.

[0079] For example, the crosstalk-aware base calling system 106 can determine the "on" and / or "off" states of the illumination indices for adjacent clusters in one or more intensity channels. As described above, in some cases, the crosstalk-aware base calling system 106 can represent the illumination states of the illumination indices in couplet format. For example, if the crosstalk-aware base calling system 106 performs adenine (A) nucleobase calls on adjacent clusters, the crosstalk-aware base calling system 106 determines that the illumination indices for the cluster of oligonucleotides are "on" in both the first intensity channel and the second intensity channel. Based on this determination, the crosstalk-aware base calling system 106 can represent the on states of the cluster of oligonucleotides in both channels as a set of illumination indices [1, 1]. As described in more detail below, the crosstalk-aware base calling system 106 can utilize the data in the set of illumination indices to determine an inter-cluster interference metric.

[0080] 5B , in some embodiments, the crosstalk-aware base calling system 106 utilizes a signal model for the target cluster 504. More specifically, the crosstalk-aware base calling system 106 can utilize a function to determine an initial intensity value (P[x,y,c,j]) for a pixel (P) representing a target cluster of oligonucleotides at a position [x,y], where (x) represents the horizontal coordinate of the pixel and (y) represents the vertical coordinate of the pixel. As the signal model indicates, the initial intensity value (P[x,y,c,j]) of the pixel representing the target cluster can include the sum of intensity values ​​from the background, the target cluster, and crosstalk emitted from adjacent clusters.

[0081] As shown in FIG. 5B, for example, the intensity value (P[x,y,c,j]) of a target cluster can be modeled as follows:

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[0083] The background intensity is

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[0086] As further shown in FIG. 5B, the sum of the intensity values ​​for the target cluster is an estimate of the amplitude of the target cluster and neighboring clusters with cluster index (i) during sequencing cycle (c) in intensity channel (j).

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[0089] As explained above, in some cases, the couplet format encoded in a target cluster may be represented by a set of illumination indices for the target cluster (e.g., [1,1], [1,0], [0,1], or [0,0]). However, as noted above, crosstalk from neighboring clusters with high intensity values ​​can inflate the intensity values ​​of a target cluster. The increased intensity values ​​of a target cluster may result in a false indication that the target cluster was on in the first or second intensity channel during sequencing.

[0090] FIG. 5B further illustrates that the signal model of the target cluster is based on the center position (x i ,y i ) can include estimates of the PSF covering various positions (x, y) relative to the center position (x, y) of the PSF response 504. For example, the crosstalk-aware base calling system 106 can i ,yi ) or the center position (x i ,y i ) can be estimated for a second location that is farther from the target cluster. As described above, the estimated PSF can indicate how crosstalk extending from adjacent clusters interferes with the intensity values ​​of the target cluster. More specifically, the estimated PSF can estimate the PSF response of the target cluster's location relative to the center of the PSF responses from adjacent clusters.

[0091] As further shown in FIG. 5B, the crosstalk-aware base calling system 106 can determine an inter-cluster interference metric 506. As described above, the inter-cluster interference metric (I i0 _ i1 ) can represent the optical interference of one cluster (represented as i1) on another cluster (represented as i0). For example, the inter-cluster interference metric (I i0 _ i1 ) can represent the optical interference from neighboring clusters on the target cluster.

[0092] Function I in Figure 5B i0 _ i1 As shown, the crosstalk-aware base calling system 106 can detect the amplitude of adjacent clusters.

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[0097] The crosstalk-aware base calling system 106 further determines an illumination index of the adjacent cluster (i1) based on the intensity value of the adjacent cluster (i1).

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[0099] In addition, as shown in FIG. 5B, the crosstalk-aware base calling system 106 determines the center position (x i1 ,y i1 ) (or region) of the PSF response, the position (x i0 ,y i0 ) can be estimated. As described above, the estimated PSF corresponding to the position of the target cluster can describe how the intensity values ​​of the neighboring clusters affect the intensity values ​​of the target cluster based on the positions of the neighboring clusters and the target cluster.

[0100] As further illustrated by FIG. 5B and the corresponding function, the crosstalk-aware base calling system 106 can subtract 508 the inter-cluster metric from the sum of the intensity values ​​of the target cluster. In particular, the crosstalk-aware base calling system 106 can remove the inter-cluster interference metric of the adjacent cluster (i1) from the sum of the intensity values ​​of the target cluster (i0). Similarly, as further illustrated by FIG. 5B, the crosstalk-aware base calling system 106 can determine and remove the inter-cluster interference metric from the adjacent cluster (i2) to the adjacent cluster (i1) from the sum of the intensity values ​​for the target cluster (i0). Thus, the crosstalk-aware base calling system 106 can determine and remove inter-cluster interference metrics for multiple adjacent clusters from the intensity values ​​of a single target cluster. For example, in some embodiments, the crosstalk-aware base calling system 106 can estimate and remove the inter-cluster interference metric of the adjacent cluster with the highest intensity closest to the target cluster. Additionally, the crosstalk-aware base calling system 106 can subtract crosstalk arising from the neighboring cluster (i1) from any other cluster position on the flow cell.

[0101] As described above, the crosstalk-aware base calling system 106 can iteratively determine inter-cluster interference metrics for crosstalk for a subset of adjacent clusters on each subset of target clusters, and remove the inter-cluster interference metrics for the subset of adjacent clusters from each subset of target clusters based on the intensity value range of the subset of adjacent clusters. For example, the crosstalk-aware base calling system 106 can determine nucleobase calls for a first subset of adjacent oligonucleotide clusters that emit the brightest signals within a top intensity value range (e.g., the top 10% are brightest). In some cases, the crosstalk-aware base calling system 106 calls the nucleobase for the brightest cluster because it is most likely to fall within the intensity value boundary associated with one of the nucleobases (e.g., A). From the nucleobase calls of the first subset of adjacent oligonucleotide clusters, the crosstalk-aware base calling system 106 determines (i) an illumination index for each cluster from the first subset of adjacent oligonucleotide clusters, and (ii) an inter-cluster interference metric for each adjacent cluster from the first subset of adjacent oligonucleotide clusters for each target cluster from the subset of target oligonucleotide clusters. The crosstalk-aware base calling system 106 further removes inter-cluster interference metrics of the first subset of adjacent oligonucleotide clusters from the sum of the intensity values ​​of the individual target clusters.

[0102] After such removal, the crosstalk-aware base calling system 106 can determine nucleic acid base calls, illumination indices, and inter-cluster interference metrics for a second subset of adjacent clusters within a second intensity value range (e.g., the top 20-30% brightest). The crosstalk-aware base calling system 106 can further remove inter-cluster interference metrics for individual adjacent clusters in the second subset of adjacent clusters from the total intensity values ​​of individual target clusters from the second subset of target oligonucleotide clusters.

[0103] 5B, after the crosstalk-aware base calling system 106 removes the inter-cluster interference metrics, the crosstalk-aware base calling system 106 can generate corrected intensity values ​​for the pixel(s) that describe the target cluster 510. As shown by FIG.

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[0109] For example, as described above, the crosstalk-aware base calling system 106 can calculate the probability that a signal falls within the intensity value boundary of a particular nucleobase (A, C, G, or T) based on a Gaussian probability distribution and expectation maximization. By removing the inter-cluster interference metric, the crosstalk-aware base calling system 106 can determine a more accurate probability that a signal falls within the intensity value boundary of a particular nucleobase (A, C, G, or T) based on the more accurate intensity value of the signal. In some embodiments, the updated probability may change the call or prediction of the nucleobase incorporated into the cluster. In other embodiments, the updated probability may not change the call or prediction of the nucleobase incorporated into the cluster, but may provide a higher base call quality metric (e.g., QUAL score) for signals from the cluster that fall within the intensity value boundary of the originally called or predicted nucleobase.

[0110] As just noted, in some cases, the crosstalk-aware base calling system 106 estimates a PSF response for a section of a nucleotide sample slide that includes the target cluster and adjacent clusters. According to one or more embodiments, Figure 6 illustrates the crosstalk-aware base calling system 106 estimating a point spread function for the intensity values ​​of the clusters.

