Determining and removing inter-cluster optical interference

Abstract

The present disclosure describes embodiments of methods, systems, and non-transitory computer-readable media that accurately estimate crosstalk added from an oligonucleotide adjacent cluster onto an oligonucleotide target cluster, and remove or subtract crosstalk emitted by the oligonucleotide adjacent cluster from the oligonucleotide target cluster. For example, the system disclosed by the invention can detect the intensity values of a target cluster and an adjacent cluster. Based on the intensity values of the neighboring clusters, the disclosed system can determine inter-cluster interference metrics that estimate crosstalk transmitted from the neighboring clusters. The disclosed system is capable of removing inter-cluster interference metrics from the intensity values of the target clusters and generating modified intensity values of the target clusters.

Description

[0001] Priority application

[0002] 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 on February 6, 2023. The entirety of the above application is hereby incorporated by reference. Background Art

[0003] In recent years, biotechnology companies and research institutions have improved the hardware and software platforms for determining the sequence of nucleotide bases (also referred to as "nucleobases") in samples. For example, some existing sequencing machines and sequencing data analysis software (collectively referred to as "existing sequencing systems") determine the individual nucleotide bases of nucleic acid sequences by using conventional Sanger sequencing or by using sequencing by synthesis (SBS). When using SBS, existing sequencing systems can monitor thousands, tens of thousands or more nucleic acid polymers synthesized in parallel to detect more accurate nucleotide base detection. For example, the camera in the SBS platform can capture images of irradiated fluorescent labels from nucleotide bases incorporated into such synthetic nucleic acid sequences (usually grouped into clusters). After capturing these images, the computing equipment from the existing system uses sequencing data analysis software to determine the nucleotide bases detected in a given image based on the light signals captured in the image data. By repeatedly incorporating nucleotide bases into oligonucleotides and capturing images of emitted light signals in various sequencing cycles, existing sequencing systems can determine the nucleotide base sequences present in the sample.

[0004] In order to increase sample throughput and efficiency, existing sequencing systems have brought multiple oligonucleotide clusters together more and more closely in the holes of flow cells or on other nucleotide sample slides. As cluster density increases, the fluorescence response (e.g., signal) from a cluster is more likely to interfere with the fluorescence response (or no response) of an adjacent cluster due to overlapping signals caused between clusters. Such overlapping signals and optical interference are generally referred to as spatial crosstalk. Existing sequencing systems attempt to reduce interfering signals by reducing optical interference to various components and implementing computational models that estimate interfering responses (e.g., DC offset, noise level, and / or point spread function) from cluster signals and depolymerize them. Unfortunately, the increase in density and optical interference between clusters makes it more difficult to estimate the point spread function (PSF) of a given cluster or nucleotide sample slide segment.

[0005] Because nucleotide sample slides carry more densely packed clusters, sequencing equipment, along with other intensity detection systems, are more likely to erroneously determine that a cluster is illuminated (rather than not illuminated) due to spatial crosstalk from neighboring clusters. Increased crosstalk, along with changes 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 within an image for a given channel. This indirect illumination within existing sequencing systems may cause base calling algorithms to determine an incorrect nucleobase call (e.g., adenine) rather than a correct nucleobase call (e.g., guanine) for a nucleobase of an oligonucleotide incorporated into one cluster during a given cycle.

[0006] Because existing sequencing systems impose limitations on the accuracy of nucleobase detection, some existing systems maintain the distance between clusters, thereby limiting the sample throughput and cluster throughput of sequencing equipment. As noted above, when existing sequencing systems increase the cluster density on the flow cell or other nucleotide sample slides, the imaging quality and the accuracy of nucleobase detection decrease, resulting in reduced data output. In order to maintain imaging quality and relatively accurate nucleobase detection, some existing sequencing systems limit the number and density of clusters on nucleotide sample slides. By avoiding excessive clustering (e.g., placing too many clusters on the flow cell) and / or insufficient clustering (e.g., placing fewer clusters on the flow cell), existing systems limit nucleotide sequencing to a narrow range of cluster densities on the flow cell, thereby reducing data output.

[0007] These problems and challenges, along with additional problems and challenges, exist in current sequencing systems. Summary of the invention

[0008] The present disclosure describes embodiments of methods, non-transient computer-readable media, and systems that can estimate the crosstalk of adjacent clusters of oligonucleotides to a target oligonucleotide cluster ("target cluster"), and when determining the modified signal of the target cluster, remove or subtract the crosstalk from the signal emitted by the target cluster. For example, the system disclosed in the present invention can detect the intensity values ​​of various oligonucleotide clusters to which labeled nucleotide bases are added. Based on the intensity values ​​of different cluster sets, the system disclosed in the present invention can determine the illumination indicator marks of one or more clusters adjacent to the target cluster. Based on the illumination indicator marks and / or other data about adjacent clusters of oligonucleotides ("adjacent clusters"), the system disclosed in the present invention determines an inter-cluster interference measure that estimates the optical interference of adjacent clusters to the target cluster. The system disclosed in the present invention can further remove the inter-cluster interference measure from the intensity value of the target cluster.

[0009] The systems disclosed herein can use such cluster-associated inter-cluster interference measures for a variety of base calling applications further described below. For example, the systems disclosed herein can more accurately determine the cluster signals and their corresponding nucleobase calls for a given sequencing cycle by (i) removing the crosstalk of neighboring clusters from the intensity value of the target cluster when determining the intensity value of the target cluster signal, and (ii) determining the nucleobase calls for the target cluster. In order to improve the accuracy and efficiency of nucleobase calls, in some cases, the systems disclosed herein repeatedly determine the crosstalk of neighboring subsets of clusters based on the range of intensity values ​​of the corresponding clusters and remove or subtract it from the target subset of clusters.

[0010] Additional features and advantages of one or more embodiments of the present disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of such exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] DETAILED DESCRIPTION Various embodiments will be described with additional specificity and detail through the use of the drawings, which are summarized below.

[0012] Figure 1 An environment is presented in which a crosstalk-aware base calling system according to one or more embodiments of the present disclosure may operate.

[0013] Figure 2 An overview of a crosstalk-aware base calling system is shown that generates a modified intensity value for a target cluster by determining an inter-cluster interference measure and removing it from the intensity value for the target cluster, in accordance with one or more embodiments of the present disclosure.

[0014] Figure 3 Shown is a schematic diagram demonstrating that optical interference between oligonucleotide clusters increases as the distance between the oligonucleotide clusters decreases according to one or more embodiments of the present disclosure.

[0015] Figure 4 A crosstalk-aware base calling system for determining an illumination indicator signature based on fluorescence responses in different channels according to one or more embodiments of the present disclosure is presented.

[0016] FIG. 5A to FIG. 5B A crosstalk-aware base calling system is presented that utilizes a linear equalizer system and generates a modified intensity value for a target cluster by determining nucleobase calls and illumination indication signatures from neighboring clusters, a signal model for the target cluster, and an inter-cluster interference metric in accordance with one or more embodiments.

[0017] Figure 6 Shown are estimated point spread functions of signal intensity values ​​from oligonucleotide clusters according to one or more embodiments of the present disclosure. 7A to 7C The effects of photointerference between oligonucleotide clusters according to one or more embodiments of the present disclosure, as well as the effects of removing photointerference of certain oligonucleotide clusters are demonstrated.

[0018] FIG. 8A to FIG. 8B Shown is a bar graph of intensity values ​​for oligonucleotide clusters with and without light interference from neighboring clusters of oligonucleotides according to one or more embodiments of the present disclosure.

[0019] Fig. 9 A series of acts for generating a modified set of strength values ​​for an oligonucleotide cluster using an inter-cluster interference measure in accordance with one or more embodiments of the present disclosure are presented.

[0020] Fig.10 A block diagram of an example computing device is presented in accordance with one or more embodiments of the present disclosure. DETAILED DESCRIPTION

[0021] The present disclosure describes one or more embodiments of a crosstalk-aware base detection system, which determines an inter-cluster interference measure representing the optical interference of an oligonucleotide cluster to a target oligonucleotide cluster, and generates a modified intensity value of a target cluster based on the inter-cluster interference measure. By determining the inter-cluster interference measure and removing it, the crosstalk-aware base detection system depolymerizes the optical interference between clusters. In order to detect and depolymerize light based on such an inter-cluster interference measure, in some specific implementations, the crosstalk-aware base detection system determines the intensity value of the signal from the oligonucleotide target cluster and the adjacent cluster of the oligonucleotide for a given sequencing cycle. Based on the intensity value of the adjacent cluster, the crosstalk-aware base detection system determines an illumination indicator mark representing whether the adjacent cluster is illuminated during a given sequencing cycle. Based on these illumination indicators, the crosstalk-aware base detection system determines an inter-cluster interference measure estimating the optical interference of the adjacent cluster to the target cluster. The crosstalk-aware base detection system can further deduct (or remove) the inter-cluster interference measure from the intensity value of the target cluster signal to create a modified intensity value of the target cluster.

[0022] As mentioned 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 target clusters and adjacent clusters in a given sequencing cycle. For example, in some cases, the crosstalk-aware base calling system detects intensity values ​​of signals emitted from each cluster on a sample nucleotide slide (including those that become target clusters and adjacent clusters) in a given sequencing cycle. In certain embodiments, clusters with higher intensity values ​​are relatively brighter, while clusters with lower intensity values ​​are relatively darker. In some cases, the crosstalk-aware base calling system uses data from brighter clusters to determine the crosstalk of brighter clusters to darker clusters.

[0023] For example, based on the detected intensity values, the crosstalk-aware base calling system can determine a subset of illumination indicators for a subset of clusters (including adjacent clusters of the target cluster). Specifically, the crosstalk-aware base calling system determines the nucleobase calls for a subset of clusters (e.g., a subset of brighter clusters that incorporate adenine), and then determines the illumination indicators for the subset of clusters based on these nucleobase calls. Such illumination indicators identify whether a given cluster is illuminated or emits a fluorescent response in a given channel (e.g., two or four channels) during a sequencing cycle, and together with the fluorescent responses in other given channels, form data for determining the nucleobase calls. In some cases, the illumination indicators together represent the illumination conditions of the clusters in multiple channels, such as a first illumination indicator indicating whether a given cluster is illuminated in a first channel during a given sequencing cycle, and a second illumination indicator indicating whether a given cluster is illuminated in a second channel during a given sequencing cycle. In contrast, in some cases, the illumination indicator can be a continuous illumination indicator that indicates the extent to which a given cluster is illuminated in a given channel.

[0024] As previously proposed, based on such illumination indication marks, the crosstalk-aware base calling system determines an inter-cluster interference measure (e.g., a crosstalk measure). As mentioned above, in some cases, the crosstalk indicates how the signal (e.g., brightness) of an adjacent cluster interferes with, manipulates, and / or changes the signal of a target cluster. Specifically, in one or more embodiments, the inter-cluster interference measure estimates the degree or extent to which light from an adjacent cluster interferes with or modifies light from a target cluster. In some cases, the crosstalk-aware base calling system can determine multiple inter-cluster interference measures, each of which estimates the light interference of a given adjacent cluster with a target cluster.