[0111] As shown in Figure 6, the estimated PSF can describe the response at a certain location or region relative to the central PSF response of a point source (e.g., a cluster of oligonucleotides). More specifically, Figure 6 shows a mathematically modeled PSF response of intensity values ​​from a cluster of oligonucleotides 602. As shown in Figure 6, the estimated PSF of the intensity values ​​of the cluster of oligonucleotides is most concentrated (e.g., brightest) at the central location or region, and the signal from the cluster of oligonucleotides decreases as the signal moves away from the central location or region of the cluster of oligonucleotides. The crosstalk-aware base calling system 106 can utilize the estimated PSF response to estimate the degree of crosstalk from neighboring clusters to the target cluster.

[0112] In some embodiments, the PSF may be estimated by utilizing a least squares (LS) or minimum mean square error (MMSE) method. For example, under the least squares (LS) approach, a detector receives a signal (y) and determines a PSF estimate (x).

[0113]

number

[0114]

number

[0115]

number

[0116]

number

[0117]

number

[0118]

number

[0119]

number

[0120] In accordance with one or more embodiments, FIGS. 7A-7C illustrate the effect of crosstalk between clusters of oligonucleotides and the elimination of optical interference for specific clusters of oligonucleotides. In particular, FIGS. 7A-7C provide, for illustrative purposes, simulated images of clusters of oligonucleotides on a nucleotide sample slide and crosstalk between the clusters of oligonucleotides. While the images in FIGS. 7A-7C show the clusters as an evenly spaced square grid, actual clusters of oligonucleotides are not uniformly dispersed on the nucleotide sample slide. Furthermore, FIGS. 7A-7C show clusters at the center of each pixel within the square grid to more clearly illustrate the effects of crosstalk. Furthermore, although FIGS. 7A-7C show a nucleotide sample slide utilizing a square grid, other embodiments of the nucleotide sample slide can utilize various shapes (e.g., diamond, hexagon, etc.).

[0121] As an overview, FIG. 7A shows an image 700 that maps the intensity values ​​of a cluster of oligonucleotides in response to optical excitation in an intensity channel. Image 700 can represent a section of a nucleotide sample slide (e.g., a flow cell) seeded with clusters of oligonucleotides. As shown in FIG. 7A, image 700 for an intensity channel includes several clusters of oligonucleotides and maps corresponding intensity values ​​to pixels. In particular, image 700 for an intensity channel uses pixels to represent the intensity value at a given location in the flow cell. The intensity value represented by each pixel is the sum of the intensity values ​​of the cluster of oligonucleotides, noise, and crosstalk from neighboring clusters.

[0122] Figure 7A also shows clusters of oligonucleotides adjacent to other clusters on a section of a nucleotide sample slide. In particular, Figure 7A shows clusters of oligonucleotides that are first adjacent, second adjacent, or third adjacent with respect to a target cluster. An adjacent cluster is first adjacent to a target cluster when such an adjacent cluster is located one cluster away from the target cluster or immediately adjacent to the target cluster with respect to other clusters. For example, the eight adjacent clusters within the first adjacent boundary 712 are first adjacent to the "off" cluster of oligonucleotide 702a because the eight adjacent clusters are next to (and close to) the "off" cluster of oligonucleotide 702a with respect to other clusters. Relatedly, an adjacent cluster is second adjacent to a target cluster when such an adjacent cluster is located two clusters away from the target cluster or next to the target cluster. For example, as shown in FIG. 7A , the 16 adjacent clusters within the second adjacent boundary 714 (and outside the first adjacent boundary 712) are second-neighbors to the "off" cluster of oligonucleotide 702a because they are located next to the "off" cluster of oligonucleotide 702a. Similarly, adjacent clusters are third-neighbors to the target cluster when such adjacent clusters are located three clusters away from the target cluster, or next to the target cluster, or next to the target cluster. As shown in FIG. 3 , the 24 adjacent clusters within the third adjacent boundary 716 (and outside the second adjacent boundary 714) are third-neighbors to the "off" cluster of oligonucleotide 702a because they are located three clusters away from (or next to) the "off" cluster of oligonucleotide 702a. As described above, in some embodiments, the crosstalk-aware base calling system 106 determines inter-cluster interference metrics for clusters that are first adjacent, second adjacent, and / or third adjacent to the target cluster.

[0123] To represent the different types of light emitted by the various clusters, Figure 7A shows different patterns representing the varying intensity values ​​of the different clusters and different circular shapes representing illumination indices of the clusters within image 700. As indicated by the darker or dimmer and lighter or lighter patterns, darker and / or dimmer pixels represent locations and / or clusters of oligonucleotides with lower intensity values, while lighter and / or brighter pixels represent locations and / or clusters of oligonucleotides with higher intensity values. Furthermore, pixels containing black circles with white borders represent "off" clusters of oligonucleotides that do not emit (or are not detected to emit) a light intensity at a particular frequency (e.g., frequency band) for a given channel, while pixels containing white circles with black borders represent "on" clusters of oligonucleotides that emit (or are detected to emit) a light intensity at a particular frequency (e.g., frequency band) for a given channel.

[0124] As discussed above, it can be difficult to nucleobase call an "off" cluster of oligonucleotides adjacent to or surrounded by an "on" cluster of oligonucleotides with a high intensity value because crosstalk from an "on" cluster of oligonucleotides with a high intensity value distorts the intensity value of the "off" cluster of oligonucleotides. In particular, Figure 7A shows how crosstalk emitted from an "on" cluster of oligonucleotides with a high intensity value increases the intensity value of a neighboring or adjacent "off" cluster of oligonucleotides. For example, in Figure 7A, the "off" cluster of oligonucleotide 702a appears to be an "on" cluster of oligonucleotides because crosstalk from the bright neighboring clusters of oligonucleotides 706a and 706b causes the pixels containing the "off" cluster of oligonucleotide 702a to appear brighter (e.g., increase the intensity value of the pixel). By making the pixels containing the "off" cluster of oligonucleotide 702a appear brighter, the likelihood of making an incorrect nucleobase call to the "off" cluster of oligonucleotide 702a increases. Furthermore, Figure 7A shows how the lower intensity values ​​of the dim "on" cluster of oligonucleotides make it difficult to determine nucleobase calls for the dim "on" cluster of oligonucleotides because they appear to have similar intensity values ​​to the intensity values ​​of the adjacent "off" cluster of oligonucleotides. Further details regarding the dim "on" cluster of oligonucleotides are provided in Figure 8A.

[0125] According to one or more embodiments, FIG. 7B illustrates the crosstalk-aware base calling system 106 first determining nucleobase calls and illumination indices for a subset of clusters as part of an ordered approach to removing crosstalk. For example, the subset of clusters of oligonucleotides highlighted by selection boundaries 708a, 708b, 708c, 708d, 708e, 708f, 708g, 708h, 708i, 708j, and 708k represents clusters of oligonucleotides emitting the highest intensity values ​​within a top intensity value range (e.g., the top 10% or top 15%). As previously discussed, the high intensity values ​​of such subsets of clusters of oligonucleotides enable the crosstalk-aware base calling system 106 to make more reliable determinations of nucleobase calls. Based on the nucleobase calls for the subset of clusters of oligonucleotides highlighted by selection boundaries 708a-708k, the crosstalk-aware base calling system 106 can more accurately determine that a cluster of oligonucleotides is "on" in an intensity channel.

[0126] Although it is easier to make nucleobase calls for the subset of clusters of oligonucleotides highlighted by selection boundaries 708a-708k, in some cases, these clusters of oligonucleotides generate the most crosstalk (e.g., optical interference), affecting neighboring clusters of oligonucleotides with lower intensity values. For example, as shown in Figure 7B, the "off" cluster of oligonucleotides 702a is surrounded by the subset of clusters of oligonucleotides highlighted by selection boundaries 708d, 708g, and 708h, which emit the highest intensity values ​​and levels of crosstalk. Crosstalk from the subset of clusters of oligonucleotides highlighted by selection boundaries 708d, 708g, and 708h increases the intensity value of the "off" cluster of oligonucleotides 702a. By increasing the intensity value of the "off" cluster of oligonucleotides 702a, the "off" cluster of oligonucleotides 702a is more likely to receive an incorrect nucleobase call without an effective method for removing the crosstalk.