[0025] After determining the inter-cluster interference measure, the crosstalk-aware base calling system can use the inter-cluster interference measure to generate a modified intensity value of the signal emitted by the cluster during the sequencing cycle. By using such a measure, the crosstalk-aware base calling system can determine the amount of inter-cluster crosstalk and then remove or subtract the crosstalk from the target cluster. To illustrate, in one or more embodiments, during the sequencing cycle, the target cluster may have a relatively dark (e.g., lower intensity) signal, while the adjacent cluster may have a relatively bright (e.g., higher intensity) signal. However, the brightness of the adjacent cluster signal may increase the brightness (e.g., intensity) of the target cluster signal, making it difficult to determine whether the cluster emits a light intensity of a specific frequency (e.g., a frequency band or spectral band) in a given channel during the sequencing cycle. In some embodiments, the crosstalk-aware base calling system can determine the inter-cluster interference measure based on an illumination indicator and other data about adjacent clusters. Based on the inter-cluster interference measure, the crosstalk-aware base calling system can eliminate light emitted from the signal of the brighter adjacent cluster from the target cluster signal (or reduce the impact of such light). Thus, in some embodiments, a crosstalk-aware base calling system can more accurately determine target cluster intensity values ​​in both channels or in each relevant channel during a sequencing cycle based on inter-cluster interference metrics, which enables more accurate nucleobase calling of target clusters.

[0026] To improve efficiency and accuracy, in some cases, the crosstalk-aware base calling system follows a specific order to determine the nucleobase calls and remove crosstalk from the clusters. For example, the crosstalk-aware base calling system can: (i) identify and determine the nucleobase calls of the brightest subset of oligonucleotide clusters (e.g., the brightest top 10%) that emit signals within the highest intensity value range, and (ii) further determine an inter-cluster interference measure that estimates the optical interference of the brightest subset of the oligonucleotide clusters with the next brightest subset of the oligonucleotide clusters (e.g., the brightest top 20% to 30%) that emit signals within a second intensity value range. As further explained below, the crosstalk-aware base calling system can similarly perform further iterative operations of determining crosstalk based on additional intensity value ranges. As an alternative to performing nucleobase calls and crosstalk removal in order according to intensity value ranges, in some embodiments, the crosstalk-aware base calling system can use a signal-to-noise ratio (SNR) measure to perform nucleobase calls and crosstalk removal for clusters in order.

[0027] Crosstalk-aware base detection systems provide several advantages over conventional sequencing platforms. Specifically, crosstalk-aware base detection systems can depolymerize the light intensity including cluster signal intensity and noise from other sources, improve the accuracy of nuclear base detection, and increase the efficiency of flow cells or nucleotide sample slides during sequencing cycles. As mentioned, crosstalk-aware base detection systems can receive signals with unmodified intensity values ​​from target clusters, wherein the unmodified intensity values ​​of the signals from the target clusters include signals from the target clusters, crosstalk (e.g., noise) from adjacent clusters, and noise (e.g., background noise or intensity fluctuations) from other sources. Crosstalk-aware base detection systems can depolymerize the light intensity consisting of target signals and noise. Specifically, crosstalk-aware base detection systems can determine an inter-cluster interference measure that estimates the light interference of adjacent clusters to the target cluster. The inter-cluster interference measure estimates the crosstalk (e.g., interfering light) of adjacent clusters from composite components (e.g., background noise, intensity values ​​of target cluster signals, crosstalk). Once the crosstalk is estimated, the crosstalk-aware base detection system can remove or subtract the crosstalk by removing the inter-cluster interference measure from the signal of the target cluster. Although existing sequencing systems often cannot determine whether the target cluster is transmitting a signal based on changes in background noise, crosstalk and / or amplitude, the inter-cluster interference measure allows the crosstalk-aware base detection system to accurately decompose the crosstalk from the background noise and / or amplitude. Unlike existing sequencing systems, in one or more embodiments, after determining the extent and source of the crosstalk, the crosstalk-aware base detection system can remove the crosstalk from the signal of the affected cluster. By removing the inter-cluster interference measure from the signal of the target cluster, the crosstalk-aware base detection system can generate a modified signal of the target cluster. Therefore, by removing the crosstalk from the signal of the target cluster and generating a more accurate signal of the target cluster, the crosstalk-aware base detection system can more accurately and reliably determine the nuclear base detection of the target cluster.

[0028] In addition to detecting and depolymerizing a portion of the signal consisting of noise and light, the crosstalk-aware base calling system also improves the accuracy of the nuclear base detection. Specifically, the crosstalk-aware base calling system can determine an inter-cluster interference measure that estimates the light interference of adjacent clusters to the target cluster, and remove the inter-cluster interference measure from the intensity value of the target cluster signal. The resulting modified intensity value represents a more accurate and / or purer signal of the target cluster. Based on the more accurate or purer cluster signal, the crosstalk-aware base calling system can also determine a more accurate or more reliable nuclear base detection of the target cluster - there is no crosstalk interference or only minimal crosstalk interference to determine the signals of these nuclear base detections. For example, the crosstalk-aware base calling system can determine that the modified intensity value of the target cluster falls within the intensity value boundary of one nuclear base instead of another nuclear base, or improves the confidence score (e.g., QU AL score) of the nuclear base detection with only a low quality score.

[0029] In addition to improving nucleotide base calling and deaggregating crosstalk from cluster signals, the crosstalk-aware base calling system also improves the efficiency of the sequencing system in performing nucleotide sequencing. By determining and removing inter-cluster interference measures and improving the signal of target clusters, the crosstalk-aware base calling system helps to group clusters into denser groups on a nucleotide sample slide. Unlike clusters in existing sequencing systems that are more restricted and divided into less dense groups, the crosstalk-aware base calling system introduces a model that removes or subtracts crosstalk and promotes more densely grouped clusters and higher throughput on the sequencing device. Therefore, by determining and removing inter-cluster interference measures, the crosstalk-aware base calling system is able to sequence nucleotide sequences of more genomic samples with improved accuracy compared to existing sequencing systems that cannot effectively adjust crosstalk for densely grouped clusters.

[0030] As demonstrated by the foregoing discussion, the present disclosure utilizes a variety of terms to describe the features and advantages of crosstalk-aware base calling systems. Additional details are now provided regarding the meaning of such terms. For example, as used herein, the term "nucleotide sample slide" refers to a plate or slide comprising oligonucleotides for sequencing nucleotide fragments of a sample. Specifically, a nucleotide sample slide can refer to a slide containing a fluid channel through which reagents and buffers can travel as part of sequencing. For example, in one or more embodiments, the nucleotide sample slide comprises a flow cell (e.g., a patterned flow cell or an unpatterned flow cell) comprising a small fluid channel and short oligonucleotides complementary to a linker sequence.

[0031] Relatedly, as used herein, the term "segment of a nucleotide sample slide" (or "nucleotide sample slide segment") refers to an area that is part of a nucleotide sample slide. Specifically, a nucleotide sample slide segment can refer to a discrete portion of a nucleotide sample slide that is different from other portions of the nucleotide sample slide. For example, a nucleotide sample slide segment can include a hole (e.g., a nanopore) of a patterned flow cell, or a discrete sub-segment (e.g., a sub-segment corresponding to a cluster) of a non-patterned flow cell. In some cases, a nucleotide sample slide segment includes a tile or sub-block having clusters of identical or similar oligonucleotides grown in parallel.

[0032] In addition, as used herein, the term "labeled nucleotide base" refers to a nucleotide base with a fluorescence or light-based indicator mark or a fluorescent dye indicator mark for nucleotide base classification. Specifically, the labeled nucleotide base can refer to a nucleotide base that is doped with a fluorescence or light-based indicator mark or a fluorescent dye indicator mark to identify a base type (e.g., adenine, cytosine, thymine, or guanine). For example, in one or more embodiments, the labeled nucleotide base includes a nucleotide base with the following fluorescent label: the fluorescent label itself or together with another fluorescent label emits a signal identifying the base type. Therefore, the nucleotide base can be identified by a dye mixture (or fluorescent label mixture) that indicates the core base type together (e.g., "on" / "on" lighting indicator mark). Based on the intensity value of the signal emitted by the labeled nucleotide base in the oligonucleotide cluster, such as a signal in 16 quadrature amplitude modulation (QAM) or pulse amplitude modulation (PAM) 4 format, the type of base (e.g., adenine, cytosine, thymine, or guanine) can be determined in certain embodiments of the crosstalk-aware base detection system.

[0033] In addition, as used herein, the term "oligonucleotide cluster" refers to a grouping containing several identical deoxyribonucleic acid (DNA) fragments bound to the surface of a flow cell. For example, in some embodiments, an oligonucleotide cluster can be composed of a template DNA strand that has been clonally amplified by bridge amplification.

[0034] In addition, as used herein, the term "signal" refers to a signal emitted, reflected or otherwise transmitted from a labeled nucleotide base or a group of labeled nucleotide bases (e.g., a labeled nucleotide base added to an oligonucleotide cluster). Specifically, a signal may refer to a signal indicating a base type. For example, a signal may include a light signal emitted or reflected from a fluorescent tag of a nucleotide base or a fluorescent tag of a plurality of nucleotide bases incorporated into an oligonucleotide. As noted above, the nucleobase incorporated into the cluster may also emit a signal (in response to a laser), which may be identified as a dye mixture (or fluorescent label mixture) that indicates the nucleobase type together (e.g., a cluster with a "on" / "on" illumination indicator). In some specific implementations, the crosstalk-sensing base detection system triggers the signal by an external stimulus (such as a laser or other light source). In some cases, the crosstalk-sensing base detection system triggers the signal by some internal stimulus. In addition, in some embodiments, the crosstalk-sensing base detection system uses a filter applied when capturing an image of a nucleotide sample slide (e.g., a nucleotide sample slide segment) to observe the signal. As suggested above, in some cases the signal comprises the aggregation of the signals provided by each labeled nucleotide base added to the individual oligonucleotides in the oligonucleotide cluster.

[0035] As used herein, the term "intensity value" refers to a value indicating a characteristic or property of a signal emitted, reflected or otherwise transmitted from a marked nucleotide base or from a group of marked nucleotide bases of an oligonucleotide cluster. Specifically, 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-aware base detection system uses different filters (or intensity channels) to capture several images of oligonucleotide clusters with marked nucleotide bases. Therefore, the intensity value of a signal can correspond to the intensity of the signal observed by a specific filter.

[0036] As used herein, the term "illumination indicator" refers to an indicator of whether an oligonucleotide cluster is illuminated by the light intensity of a specific frequency band or emits the light intensity of a specific frequency band during a sequencing cycle. Specifically, the illumination indicator indicates whether an oligonucleotide cluster (or the extent of the following): (i) contains a labeled nucleotide of a specific light intensity that emits a specific frequency (e.g., a frequency band) to be illuminated (e.g., turned on or activated), or (ii) does not contain a labeled nucleotide base that is not illuminated by the specific light intensity of a specific frequency (e.g., a frequency band) in an intensity channel during sequencing (e.g., disconnected or inactivated). In some cases, the illumination indicator can be in a paired format. For example, if an oligonucleotide cluster is incorporated with a nuclear base having a fluorescent tag or other marker, these fluorescent tags or other markers (in response to light or laser) irradiate or emit the light intensity of a specific frequency (e.g., a frequency band) in a channel during a sequencing cycle, then the "on" or "illumination" state of the illumination indicator can be represented by 1. In contrast, if the oligonucleotide cluster has no (or only very few) fluorescent tags or other labeled nucleobases that illuminate or emit light intensity of a specific frequency (e.g., frequency band) in a channel (in response to light or laser) during a sequencing cycle, the "off" or "not illuminated" state of the illumination indicator mark can be represented by 0. For example, [1,1] can indicate an illumination indicator mark in which the oligonucleotide cluster is illuminated in two different channels. Although the specification and drawings depict illumination indicators in different channels (e.g., two channels or four channels), the crosstalk-aware base calling system can simultaneously detect signals from clusters in such different channels.

[0037] In contrast, if the oligonucleotide polyclonal cluster incorporates nucleobases with different fluorescent tags or other labels that (in response to light or laser light) illuminate or emit light in different spectral bands in a given channel during a sequencing cycle, the state of the illumination indicator will not be completely "on" or "off" (or not completely "illuminated" or "not illuminated"). In some cases, such mixed signals from the oligonucleotide polyclonal cluster are filtered out and discarded based on the intensity value boundaries of different types of nucleobases.