[0127] While Figures 7A-7B illustrate the effect of crosstalk between clusters of oligonucleotides, Figure 7C illustrates the effect of removing crosstalk from a particular cluster of oligonucleotides. As shown in Figure 7C, the crosstalk-aware base calling system 106 removes crosstalk from the intensity values ​​of various target clusters, from subsets of clusters of oligonucleotides highlighted by selection boundaries 708a-708k. To illustrate the intensity values ​​of light emitted by the target clusters without the light emitted by the subsets of clusters of oligonucleotides highlighted by selection boundaries 708a-708k, Figure 7C depicts an image 700 with a pattern indicating that the light emitted by such subsets of clusters has been removed. Before removing the crosstalk, as discussed above, the crosstalk-aware base calling system 106 can (i) determine nucleic acid base calls and determine a set of illumination indices for the subset of clusters of oligonucleotides highlighted by the selection boundaries 708a-708k, (ii) determine inter-cluster interference metrics for each cluster of the subset of clusters of oligonucleotides highlighted by the selection boundaries 708a-708k, and (iii) remove the inter-cluster interference metrics of the subset of clusters of oligonucleotides from other adjacent clusters of oligonucleotides that have dimmer intensity values.

[0128] When the inter-cluster interference metric is removed, image 700 shows dimmer, more accurate intensity values ​​of the "on" and "off" clusters of oligonucleotides. For example, crosstalk-aware base calling system 106 cancels out or removes crosstalk emitted by clusters of oligonucleotides 710a and 710b, which interferes with the relatively low intensity value of the target cluster of oligonucleotides. As suggested by FIG. 7C, the intensity value of the "off" cluster of oligonucleotides 702a more clearly indicates that the cluster of oligonucleotides 702a is "off" in a particular channel. Thus, the intensity value of the cluster of oligonucleotides 702a more closely resembles the intensity value of the cluster of oligonucleotides 702b, both of which do not emit light intensity at a particular frequency (e.g., frequency band) within the channel captured by image 700.

[0129] 7C, by removing crosstalk from adjacent clusters of oligonucleotides with the highest intensity values ​​on a target cluster, crosstalk-aware base calling system 106 determines a corrected intensity value for the cluster of oligonucleotides 704, thereby revealing that the cluster of oligonucleotides 704 is "on" or emits light intensity at a particular frequency (e.g., frequency band) in an intensity channel during sequencing. Thus, crosstalk-aware base calling system 106 (i) can make more accurate nucleic acid base calls based on the more accurate and corrected intensity value of the target cluster of oligonucleotides, and (ii) can more reliably determine that a given cluster of oligonucleotides is "on" or emits light intensity at a particular frequency (e.g., frequency band) in a given channel during a sequencing cycle.

[0130] As described above, the crosstalk-aware base calling system 106 improves the accuracy with which illumination indices (and corresponding nucleic acid base calls) can be determined by determining and removing inter-cluster interference metrics. According to one or more embodiments, Figures 8A-8B show histograms of intensity values ​​for clusters of oligonucleotides with and without crosstalk from neighboring clusters.

[0131] For example, as shown in FIG. 8A , a cluster of oligonucleotides with higher intensity values ​​806, indicated by black values ​​in the histogram, represents a cluster with intensity values ​​for which accurate nucleobase calls can be more easily determined based on a clearly "on" illumination indicator. The relatively high (or brightest) intensity values ​​reduce the likelihood that the crosstalk-aware base calling system 106 will make an inaccurate nucleobase call due to crosstalk from adjacent clusters. Thus, the crosstalk-aware base calling system 106 can more easily determine whether a cluster of oligonucleotides with relatively higher intensity values ​​is "on." Conversely, a cluster of oligonucleotides with lower intensity values ​​802, indicated by white values ​​on the histogram, represents a cluster with intensity values ​​for which accurate nucleobase calls can more easily be determined based on a clearly "off" illumination indicator. Thus, in the illustrated embodiment, the crosstalk-aware base calling system 106 can more easily determine whether a cluster of oligonucleotides with lower intensity values ​​802 is "off."

[0132] However, as further shown in FIG. 8A , the histogram includes regions of overlapping intensity values ​​804, indicated by black and white striped values, where determining nucleobase calls and illumination indices is difficult. For example, if the increased intensity values ​​of an "off" cluster of oligonucleotides overlap with the intensity values ​​of an "on" cluster of oligonucleotides, the clusters with overlapping intensity values ​​804 may prove difficult to determine accurate nucleobase calls and illumination indices. As discussed above, in some cases, crosstalk from a bright, neighboring cluster of oligonucleotides increases the intensity value of a dim "off" oligonucleotide cluster, creating an "off" cluster of oligonucleotides that appears "on" or exhibits intensity values ​​that may or may not be "on." In addition, as discussed above, some "on" clusters of oligonucleotides may not actually emit light intensity at a particular frequency (e.g., frequency band) within an intensity channel with a high intensity value, and may appear "off" within the intensity channel.

[0133] In some cases, existing sequencing systems specify an intensity value threshold to determine whether the intensity values ​​of a given cluster of oligonucleotides indicate that the given cluster emits light intensity at a particular frequency (e.g., a frequency band) within an intensity channel. However, as shown in FIG. 8A, the intensity value threshold rarely accurately determines whether clusters of oligonucleotides exhibiting overlapping intensity values ​​804 should have an "on" or "off" illumination indices for a given intensity channel. Thus, the histogram shown in FIG. 8A demonstrates that existing sequencing systems using intensity value thresholds (without an effective method for removing or reducing crosstalk) are unable to accurately resolve illumination indices and corresponding nucleobase calls for clusters of oligonucleotides exhibiting overlapping intensity values ​​804.

[0134] According to one or more embodiments, FIG. 8B illustrates that by determining and removing an inter-cluster interference metric representing crosstalk emitted from adjacent clusters of oligonucleotides, the crosstalk-aware base calling system 106 determines more accurate corrected intensity values ​​(and corresponding nucleobase calls) for the target cluster. For example, FIG. 8B illustrates corrected (or more accurate) intensity values ​​for a cluster of oligonucleotides. As shown in FIG. 8B, the corrected intensity values ​​808 of the "off" clusters of oligonucleotides, represented as white values, do not overlap with the corrected intensity values ​​810 of the "on" clusters of oligonucleotides, represented as black values. By clearly separating the corrected intensity values ​​808 of the "off" clusters of oligonucleotides from the corrected intensity values ​​810 of the "on" clusters of oligonucleotides, the crosstalk-aware base calling system 106 can apply intensity value ranges to clearly distinguish between the "on" and "off" clusters of oligonucleotides and more accurately determine nucleobase calls for such clusters of oligonucleotides.

[0135] 1-8B, corresponding text, and examples provide several different methods, systems, devices, and non-transitory computer-readable media for a crosstalk-aware base calling system 106. In addition to the above, one or more embodiments can also be described in terms of a flowchart including operations for achieving a particular result, such as that shown in FIG. 9. FIG. 9 may be performed with more or fewer operations. Furthermore, operations may be performed in a different order. Furthermore, operations described herein may be repeated or performed in parallel with each other or with different occurrences of the same or similar operations.

[0136]

[0023] Figure 9 illustrates a flowchart of a series of operations 900 for generating a quality metric for nucleobase calling using an inter-cluster interference metric, according to one or more embodiments. While Figure 9 illustrates operations according to one embodiment, alternative embodiments may omit, add, reorder, and / or modify any of the operations shown in Figure 9. In some implementations, the operations of Figure 9 are performed as part of a method. In some cases, a non-transitory computer-readable medium stores instructions that, when executed by at least one processor, cause a computing device to perform the operations of Figure 9. In some implementations, a system performs the operations of Figure 9. For example, in one or more cases, the system includes at least one processor and a non-transitory computer-readable medium containing instructions, the instructions, when executed by the at least one processor, cause the system to perform the operations of Figure 9.

[0137] The series of operations 900 includes an operation 902 for detecting a set of intensity values ​​from a first cluster and a second cluster. For example, operation 902 can include detecting intensity values ​​from a first signal from the first cluster and a second signal from the second cluster.

[0138] Additionally, the series of operations 900 includes an operation 904 of determining a set of lighting indices for a first cluster. For example, operation 904 may include determining nucleobase calls for the first cluster and determining a set of lighting indices for the first cluster based on the nucleobase calls.

[0139] Further, the series of operations 900 includes an operation 906 of determining an inter-cluster interference metric. For example, operation 906 may include estimating a degree of crosstalk from a first cluster to a second cluster by multiplying the estimated amplitude of the first cluster, the set of illumination indices for the first cluster, and the point spread function response.

[0140] The series of operations 900 further includes operation 908 of generating modified intensity values ​​for the second cluster by removing the inter-cluster interference metric. In particular, operation 908 can include generating a modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric from the second set of intensity values ​​and subtracting the inter-cluster interference metric from a sum of the intensity values ​​of the second cluster for the sequencing cycle.