[0038] Although the present disclosure frequently uses illumination indicators in the form of "on" or "off" (or corresponding "1" or "0"), the illumination indicator can be channel-specific and is not designed to indicate the presence or absence of background noise or other light. Therefore, the "off" or "0" indicator does not indicate the absence of light, but rather estimates that a particular cluster has not been incorporated (or has only been incorporated very little) with a fluorescent tag or another labeled nucleobase that illuminates or emits (in response to light or laser) a specific frequency (e.g., frequency band) of light intensity in a particular channel during a sequencing cycle. Therefore, the illumination indicator can take other formats. As an alternative to the paired format, in some embodiments, the illumination indicator can be continuous, which indicates the extent to which a given cluster is illuminated during a sequencing cycle. For example, such a continuous illumination indicator can take the form of a measure or score (e.g., between 0 and 1) indicating the extent to which a cluster is illuminated by light emitted by a specific type of nucleotide incorporated into the cluster during a sequencing cycle.

[0039] In addition, as used herein, the term "intercluster interference measure" refers to a measure or quantification of the light interference or modification from one oligonucleotide cluster of light from another oligonucleotide cluster. Specifically, the intercluster interference measure can refer to the degree, amount and / or scope of the interference of the light signal from one oligonucleotide cluster to another oligonucleotide cluster.

[0040] As used herein, the term "nucleotide base call" refers to the determination or prediction of a specific nucleobase (or nucleobase pair) of a genomic coordinate of an oligonucleotide (e.g., a nucleotide read) or a sample genome during a sequencing cycle. Specifically, a nucleobase call can indicate a determination or prediction of the type of nucleobase that has been incorporated into an oligonucleotide on a nucleotide sample slide (e.g., a read-based nucleobase call). In some cases, for a nucleotide read, a nucleobase call includes determining or predicting a nucleobase based on an intensity value generated by a fluorescently labeled nucleotide of an oligonucleotide added to a nucleotide sample slide (e.g., in a cluster of a flow cell). As mentioned above, a single nucleobase call can be an adenine (A) call, a cytosine (C) call, a guanine (G) call, a thymine (T) call, or a uracil (U) call.

[0041] Additionally, as used herein, the term "sequencing cycle" (or "cycle") refers to the repetition of adding a nucleotide base to or incorporating an oligonucleotide or the repetition of adding a nucleotide base to or incorporating an oligonucleotide in parallel. Specifically, a cycle can include repeatedly collecting and analyzing one or more images, which have data indicating each nucleotide base added or incorporated into an oligonucleotide or added or incorporated into a plurality of oligonucleotides in parallel. Therefore, a cycle can be repeated as a part of nucleic acid polymer sequencing. For example, in one or more embodiments, each sequencing cycle relates to a single read in which only a DNA or RNA chain is read in a single direction or a double-ended read in which a DNA or RNA chain is read from two ends. In addition, in some cases, each sequencing cycle relates to a camera taking an image of multiple parts of a nucleotide sample slide or a nucleotide sample slide to generate image data for determining the specific core base added or incorporated into a specific oligonucleotide. After the image capture stage, the sequencing system can remove certain fluorescent labels from the nucleotide bases incorporated, and perform another sequencing cycle until the nucleic acid polymer has been completely sequenced. In one or more embodiments, a sequencing cycle includes a cycle within a sequencing by synthesis (SBS) run.

[0042] Additionally, as used herein, the term "nucleotide base call data" refers to a digital file, image data, or other digital information indicating the sequence of individual nucleotide bases or nucleotide bases of a nucleic acid polymer. Specifically, the nucleotide base call data may include intensity values ​​(e.g., color or light intensity values ​​of individual clusters) of an image captured by a camera from a nucleotide sample slide or other data indicating the sequence of individual nucleotide bases or nucleotide bases of a nucleic acid polymer. In addition or in lieu of the intensity values, the nucleotide base call data may include chromatographic peaks or current changes indicating individual core bases in the sequence. Additionally, in some embodiments, the nucleotide base call data include individual nucleotide base calls identifying individual nucleotide bases (e.g., A, T, C, or G). For example, the nucleotide base call data may include data of nucleotide base calls in a nucleic acid polymer sequence, the number of nucleotide base calls corresponding to a specific base (e.g., adenine, cytosine, thymine, or guanine), as organized in a digital file (such as a binary base call (BCL) file). Additionally, the nucleotide base call data may 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 instrument utilizing sequencing by synthesis (SBS).

[0043] Additional details regarding the crosstalk-aware base calling system will now be provided in conjunction with illustrative drawings that depict example embodiments and implementations of the crosstalk-aware base calling system. For example, Figure 1A 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 is shown. 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. Figure 1 One embodiment of a crosstalk-aware base calling system 106 is shown, but alternative embodiments and configurations are possible.

[0044] like Figure 1 As further shown in FIG. 1 , server device 102, user client device 108, and sequencing device 114 are connected via network 112. Each component of environment 100 can communicate via network 112. Network 112 includes any suitable network over which computing devices can communicate. Fig.10 The example network is discussed in more detail.

[0045] like Figure 1 , environment 100 includes a sequencing device 114. The sequencing device 114 includes a device for sequencing a full genome or other nucleic acid polymer. In some embodiments, the sequencing device 114 analyzes samples to generate data directly or indirectly on the sequencing device 114 using the computer-implemented methods and systems described herein. In one or more embodiments, the sequencing device 114 utilizes sequencing by synthesis (SBS) to sequence a full genome or other nucleic acid polymer. As shown in the figure, in some embodiments, the sequencing device 114 bypasses the network 112 and communicates directly with the user client device 108.