[0141] In some cases, the series of operations includes an additional operation of determining a set of illumination metrics further based on amplitudes for the first set of intensity values ​​and an estimated point spread function for the section of the nucleotide sample slide including the first cluster of oligonucleotides, and determining an inter-cluster interference metric further based on the estimated point spread function.

[0142] In one or more embodiments, the series of operations 900 further includes an additional operation of estimating a point spread function using the location of the second cluster of oligonucleotides or a different cluster of oligonucleotides as a point, including a region that includes the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

[0143] In some cases, the series of operations 900 includes further operations in which a first position within the nucleotide sample slide for a first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second position within the nucleotide sample slide for a second cluster of oligonucleotides.

[0144] Further, in one or more embodiments, the series of operations 900 includes the additional operations of determining, for the sequencing cycle, nucleobase calls for a first cluster of oligonucleotides based on a first set of intensity values ​​and intensity value boundaries for the nucleobases, and determining a set of illumination indices further based on the nucleobase calls for the first cluster of oligonucleotides.

[0145] In some embodiments, the series of operations 900 also includes the additional operation of determining nucleobase calls for the first cluster of oligonucleotides based on the intensity values ​​from the first set of intensity values ​​corresponding to the first channel and the intensity values ​​from the first set of intensity values ​​corresponding to the second channel, and generating a modified second set of intensity values ​​by subtracting a value for the inter-cluster interference metric from the intensity values ​​from the second set of intensity values ​​corresponding to the first channel or the intensity values ​​from the second set of intensity values ​​corresponding to the second channel. In some cases, the series of operations 900 includes generating the modified second set of intensity values ​​by subtracting a value for the inter-cluster interference metric from the intensity values ​​from the second set of intensity values ​​corresponding to the first channel or the intensity values ​​from the second set of intensity values ​​corresponding to the second channel. Thus, in certain embodiments, the inter-cluster interference metric may be removed or canceled from the intensity values ​​in both the first channel and the second channel.

[0146] Additionally, in other embodiments, the series of operations 900 may include the additional operations of determining a first illumination index indicating whether a first cluster of oligonucleotides is illuminated or not illuminated in a first channel during a sequencing cycle, determining a second illumination index indicating whether a second cluster of oligonucleotides is illuminated or not illuminated in a second channel during a sequencing cycle, or determining a first continuous illumination index indicating the degree to which the first cluster of oligonucleotides is illuminated in the first channel during a sequencing cycle, and determining a second continuous illumination index indicating the degree to which the first cluster of oligonucleotides is illuminated in the second channel during a sequencing cycle.

[0147] In one or more cases, the series of operations 900 includes an additional operation of determining, for a sequencing cycle, based on a modified second set of intensity values, an adjusted set of illumination indices representing whether a second cluster of oligonucleotides is illuminated during the sequencing cycle, the adjusted set of illumination indices being different from the initial set of illumination indices corresponding to the second set of intensity values.

[0148] In some implementations, the series of operations 900 further includes an additional operation of determining, for the sequencing cycle and based on the modified second set of intensity values, nucleobase calls for a second cluster of oligonucleotides that differ from the nucleobases corresponding to the second set of intensity values.

[0149] In an additional embodiment, the series of operations 900 includes the additional operations of detecting a third set of intensity values ​​for a third signal from a third cluster of oligonucleotides for the sequencing cycle; determining an additional set of illumination indices representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determining an additional inter-cluster interference metric estimating optical interference from the third cluster of oligonucleotides to the second cluster of oligonucleotides based on the additional set of illumination indices; and generating a modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides for the sequencing cycle by removing the inter-cluster interference metric and the additional inter-cluster interference metric from the second set of intensity values.

[0150] Furthermore, in one or more embodiments, the series of operations 900 further includes the additional operations of determining that a first set of intensity values ​​for a first signal from a first cluster of oligonucleotides is within the intensity value range, determining that a second set of intensity values ​​for a second signal from a second cluster of oligonucleotides is not within the intensity value range, and generating a modified second set of intensity values ​​by removing from the second set of intensity values ​​an inter-cluster interference metric that estimates optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides based on the first set of intensity values ​​being within the intensity value range and the second set of intensity values ​​being not within the intensity value range. Alternatively, the series of operations 900 includes generating a modified second set of intensity values ​​by removing from the second set of intensity values ​​an inter-cluster interference metric that estimates optical interference from one or more pixels depicting the first cluster of oligonucleotides on one or more pixels depicting the second cluster of oligonucleotides based on the first set of intensity values ​​being within the intensity value range and the second set of intensity values ​​being not within the intensity value range.

[0151] In some cases, the series of operations 900 includes the further operations of determining a first nucleobase call for a first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values ​​within the intensity value range based on the first set of intensity values, and determining a second nucleobase call for a second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values ​​not within the intensity value range based on a revised second set of intensity values.

[0152] In one or more embodiments, the crosstalk-aware base calling system 106 detects a first set of intensity values ​​by detecting a first intensity value for a first signal from a first cluster of oligonucleotides in a single channel, detects a second set of intensity values ​​by detecting a second intensity value for a second signal from a second cluster of oligonucleotides in a single channel, and determines a set of illumination indices by determining a single illumination indices representing whether the first cluster of oligonucleotides is illuminated in the single channel during a sequencing cycle.

[0153] The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. Particularly applicable techniques involve attaching nucleic acids to fixed positions within an array so that their relative positions do not change, and repeatedly imaging the array. For example, embodiments in which images are obtained in different color channels corresponding to different labels used to distinguish one nucleotide base type from another are particularly applicable. In some embodiments, the process of determining the nucleotide sequence of a target nucleic acid (i.e., a nucleic acid polymer) can be an automated process. A preferred embodiment involves sequencing-by-synthesis (SBS) techniques.

[0154] SBS technology generally involves the enzymatic extension of nascent nucleic acid chain by repeatedly adding nucleotide to template chain.In the traditional method of SBS, a single nucleotide monomer can be provided to target nucleic acid in the presence of polymerase in each delivery.However, in the method described herein, in the presence of polymerase during delivery, two or more types of nucleotide monomers can be provided to target nucleic acid.

[0155] The SBS techniques described below can utilize single-read sequencing or paired-end sequencing. In single-read sequencing, the sequencer reads a fragment from one end to the other to generate a base-paired sequence. In contrast, during paired-end sequencing, the sequencer starts with one read, finishes reading a specified read length in the same direction, and starts another read from the opposite end of the fragment.

[0156] SBS can utilize nucleotide monomers with terminator moieties or nucleotide monomers lacking any terminator moiety. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides, as described in more detail below. In methods using nucleotide monomers without terminators, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. In SBS techniques utilizing nucleotide monomers with terminator moieties, the terminators can be effectively irreversible under the sequencing conditions used, as in conventional Sanger sequencing using dideoxynucleotides, or the terminators can be reversible, as in the sequencing method developed by Solexa (now Illumina, Inc.).

[0157] SBS techniques can use nucleotide monomers that have a label moiety or lack a label moiety. Therefore, incorporation events can be detected based on the properties of the label, such as the fluorescence of the label, the properties of the nucleotide monomer, such as molecular weight or charge, or by-products of nucleotide incorporation, such as the release of pyrophosphate. In embodiments in which two or more different nucleotides are present in the sequencing reagent, the different nucleotides can be distinguishable from one another, or alternatively, the two or more different labels can be distinguishable under the detection technique used. For example, the different nucleotides present in the sequencing reagent can have different labels, which can be distinguished using appropriate optical systems, as exemplified by the sequencing method developed by Solexa (now Illumina, Inc.).

[0158] A preferred embodiment includes pyrosequencing technology, which detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M., and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375),363, U.S. Patent No. 6,210,891, U.S. Patent No. 6,258,568, and U.S. Patent No. 6,274,320, the disclosures of which are incorporated herein by reference in their entireties. In pyrosequencing, released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurase, and the level of generated ATP is detected via luciferase-generated photons. The nucleic acid to be sequenced can be attached to features in an array, and the array can be imaged to capture the chemiluminescent signal generated by the incorporation of nucleotides into the array features. Images can be obtained after treating the array with a specific nucleotide type (e.g., A, T, C, or G). Images obtained after the addition of each nucleotide type differ in terms of which features in the array are detected. These differences in the images reflect the different sequence content of the features on the array. However, the relative positions of each feature remain unchanged in the image. Images can be stored, processed, and analyzed using the methods described herein.For example, images obtained after treating the array with each different nucleotide type can be processed in the same manner as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.

[0159] In another exemplary type of SBS, cycle sequencing is achieved by stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels, as described, for example, in International Publication No. 04 / 018497 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated by reference. This approach has been commercialized by Solexa (now Illumina Inc.) and is also described in International Publication Nos. 91 / 06678 and 07 / 123,744, each of which is incorporated by reference herein. The availability of fluorescently labeled terminators, both of which can be reversed and from which the fluorescent label is cleaved, facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

[0160] Preferably, in reversible terminator-based sequencing embodiments, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example, by cleavage or degradation. Images can be captured after incorporation of the label into arrayed nucleic acid features. In certain embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array, each nucleotide type bearing a spectrally distinct label. Four images can then be obtained, each using a detection channel selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, with images of the array being obtained between each addition step. In such embodiments, each image shows nucleic acid features incorporating a particular type of nucleotide. Because the sequence content of each feature varies, different features are present or absent in different images. However, the relative positions of the features remain unchanged within the image. Images obtained from such reversible terminator SBS methods can be stored, processed, and analyzed as described herein. Following the image capture step, the label can be removed, and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removal of the label after detection in a particular cycle and before the subsequent cycle has the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described below.

[0161] In certain embodiments, some or all of the nucleotide monomers can contain reversible terminators. In such embodiments, the reversible terminator / cleavable fluorophore can comprise a fluorophore attached to the ribose moiety via a 3' ester bond (Metzker, Genome Res. 15:1767-1776 (2005), incorporated herein by reference). Other approaches separate the terminator chemistry from the cleavage of the fluorescent label (Ruparel et al., Proc Natl Acad Sci USA 102:5932-7 (2005), incorporated herein by reference in its entirety). Ruparel et al. describe the development of a reversible terminator that uses a small 3' allyl group to block extension but can be easily deblocked by brief treatment with a palladium catalyst. The fluorophore was attached to the base via a photocleavable linker that can be easily cleaved by 30 seconds of exposure to long-wavelength UV light. Therefore, either disulfide reduction or photocleavage can be used as the cleavable linker. Another approach to reversible termination is the use of a natural terminator followed by the placement of a bulky dye on the dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and / or electrostatic hindrance. The presence of one incorporation event prevents further incorporation unless the dye is removed. Cleavage of the dye removes the fluorophore, effectively reversing the terminus. Examples of modified nucleotides are also described in U.S. Pat. Nos. 7,427,673 and 7,057,026, the disclosures of which are incorporated herein by reference in their entireties.

[0162] Additional exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Application Publication No. 2007 / 0166705, U.S. Patent Application Publication No. 2006 / 0188901, U.S. Patent No. 7,057,026, U.S. Patent Application Publication No. 2006 / 0240439, U.S. Patent Application Publication No. 2006 / 0281109, WO 05 / 065814, U.S. Patent Application Publication No. 2005 / 0100900, WO 06 / 064199, WO 07 / 010,251, U.S. Patent Application Publication No. 2012 / 0270305, and U.S. Patent Application Publication No. 2013 / 0260372, the disclosures of which are incorporated herein by reference in their entireties.

[0163] Some embodiments may utilize detection of four different nucleotides using fewer than four different labels. For example, SBS may be performed using the methods and systems described in the incorporated document, U.S. Patent Application Publication No. 2013 / 0079232. As a first example, pairs of nucleotide types may be detected at the same wavelength but may be distinguished based on differences in intensity between one member of the pair and the other, or based on a change to one member of the pair (e.g., via chemical, photochemical, or physical modification) that results in the appearance or disappearance of a distinct signal compared to the signal detected for the other member of the pair. As a second example, three of the four different nucleotide types may be detected under certain conditions, while the fourth nucleotide type may be devoid of a detectable label under those conditions or may be minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid may be determined based on the presence of their respective signals, and incorporation of the fourth nucleotide type into a nucleic acid may be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type can include a label that is detected in two different channels, while the other nucleotide type is detected in one or less of the channels. The three exemplary configurations described above are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples is a fluorescence-based SBS method that uses a first nucleotide type (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type (e.g., dTTP having at least one label that is detected in both channels when excited by the first and / or second excitation wavelength), and a fourth nucleotide type (e.g., unlabeled dGTP) that is not detected in any channel or minimally detected in any channel.

[0164] Furthermore, as described in incorporated U.S. Patent Application Publication No. 2013 / 0079232, sequencing data can be obtained using a single channel. In such so-called single-dye sequencing approaches, a first nucleotide type is labeled but the label is removed after the first image is generated, and a second nucleotide type is labeled only after the first image is generated. A third nucleotide type retains its label in both the first and second images, and a fourth nucleotide type remains unlabeled in both images.

[0165] Some embodiments may utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify their incorporation. The oligonucleotides typically have different labels that correlate with the identity of specific nucleotides in the sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained after treating an array of nucleic acid features with labeled sequencing reagents. Each image shows nucleic acid features incorporating a specific type of label. Because the sequence content of each feature varies, different features may or may not be present in different images, but the relative positions of the features remain constant within the image. Images obtained from ligation-based sequencing methods can be stored, processed, and analyzed as described herein. Exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties.

[0166] Some embodiments can utilize nanopore sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope." Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, GV & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K., "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR, "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties.)The data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein. Specifically, the data can be processed as images according to the exemplary processing of optical and other images described herein.

[0167] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing polymerase and a γ-phosphate-labeled nucleotide, for example, as described in U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (each of which is incorporated herein by reference), or nucleotide incorporation can be detected using zero-mode waveguides, for example, as described in U.S. Patent No. 7,315,019 (each of which is incorporated herein by reference), and fluorescent nucleotide analogs and engineered polymerases, for example, as described in U.S. Patent No. 7,405,281 and U.S. Patent Application Publication No. 2008 / 0108082 (each of which is incorporated herein by reference). Illumination can be restricted to a zeptoliter-scale volume around the surface-tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, MJ et al., "Zero-mode waveguides for single-molecule analysis at high concentrations." Science, 299, 682-686 (2003); Lundquist, PM et al., "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al., "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed, and analyzed as described herein.

[0168] Some SBS embodiments involve the detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on the detection of released protons can use commercially available electrical detectors and related technology from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or can use the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082 (A1), 2009 / 0127589 (A1), 2010 / 0137143 (A1), or 2010 / 0282617 (A1), each of which is incorporated herein by reference. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be easily adapted to substrates used to detect protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons used to detect protons.

[0169] The SBS method described above can be advantageously performed in a multiplex format, allowing multiple different target nucleic acids to be manipulated simultaneously. In certain embodiments, the different target nucleic acids can be processed in a common reaction vessel or on the surface of a specific substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids are typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent binding, binding to beads or other particles, or binding to a surface-bound polymerase or other molecule. The array can contain a single copy of the target nucleic acid at each site (also referred to as a feature), or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be generated by amplification methods such as bridge amplification or emulsion PCR, which are described in more detail below.

[0170] The methods described herein can be used to detect, for example, at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 , 5,000,000 features / cm 2 Arrays having features of any of a variety of densities can be used, including 1000, ...2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000

[0171] An advantage of the methods described herein is that they provide rapid and efficient detection of multiple target nucleic acids in parallel. Accordingly, the present disclosure provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art, such as those exemplified above. Accordingly, the integrated system of the present disclosure can include fluidic components capable of delivering amplification and / or sequencing reagents to one or more immobilized DNA fragments, including components such as pumps, valves, reservoirs, and fluid lines. A flow cell can be configured and / or used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent No. 2010 / 0111768(A1) and U.S. Patent Application No. 13 / 273,666, each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used in the amplification and detection methods. Taking a nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system can be used to deliver sequencing reagents in the amplification methods described herein and in the sequencing methods exemplified above. Alternatively, an integrated system may include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and sequencing the nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and the apparatus described in U.S. Patent Application No. 13 / 273,666, which is incorporated herein by reference.