[0046] like Figure 1 As further described, environment 100 includes server device 102. Server device 102 can generate, receive, analyze, store, receive and transmit electronic data, such as data for sequencing nucleic acid polymers. Server device 102 can receive data from sequencing device 114. For example, server device 102 can collect and / or receive sequencing data, including nucleotide base call data, quality data, and other data related to sequencing nucleic acid polymers. Server device 102 can also communicate with user client device 108. Specifically, server device 102 can send read data, nucleic acid polymer sequence, error data and other information to user client device 108. In some embodiments, server device 102 includes distributed servers, wherein server device 102 includes many server devices distributed across network 112 and located at different physical locations. Server device 102 may include content server, application server, communication server, network hosting server or another type of server.

[0047] like Figure 1As further shown in , the server device 102 may include a sequencing system 104. Generally speaking, the sequencing system 104 analyzes the sequencing data received from the sequencing device 114 to determine the nucleotide sequence of the whole genome sample 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. For example, the sequencing system 104 can receive data of nucleotide reads from the sequencing device 114, and then the sequencing system 104 generates variant calls (or other nucleobase calls) of the genomic sample from these nucleotide reads. In some embodiments, the sequencing system 104 determines the sequence of nucleotide bases in DNA and / or RNA.

[0048] like Figure 1 As further shown, the sequencing device 114 includes a crosstalk-aware base calling system 106. Generally speaking, the crosstalk-aware base calling system 106 determines an inter-cluster interference measure to modify or correct the signal for the estimated optical interference of the neighboring clusters on the target cluster. More specifically, in some embodiments, the crosstalk-aware base calling system 106 detects intensity values ​​of the target cluster and the neighboring clusters in a given sequencing cycle. The crosstalk-aware base calling system 106 determines the nuclear base calls and illumination indications of the neighboring clusters. The crosstalk-aware base calling system 106 further determines an inter-cluster interference measure of the crosstalk of the neighboring clusters on the target cluster. The crosstalk-aware base calling system 106 further generates a modified intensity value for the target cluster by removing the inter-cluster interference measure from the target cluster intensity value.

[0049] Figure 1 The environment 100 shown in FIG. 1 also includes a user client device 108. The user client device 108 can generate, store, receive, and send digital data. Specifically, the user client device 108 can receive sequencing data from the sequencing device 114. In addition, 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.

[0050] Figure 1 The user client device 108 shown in the figure may include various types of client devices. For example, in some embodiments, the user client device 108 includes a non-mobile device, such as a desktop computer or server, or other types of client devices. In still other embodiments, the user client device 108 includes a mobile device, such as a laptop, a tablet computer, a mobile phone, a smart phone, etc. Additional details about the user client device 108 are described below in conjunction with Fig.10 Discuss.

[0051] like Figure 1As further shown, the user client device 108 includes a sequencing application 110. The sequencing application 110 can 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 can 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 the sequencing data. In addition, the sequencing application 110 can include instructions that, when executed, cause the user client device 108 to provide a visualization of read stacks or read alignments of nucleotide reads of a genomic sample.

[0052] like Figure 1 As further shown, the crosstalk-aware base calling system 106 can 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 by the user client device 108 (e.g., by being located entirely or partially thereon). In 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 across the server device 102, the user client device 108, and the sequencing device 114 in a variety of different ways. In one example, the crosstalk-aware base calling system 106 is partially located on the sequencing device 114 as well as the server device 102. In particular, the crosstalk-aware base calling system 106 can determine an inter-cluster interference metric of crosstalk from neighboring clusters on the sequencing device 114 to the target cluster, and modify the intensity value of the target cluster by removing the inter-cluster interference metric as part of the server device 102.

[0053] although Figure 1 The components of the environment 100 are shown communicating via the network 112, but in some embodiments, the components of the environment 100 bypass the network and communicate directly with each other. For example, and as previously described, the user client device 108 can communicate directly with the sequencing device 114. In addition, the user client device 108 can bypass the network 112 and communicate directly with the crosstalk-aware base calling system 106. In addition, the crosstalk-aware base calling system 106 can access one or more databases housed on the server device 102 or other locations in the environment 100.

[0054] The following paragraphs provide further details about the crosstalk-aware base calling system 106. According to one or more embodiments, Figure 2 A schematic diagram of a crosstalk-aware base calling system 106 that generates inter-cluster interference metrics and modifies target cluster strength values ​​is depicted. Figure 2As shown in the schematic diagram, the crosstalk-aware base detection system 106 performs a series of actions, including: an action 202 of detecting intensity values ​​of a target cluster and adjacent clusters; an action 204 of determining nuclear base detection and illumination indicator labels of adjacent clusters; an action 206 of determining an inter-cluster interference measure of crosstalk from adjacent clusters to the target cluster; and an action 208 of generating a modified intensity value of the target cluster by removing the inter-cluster interference measure.

[0055] As just mentioned, Figure 2 An action 202 of detecting intensity values ​​of a target cluster and adjacent clusters is shown. In some embodiments, the crosstalk-aware base calling system 106 can detect a set of intensity values ​​of a target cluster and a set of intensity values ​​of adjacent clusters by laser (e.g., light) excitation and imaging. During a sequencing cycle, the crosstalk-aware base calling system 106 can direct a light source with a specified wavelength to a nucleotide sample slide (or a portion of a nucleotide sample slide) and capture images of multiple clusters of emission signals within the nucleotide sample slide. In some embodiments, the crosstalk-aware base calling system 106 captures multiple images of multiple clusters of emission signals. For example, the crosstalk-aware base calling system 106 can capture multiple images using various filters or intensity channels. To illustrate, in some embodiments, the crosstalk-aware base calling system 106 utilizes a dual-channel implementation, capturing two images of a segment of a nucleotide sample slide per sequencing cycle. Specifically, 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 image and the second image may capture the intensity of emission signals from the target cluster and the neighboring clusters corresponding to the filters.