[0172] The sequencing system described above sequences nucleic acid polymers present in a sample received by the sequencing device. As defined herein, "sample" and its derivatives are used in the broadest sense and include any sample, culture, etc. suspected of containing a target. In some embodiments, a sample includes DNA, RNA, PNA, LNA, chimeric, or hybrid forms of nucleic acid. A sample can include any biological, clinical, surgical, agricultural, air, or water sample containing one or more nucleic acids. The term also includes any isolated nucleic acid sample, such as genomic DNA, fresh-frozen, or formalin-fixed, paraffin-embedded nucleic acid sample. It is also contemplated that a sample can be derived from a single individual, a collection of nucleic acid samples from genetically related members, nucleic acid samples from genetically unrelated members, nucleic acid samples from a single individual (matched), such as a tumor sample and a normal tissue sample, or a sample from a single source containing two different forms of genetic material, such as maternal and fetal DNA obtained from a maternal subject, or the presence of contaminating bacterial DNA in a sample containing plant or animal DNA. In some embodiments, the source of nucleic acid material can include nucleic acid obtained from a newborn, such as is typically used for newborn screening.

[0173] A nucleic acid sample can include high molecular weight material, such as genomic DNA (gDNA). A sample can include low molecular weight material, such as nucleic acid molecules obtained from FFPE or archived DNA samples. In another embodiment, the low molecular weight material includes enzymatically or mechanically fragmented DNA. A sample can include cell-free circulating DNA. In some embodiments, a sample can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissection, surgical resection, and other clinical or laboratory samples. In some embodiments, a sample can be an epidemiological, agricultural, forensic, or pathogenic sample. In some embodiments, a sample can include nucleic acid molecules obtained from animals, such as humans or mammalian sources. In another embodiment, a sample can include nucleic acid molecules obtained from non-mammalian sources, such as plants, bacteria, viruses, or fungi. In some embodiments, the source of the nucleic acid molecule can be an archived or extinct sample or species.

[0174] Additionally, the methods and compositions disclosed herein may be useful for amplifying nucleic acid samples with low-quality nucleic acid molecules, such as degraded and / or fragmented genomic DNA from forensic samples. In one embodiment, a forensic sample may include nucleic acids obtained from a crime scene, from missing person DNA data, from a laboratory associated with a forensic investigation, or may include forensic samples obtained by law enforcement, one or more military services, or any such personnel. A nucleic acid sample may be crude DNA, including purified samples or lysates, derived from, for example, oral swabs, paper, cloth, or other substrates that may be impregnated with saliva, blood, or other bodily fluids. Thus, in some embodiments, a nucleic acid sample may contain small amounts of DNA or fragmented DNA fragments, such as genomic DNA. In some embodiments, target sequences may be present in one or more bodily fluids, including, but not limited to, blood, sputum, plasma, semen, urine, and serum. In some embodiments, target sequences may be obtained from hair, skin, tissue samples, autopsies, or remains of victims. In some embodiments, nucleic acids containing one or more target sequences may be obtained from deceased animals or humans. In some embodiments, the target sequence can comprise nucleic acid obtained from non-human DNA, such as microbial, plant, or entomological DNA. In some embodiments, the target sequence or amplified target sequence is intended for human identification. In some embodiments, the present disclosure generally relates to methods for identifying characteristics of forensic samples. In some embodiments, the present disclosure generally relates to human identification methods using one or more target-specific primers disclosed herein or one or more target-specific primers designed using the primer design criteria outlined herein. In one embodiment, a forensic sample or human identification sample containing at least one target sequence can be amplified using any one or more of the target-specific primers disclosed herein or using the primer criteria outlined herein.

[0175] Components of the crosstalk-aware base calling system 106 can include software, hardware, or both. For example, components of the crosstalk-aware base calling system 106 can include one or more instructions stored on a non-transitory computer-readable storage medium and executable by a processor of one or more computing devices (e.g., user client device 108). When executed by one or more processors, the computer-executable instructions of the crosstalk-aware base calling system 106 can cause the computing device to perform the fault source identification methods described herein. Alternatively, components of the crosstalk-aware base calling system 106 can include hardware, such as a dedicated processing device, for performing a particular function or group of functions. Additionally or alternatively, components of the crosstalk-aware base calling system 106 can include a combination of computer-executable instructions and hardware.

[0176] Furthermore, components of the crosstalk-aware base calling system 106 that perform the functions described herein with respect to the crosstalk-aware base calling system 106 may be implemented, for example, as part of a standalone application, as a module of an application, as a plug-in to an application, as library function(s) that can be called by other applications, and / or as a cloud computing model. Thus, components of the crosstalk-aware base calling system 106 may be implemented as part of a standalone application on a personal computing device or a mobile device. Additionally or alternatively, components of the crosstalk-aware base calling system 106 may be implemented in any application that provides sequencing services, including, but not limited to, Illumina BaseSpace, Illumina DRAGEN, or Illumina TruSight software. "Illumina," "BaseSpace," "DRAGEN," and "TruSight" are registered trademarks or trademarks of Illumina, Inc. in the United States and / or other countries.

[0177] Embodiments of the present disclosure may include or utilize special purpose or general purpose computers, including computer hardware such as, for example, one or more processors and system memory, as discussed in more detail below. Embodiments within the scope of the present disclosure also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. In particular, one or more of the processes described herein may be embodied in a non-transitory computer-readable medium and implemented at least in part as instructions executable by one or more computing devices (e.g., any of the media content access devices described herein). Generally, a processor (e.g., a microprocessor) receives instructions from a non-transitory computer-readable medium (e.g., memory, etc.) and executes those instructions, thereby performing one or more processes, including one or more of the processes described herein.

[0178] Computer-readable media may be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer-readable media that store computer-executable instructions are non-transitory computer-readable storage media (devices). Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example, and not limitation, embodiments of the present disclosure may include at least two distinctly different kinds of computer-readable media: non-transitory computer-readable storage media (devices) and transmission media.

[0179] Non-transitory computer-readable storage media (devices) include RAM, ROM, EEPROM, CD-ROM, (e.g., RAM-based) solid-state drives (SSD), flash memory, phase-change memory (PCM), other types of memory, other optical disk storage, magnetic disk storage or other magnetic storage, or any other medium that can be used to store desired program code means in the form of computer-executable instructions or data structures and that can be accessed by a general-purpose or special-purpose computer.

[0180] A "network" is defined as one or more data links that enable the transport of electronic data between computer systems and / or modules and / or other electronic devices. When information is transferred or provided to a computer over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless), the computer properly views the connection as a transmission medium. Transmission media can be used to transport desired program code means in the form of computer-executable instructions or data structures and can include networks and / or data links that can be accessed by a general-purpose or special-purpose computer. Combinations of the above should also be included within the scope of computer-readable media.

[0181] Furthermore, upon reaching various computer system components, program code means in the form of computer-executable instructions or data structures may be automatically transferred from transmission media to non-transitory computer-readable storage media (devices) (or vice versa). For example, computer-executable instructions or data structures received over a network or data link may be buffered in RAM within a network interface module (e.g., a NIC) and then eventually transferred to computer system RAM and / or to less volatile computer storage media (devices) within the computer system. Thus, it should be understood that non-transitory computer-readable storage media (devices) may be included in computer system components that also (or even primarily) utilize transmission media.

[0182] Computer-executable instructions include, for example, instructions and data that, when executed by a processor, cause a general-purpose computer, a special-purpose computer, or a special-purpose processing device to perform a certain function or group of functions. In some embodiments, computer-executable instructions are executed on a general-purpose computer, transforming the general-purpose computer into a special-purpose computer that implements elements of the present disclosure. Computer-executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code. While the subject matter has been described in language specific to structural features and / or methodological acts, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the described features or acts described above. Rather, the described features and acts are disclosed as example forms of implementing the claims.

[0183] Those skilled in the art will appreciate that the present disclosure may be implemented in networked computing environments having many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, cellular phones, PDAs, tablets, pagers, routers, switches, etc. The present disclosure may also be implemented in distributed system environments where tasks are performed by both local and remote computer systems that are linked through a network (either by hardwired data links, wireless data links, or a combination of hardwired and wireless data links). In a distributed system environment, program modules may be located in both local and remote memory storage devices.

[0184] Embodiments of the present disclosure may also be implemented in a cloud computing environment. As used herein, "cloud computing" is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources. For example, cloud computing may be adopted in markets to provide ubiquitous, convenient, on-demand access to a shared pool of configurable computing resources. The shared pool of configurable computing resources can be quickly configured through virtualization, exposed with low management effort or service provider interaction, and then scaled accordingly.

[0185] Cloud computing models can consist of various characteristics, such as, for example, on-demand self-service, wide area network access, resource pooling, rapid elasticity, and measured service. Cloud computing models can also expose various service models, such as, for example, Software as a Service (SaaS), Platform as a Service (PaaS), and Infrastructure as a Service (IaaS). Cloud computing models can also be deployed using different deployment models, such as private cloud, community cloud, public cloud, and hybrid cloud. As used herein and in the claims, a "cloud computing environment" is an environment in which cloud computing is employed.

[0186] 10 shows a block diagram of a computing device 1000 that may be configured to perform one or more of the processes described above. It will be understood that one or more computing devices, such as computing device 1000, may implement the crosstalk-aware base calling system 106 and the sequencing system 104. As shown by FIG. 10, computing device 1000 may include a processor 1002, a memory 1004, a storage device 1006, an I / O interface 1008, and a communication interface 1010, which may be communicatively coupled by a communication infrastructure 1012. In certain embodiments, computing device 1000 may include fewer or more components than those shown in FIG. 10. The following paragraphs describe in more detail the components of computing device 1000 shown in FIG. 10.

[0187] In one or more embodiments, the processor 1002 includes hardware for executing instructions, such as those comprising a computer program. By way of example and not limitation, to execute instructions for dynamically modifying a workflow, the processor 1002 may retrieve (or fetch) instructions from an internal register, an internal cache, memory 1004, or storage device 1006, decode them, and execute them. The memory 1004 may be volatile or non-volatile memory used to store data, metadata, and programs for execution by the processor. The storage device 1006 includes a storage device, such as a hard disk, flash disk drive, or other digital storage device, for storing data or instructions for implementing the methods described herein.

[0188] The I / O interface 1008 enables a user to provide input to, receive output from, and otherwise transfer data to and receive data from the computing device 1000. The I / O interface 1008 may include a mouse, a keypad or keyboard, a touchscreen, a camera, an optical scanner, a network interface, a modem, other known I / O devices, or a combination of such I / O interfaces. The I / O interface 1008 may include one or more devices for presenting output to a user, including, but not limited to, a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., a display driver), one or more audio speakers, and one or more audio drivers. In a particular embodiment, the I / O interface 1008 is configured to provide graphical data to a display for presentation to a user. The graphical data may represent one or more graphical user interfaces and / or any other graphical content that may be useful in a particular implementation.

[0189] Communications interface 1010 may include hardware, software, or both. In any case, communications interface 1010 may provide one or more interfaces for communications (e.g., packet-based communications, etc.) between computing device 1000 and one or more other computing devices or networks. By way of example and not limitation, communications interface 1010 may include a network interface controller (NIC) or network adapter for communicating with an Ethernet or other wired-based network, or a wireless NIC (WNIC) or wireless adapter for communicating with a wireless network such as Wi-Fi.

[0190] Additionally, the communication interface 1010 can facilitate communication with various types of wired or wireless networks. The communication interface 1010 can also facilitate communication using various communication protocols. The communication infrastructure 1012 can also include hardware, software, or both that couple components of the computing device 1000 to one another. For example, the communication interface 1010 can use one or more networks and / or protocols to enable multiple computing devices connected by a particular infrastructure to communicate with each other to perform one or more aspects of the processes described herein. By way of example, a sequencing process can enable multiple devices (e.g., client devices, sequencing devices, and server devices) to exchange information such as sequencing data and error notifications.

[0191] In the foregoing specification, the present disclosure has been described with reference to specific exemplary embodiments thereof. Various embodiments and aspects of the present disclosure will be described with reference to the details discussed herein, and the accompanying drawings illustrate various embodiments. The above description and drawings are illustrative of the present disclosure and should not be construed as limiting the disclosure. Numerous specific details are set forth to provide a thorough understanding of various embodiments of the present disclosure.

[0192] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects as illustrative only and not restrictive. For example, methods described herein may be implemented using fewer or more steps / actions, or the steps / actions may be performed in a different order. Additionally, steps / actions described herein may be repeated or performed in parallel with each other, or with different occurrences of the same or similar steps / actions. The scope of the present application is therefore indicated by the appended claims, rather than the foregoing description. All changes that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

Claims

1. 1. A system comprising: at least one processor; When executed by the at least one processor, the system: detecting a first set of intensity values ​​for a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​for a second signal from a second cluster of oligonucleotides for a sequencing cycle; determining a set of illumination indicia representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determining an inter-cluster interference metric estimating optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides based on the set of illumination indices; and a non-transitory computer-readable medium comprising instructions to generate, for the sequencing cycle, a modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric from the second set of intensity values.

2. When executed by the at least one processor, the system: determining the set of illumination indicia further based on amplitudes for the first set of intensity values ​​and an estimated point spread function for a section of the nucleotide sample slide containing the first cluster of oligonucleotides; The system of claim 1 , further comprising instructions for determining the inter-cluster interference metric further based on the estimated point spread function.

3. 3. The system of claim 2, wherein the estimated point spread function uses the positions of the second cluster of oligonucleotides or a different cluster of oligonucleotides as points and includes a region that includes the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

4. 2. The system of claim 1, wherein a first position within the nucleotide sample slide for the first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second position within the nucleotide sample slide for the second cluster of oligonucleotides.

5. When executed by the at least one processor, the system: determining nucleobase calls for said first cluster of oligonucleotides based on said first set of intensity values ​​and intensity value boundaries for the nucleobases for said sequencing cycle; 10. The system of claim 1, further comprising instructions to determine the set of illumination indices further based on the nucleobase calls for the first cluster of oligonucleotides.

6. When executed by the at least one processor, the system: determining the nucleobase calls for the first cluster of oligonucleotides based on intensity values ​​from the first set of intensity values ​​corresponding to a first channel and intensity values ​​from the first set of intensity values ​​corresponding to a second channel; 6. The system of claim 5, further comprising instructions to generate the modified second set of intensity values ​​by subtracting a value of the inter-cluster interference metric from an intensity value from the second set of intensity values ​​corresponding to the first channel or from an intensity value from the second set of intensity values ​​corresponding to the second channel.

7. When executed by the at least one processor, the system: determining a first illumination indicia indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; determining a second illumination indicia indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or 2. The system of claim 1, further comprising instructions to determine the set of illumination indices by: determining a first continuous illumination indicia indicative of the degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicia indicative of the degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

8. 2. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to determine, for the sequencing cycle, based on the modified second set of intensity values, an adjusted set of illumination indices that indicate whether the second cluster of oligonucleotides is illuminated during the sequencing cycle, the adjusted set of illumination indices being different from an initial set of illumination indices corresponding to the second set of intensity values.

9. 2. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to determine, for the sequencing cycle, nucleobase calls for the second cluster of oligonucleotides that differ from the nucleobases corresponding to the second set of intensity values ​​based on the modified second set of intensity values.

10. When executed by the at least one processor, the system: detecting a third set of intensity values ​​for a third signal from a third cluster of oligonucleotides for said sequencing cycle; determining an additional set of illumination indicia representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determining an additional inter-cluster interference metric estimating optical interference from the third cluster of oligonucleotides relative to the second cluster of oligonucleotides based on the additional set of illumination indices; 2. The system of claim 1, further comprising instructions to generate the modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric and the additional inter-cluster interference metric from the second set of intensity values ​​for the sequencing cycle.

11. When executed by the at least one processor, the system: determining that the first set of intensity values ​​for the first signal from the first cluster of oligonucleotides is within an intensity value range; determining that the second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is not within the intensity value range; 2. The system of claim 1, further comprising instructions to generate the modified second set of intensity values ​​by removing the inter-cluster interference metric that estimates optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides from the second set of intensity values ​​based on the first set of intensity values ​​being within the intensity value range and the second set of intensity values ​​not being within the intensity value range.

12. When executed by the at least one processor, the system: determining a first nucleobase call for said first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values ​​within said intensity value range based on said first set of intensity values; 12. The system of claim 11, further comprising instructions to determine a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values ​​not within the intensity value range based on the modified second set of intensity values.

13. When executed by the at least one processor, the system: detecting the first set of intensity values ​​by detecting a first intensity value for the first signal from a first cluster of oligonucleotides in a single channel; 2. The system of claim 1, further comprising instructions to detect the second set of intensity values ​​by detecting a second intensity value for the second signal from a second cluster of oligonucleotides in the single channel, and to determine the set of illumination indices by determining a single illumination indicia representative of whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

14. When executed by at least one processor, the computing device detecting a first set of intensity values ​​for a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​for a second signal from a second cluster of oligonucleotides for a sequencing cycle; determining a set of illumination indicia representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determining an inter-cluster interference metric estimating optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides based on the set of illumination indices; A non-transitory computer-readable medium storing instructions to generate, for the sequencing cycle, a modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric from the second set of intensity values.