[0056] However, the crosstalk-aware base calling system 106 can use an alternative channel-based method to implement sequencing operations. In some specific implementations, the crosstalk-aware base calling system 106 utilizes a four-channel implementation and captures four different images of the flow cell segment. Similar to the dual-channel implementation, the crosstalk-aware base calling system 106 can use different filters to capture each image of the four-channel implementation. Each image can capture the intensity of the emission signal based on the filter used for the image. Therefore, in some cases, each of the four images depicts the emitted signal with different intensities. In addition, the crosstalk-aware base calling system 106 can utilize a single-channel implementation and capture an image of the nucleotide sample slide segment, and then use a specific filter to capture the intensity of the emission signal. In other embodiments, the crosstalk-aware base calling system 106 can utilize a single-channel implementation and capture an image of the nucleotide sample slide segment (or a three-channel implementation, and capture three images), and then capture the intensity value of the emission signal by utilizing a specific filter.

[0057] Based on the captured images of the intensity (e.g., color intensity and / or light intensity) of the signals emitted by the target cluster and the neighboring clusters, the crosstalk-aware base calling system 106 can measure the signal intensity of the target cluster and the neighboring clusters and provide signal intensity values ​​(e.g., wavelength and / or brightness) of the target cluster and the neighboring clusters. For example, when two intensity channels are utilized, the crosstalk-aware base calling system 106 can measure the wavelengths of the signals emitted by the target cluster and the neighboring clusters in the first channel and the second channel.

[0058] like Figure 2 Further indicated, the crosstalk-aware base detection system 106 can perform an action 204 of determining the nucleobase detection and illuminating the indicator mark of the adjacent cluster. As previously mentioned, the emission signal of the cluster can indicate the type of nucleotide base. For example, in some embodiments, the crosstalk-aware base detection system 106 analyzes (e.g., simultaneously) the intensity values ​​of the signal from a given cluster in two channels, or in each channel of multiple channels, to determine the nucleobase detection. In some embodiments, based on the signal intensity values ​​of the cluster in each channel, the crosstalk-aware base detection system 106 can use expectation maximization and Gaussian probability distribution to calculate the probability that the signal falls within the intensity value boundary of a specific base (A, C, G, or T). Then, the crosstalk-aware base detection system 106 can detect the nucleobase incorporated into the cluster by selecting the intensity value boundary of the nucleobase with the highest probability. For example, based on the intensity value emitted by the cluster signal, the crosstalk-aware base detection system 106 can determine the intensity value boundary of the nucleobase with the highest probability that the cluster is adenine (A).

[0059] After determining the nucleobase call of the cluster, in some embodiments, the crosstalk-aware base calling system 106 determines an illumination indicator for the cluster. For example, based on the nucleobase call, the crosstalk-aware base calling system 106 can determine whether the cluster is "on" (e.g., illuminated or actively emitting a light intensity of a particular frequency) or "off" (e.g., not illuminated or not emitting a light intensity of a particular frequency) in a given intensity channel during a sequencing cycle. For example, if the nucleobase call of 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 are "on" (or the cluster emits light in both the first channel and the second channel) during the sequencing cycle.

[0060] While the previous embodiment describes the crosstalk-aware base calling system 106 determining the nucleobase calls before determining the illumination indicators, in some embodiments, the crosstalk-aware base calling system 106 can perform these actions in the reverse order. For example, in some embodiments, the crosstalk-aware base calling system 106 can determine whether the illumination indicators for a given channel are "on" or "off" during a sequencing cycle, and determine the nucleobase calls for the clusters based on these illumination indicators.

[0061] In some cases, the crosstalk-aware base calling system 106 can represent the illumination indicator states of adjacent clusters in intensity channels as a set of illumination indicators in a paired format. For example, in some embodiments, the crosstalk-aware base calling system 106 determines that the nucleobase call of an adjacent cluster is adenine (A), and therefore determines the corresponding illumination indicators of the adjacent cluster in two different channels as on / on or [1,1]. Figure 2 As indicated, in some embodiments, the illumination indication marks for nucleobase detection as cytosine (C), thymine (T), and guanine (G) can be represented as on / off or [1,0], off / on or [0,1], off / off or [0,0], respectively.

[0062] like Figure 2 As further shown, after determining the nuclear base calls and the set of illumination indicators of the neighboring clusters, the crosstalk-aware base calling system 106 performs an act 206 of determining an inter-cluster interference measure of the crosstalk of the neighboring clusters to the target cluster. For example, the crosstalk-aware base calling system 106 determines the inter-cluster interference measure based on the set of illumination indicators of the neighboring clusters. As further described below, in some embodiments, the crosstalk-aware base calling system 106 determines the inter-cluster interference measure based on the amplitude of the neighboring clusters, the set of illumination indicators encoded for the neighboring clusters, and an estimated point spread function response from the neighboring cluster positions to the target cluster positions. Based on the estimated amplitude, illumination indicators, and point spread functions of the neighboring clusters, the crosstalk-aware base calling system 106 can measure the amount of crosstalk (e.g., optical interference) of the neighboring clusters to the target cluster.

[0063] In some cases, the crosstalk-aware base calling system 106 utilizes an inter-cluster interference metric as part of a function that subtracts crosstalk from a target cluster. Figure 5B and the corresponding paragraphs below provide information on how the crosstalk-aware base calling system 106 estimates and utilizes the magnitudes of neighboring clusters according to one or more embodiments. Illuminated indicators of adjacent clusters and the point spread function response from the neighboring clusters to the target cluster To determine the inter-cluster interference measure (I i0_i1 ). In some embodiments, crosstalk can be modeled as light interference on the target cluster. In other embodiments, crosstalk can be modeled as light interference on pixels associated with the target cluster.

[0064] After determining the inter-cluster interference metric, the crosstalk-aware base calling system 106 performs an action 208 of generating a modified intensity value for the target cluster by removing the inter-cluster interference metric. Based on the modified intensity value for 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 modified intensity value for the target cluster results in a guanine (G) nucleobase call, while the unmodified intensity value for the target cluster initially results in a cytosine (C) nucleobase call.