15. When executed by the at least one processor, causes the computing device to determine the set of illumination indices further based on amplitudes for the first set of intensity values ​​and an estimated point spread function for a section of a nucleotide sample slide containing the first cluster of oligonucleotides; The non-transitory computer-readable medium of claim 14 , further comprising instructions for determining the inter-cluster interference metric further based on the estimated point spread function.

16. 16. The non-transitory computer-readable medium of claim 15, wherein the estimated point spread function uses the locations of the second cluster of oligonucleotides or a different cluster of oligonucleotides as points and includes a region that includes the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

17. 15. The non-transitory computer-readable medium of claim 14, wherein a first location within a nucleotide sample slide for the first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second location within the nucleotide sample slide for the second cluster of oligonucleotides.

18. when executed by the at least one processor, causes the computing device to determine, for the sequencing cycle, nucleobase calls for the first cluster of oligonucleotides based on the first set of intensity values ​​and intensity value boundaries for nucleobases; 15. The non-transitory computer-readable medium of claim 14, further comprising instructions to determine the set of illumination indicia further based on the nucleobase calls for the first cluster of oligonucleotides.

19. when executed by the at least one processor, causes the computing device to determine the nucleobase call for the first cluster of oligonucleotides based on an intensity value from the first set of intensity values ​​corresponding to a first channel and an intensity value from the first set of intensity values ​​corresponding to a second channel; 20. The non-transitory computer-readable medium of claim 18, further comprising instructions to generate the modified second set of intensity values ​​by subtracting a value of the inter-cluster interference metric from an intensity value from the second set of intensity values ​​corresponding to the first channel or from an intensity value from the second set of intensity values ​​corresponding to the second channel.

20. When executed by the at least one processor, the computing device: determining a first illumination indicia indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; determining a second illumination indicia indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or 15. The non-transitory computer-readable medium of claim 14, further comprising instructions to determine the set of illumination indices by: determining a first continuous illumination indicia indicative of the degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicia indicative of the degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

21. 15. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to determine, for the sequencing cycle based on the modified second set of intensity values, an adjusted set of illumination indices that indicate whether the second cluster of oligonucleotides is illuminated during the sequencing cycle, the adjusted set of illumination indices being different from an initial set of illumination indices corresponding to the second set of intensity values.

22. 15. The non-transitory computer-readable medium of Claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to determine, for the sequencing cycle and based on the modified second set of intensity values, nucleobase calls for the second cluster of oligonucleotides that differ from the nucleobases corresponding to the second set of intensity values.

23. When executed by the at least one processor, the method causes the computing device to detect a third set of intensity values ​​for a third signal from a third cluster of oligonucleotides for the sequencing cycle; determining an additional set of illumination indicia representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determining an additional inter-cluster interference metric estimating optical interference from the third cluster of oligonucleotides relative to the second cluster of oligonucleotides based on the additional set of illumination indices; 15. The non-transitory computer-readable medium of claim 14, further comprising instructions to generate the modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric and the additional inter-cluster interference metric from the second set of intensity values ​​for the sequencing cycle.

24. When executed by the at least one processor, the method causes the computing device to determine that the first set of intensity values ​​for the first signal from the first cluster of oligonucleotides is within an intensity value range; determining that the second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is not within the intensity value range; 15. The non-transitory computer-readable medium of claim 14, further comprising instructions to generate the modified second set of intensity values ​​by removing the inter-cluster interference metric that estimates optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides from the second set of intensity values ​​based on the first set of intensity values ​​being within the intensity value range and the second set of intensity values ​​not being within the intensity value range.

25. When executed by the at least one processor, the computing device: determining a first nucleobase call for said first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values ​​within said intensity value range based on said first set of intensity values; 25. The non-transitory computer-readable medium of claim 24, further comprising instructions to determine a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values ​​that are not within the intensity value range based on the modified second set of intensity values.

26. When executed by the at least one processor, the method causes the computing device to detect the first set of intensity values ​​by detecting a first intensity value for the first signal from a first cluster of oligonucleotides in a single channel; 15. The non-transitory computer-readable medium of claim 14, further comprising instructions to detect the second set of intensity values ​​by detecting a second intensity value for the second signal from a second cluster of oligonucleotides in the single channel, and to determine the set of illumination indices by determining a single illumination indicia representative of whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

27. 1. A method comprising: detecting, for a sequencing cycle, a first set of intensity values ​​for a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​for a second signal from a second cluster of oligonucleotides; determining a set of illumination indicia representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determining an inter-cluster interference metric estimating optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides based on the set of illumination indices; generating a modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric from the second set of intensity values ​​for the sequencing cycle.

28. determining the set of illumination indicia further based on amplitudes for the first set of intensity values ​​and an estimated point spread function for a section of a nucleotide sample slide containing the first cluster of oligonucleotides; determining the inter-cluster interference metric further based on the estimated point spread function; 28. The method of claim 27, further comprising:

29. 29. The method of claim 28, wherein the estimated point spread function uses the positions of the second cluster of oligonucleotides or a different cluster of oligonucleotides as points and includes a region that includes the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

30. 28. The method of claim 27, wherein a first location within a nucleotide sample slide for the first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second location within the nucleotide sample slide for the second cluster of oligonucleotides.

31. determining nucleobase calls for said first cluster of oligonucleotides based on said first set of intensity values ​​and intensity value boundaries for the nucleobases for said sequencing cycle; determining the set of illumination indices further based on the nucleobase calls for the first cluster of oligonucleotides; 28. The method of claim 27, further comprising:

32. determining the nucleobase calls for the first cluster of oligonucleotides based on intensity values ​​from the first set of intensity values ​​corresponding to a first channel and intensity values ​​from the first set of intensity values ​​corresponding to a second channel; generating the modified second set of intensity values ​​by subtracting the value of the inter-cluster interference metric from an intensity value from the second set of intensity values ​​corresponding to the first channel or from an intensity value from the second set of intensity values ​​corresponding to the second channel; 32. The method of claim 31 further comprising:

33. Determining the set of lighting indicia includes: determining a first illumination indicia indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; determining a second illumination indicia indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or 28. The method of claim 27, comprising: determining a first continuous illumination index indicative of the degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination index indicative of the degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

34. 28. The method of claim 27, further comprising determining, for the sequencing cycle based on the modified second set of intensity values, an adjusted set of illumination indices representing whether the second cluster of oligonucleotides is illuminated during the sequencing cycle, the adjusted set of illumination indices different from the initial set of illumination indices corresponding to the second set of intensity values.

35. 28. The method of Claim 27, further comprising determining, for said sequencing cycle, nucleobase calls for said second cluster of oligonucleotides that differ from the nucleobases corresponding to said second set of intensity values ​​based on said modified second set of intensity values.

36. detecting a third set of intensity values ​​for a third signal from a third cluster of oligonucleotides for said sequencing cycle; determining an additional set of illumination indicia representing whether a third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determining an additional inter-cluster interference metric estimating optical interference from the third cluster of oligonucleotides relative to the second cluster of oligonucleotides based on the additional set of illumination indices; generating the modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides by removing the inter-cluster interference metric and the additional inter-cluster interference metric from the second set of intensity values ​​for the sequencing cycle; 28. The method of claim 27, further comprising:

37. determining that the first set of intensity values ​​for the first signal from the first cluster of oligonucleotides is within an intensity value range; determining that the second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is not within the intensity value range; generating the modified second set of intensity values ​​by removing an inter-cluster interference metric from the second set of intensity values ​​that estimates optical interference from the first cluster of oligonucleotides to the second cluster of oligonucleotides based on the first set of intensity values ​​being within the intensity value range and the second set of intensity values ​​not being within the intensity value range; 28. The method of claim 27, further comprising:

38. determining a first nucleobase call for the first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values ​​within the intensity value range based on the first set of intensity values; determining a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values ​​not within the intensity value range based on the modified second set of intensity values; 38. The method of claim 37, further comprising:

39. detecting the first set of intensity values ​​includes detecting a first intensity value for the first signal from a first cluster of oligonucleotides in a single channel; detecting the second set of intensity values ​​includes detecting second intensity values ​​for the second signal from a second cluster of oligonucleotides in the single channel, and determining the set of illumination indicia includes determining a single illumination indicia representative of whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle; 28. The method of claim 27, comprising:

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