[0065] As previously mentioned, the crosstalk-aware base calling system 106 can follow a particular cluster order to determine illumination indicators and crosstalk in a given sequencing cycle. For example, the crosstalk-aware base calling system 106 can identify a first subset of oligonucleotide clusters that emit the brightest signals within a range of highest intensity values ​​(e.g., the brightest top 10%). The crosstalk-aware base calling system 106 then determines: (i) the nucleobase calls of the first subset of oligonucleotide clusters, and (ii) an inter-cluster interference measure that estimates the interference of clusters from the first subset of oligonucleotide clusters to a second subset of oligonucleotide clusters that emit signals within a second range of intensity values ​​(e.g., the brightest top 20% to 30%). Based on the additional range of intensity values, the remaining cluster orders can follow the same or similar pattern. In some cases, for example, the crosstalk-aware base calling system 106 determines: (i) nuclear base calls for a second subset of oligonucleotide clusters, and (ii) an inter-cluster interference measure that estimates interference of clusters from the second subset of oligonucleotide clusters with a third subset of oligonucleotide clusters that emit signals within a third range of intensity values ​​(e.g., the brightest top 30% to 40%).

[0066] As just described, the crosstalk-aware base calling system 106 can determine the nuclear base calls, illumination indicators, and crosstalk in a given sequencing cycle in a certain order based on a range of intensity values. As an alternative to using a range of intensity values, the crosstalk-aware base calling system 106 can (i) identify a first subset of oligonucleotide clusters based on a DC offset and an amplitude of a signal emitted by the cluster, and (ii) determine an inter-cluster interference measure that estimates the interference of clusters from the first subset of oligonucleotide clusters to a second subset of oligonucleotide clusters that emit signals. For example, the crosstalk-aware base calling system 106 can (i) identify a first subset of oligonucleotide clusters that exhibit a combination of a DC offset and an amplitude within a first threshold difference of a received intensity value for a given cluster, and (ii) identify a second subset of oligonucleotide clusters that exhibit a combination of a DC offset and an amplitude within a second threshold difference of a received intensity value for a given cluster.

[0067] Figure 2An overview of the actions performed by the crosstalk-aware base calling system 106 is provided as part of generating a modified intensity value for a target cluster by removing or subtracting crosstalk from neighboring clusters to the target cluster using an inter-cluster interference metric. According to one or more embodiments, Figure 3 An example of inter-cluster crosstalk (e.g., optical interference) increasing as the distance between clusters decreases is shown. Specifically, Figure 3 A 1D cross-section of a 2D nucleotide sample slide containing three oligonucleotide clusters is depicted to show how the distance between clusters affects inter-cluster crosstalk.

[0068] As discussed previously, some existing sequencing systems place limits on the number and density of oligonucleotide clusters in the flow cell in order to maintain accurate nucleobase calling. Figure 3 As shown, when there is a sufficiently large distance between the oligonucleotide adjacent cluster 302, the oligonucleotide central cluster 304 and the oligonucleotide adjacent cluster 306, the signal of the oligonucleotide central cluster 304 with a relatively high intensity value does not overlap (or only minimally overlaps) with the signals of the oligonucleotide adjacent cluster 302 and the oligonucleotide adjacent cluster 306 with relatively low intensity values.

[0069] Because the overlap between the signals of the oligonucleotide adjacent cluster 302, the oligonucleotide central cluster 304, and the oligonucleotide adjacent cluster 306 is relatively small, existing sequencing systems can more easily detect the interference of the signal of the oligonucleotide central cluster 304 on the intensity values ​​of the signals emitted by the oligonucleotide adjacent cluster 302 and the oligonucleotide adjacent cluster 306. With more accurate or purer intensity values ​​of the oligonucleotide adjacent cluster 302 and the oligonucleotide adjacent cluster 306, the crosstalk-aware base calling system 106 can more accurately perform nuclear base calling and determine whether the oligonucleotide adjacent cluster 302 and the oligonucleotide adjacent cluster 306 are "switched on" (e.g., illuminated or emitting light intensity at a specific frequency) or "off" (e.g., not illuminated or emitting light intensity at a specific frequency) in certain intensity channels during a sequencing cycle.

[0070] like Figure 3 As further depicted, as the density between oligonucleotide clusters increases, accurately determining the intensity values ​​of oligonucleotide clusters emitting relatively low (e.g., relatively dim) intensity values ​​and nucleobase detection becomes more difficult because light interference from oligonucleotide clusters emitting relatively high (e.g., relatively bright) intensity values ​​affects the intensity values ​​of the darker oligonucleotide clusters.

[0071] like Figure 3As shown, for example, the reduced distance between the oligonucleotide adjacent clusters 308, the oligonucleotide central cluster 310, and the oligonucleotide adjacent clusters 312 causes an increase in crosstalk that interferes with the relatively low intensity values ​​of the oligonucleotide adjacent clusters 308 and the oligonucleotide adjacent clusters 312. More specifically, the optical signal emitted from the oligonucleotide central cluster 310 interferes with or makes it more difficult to detect the intensity values ​​of the oligonucleotide adjacent clusters 308 and the oligonucleotide adjacent clusters 312. Due to optical interference, and the increased likelihood of detecting the intensity values ​​of the oligonucleotide central cluster 310 that are incorrectly attributed to the oligonucleotide adjacent clusters 308 and the oligonucleotide adjacent clusters 312, existing sequencing systems are generally unable to accurately perform nucleobase calls on the oligonucleotide adjacent clusters 308 and the oligonucleotide adjacent clusters 312.

[0072] As noted above, the crosstalk-aware base calling system 106 can determine the nucleobase calls and corresponding illumination indicators. According to one or more embodiments, Figure 4 It is shown that in a given sequencing cycle, the crosstalk-aware base calling system 106 determines the nucleobase calls and corresponding sets of illumination indicators for oligonucleotide clusters in different channels. As mentioned above, the illumination indicator indicates whether and / or to what extent a cluster provides a fluorescent response in a specific intensity channel during sequencing.

[0073] Specifically, Figure 4 The on / off state of the illumination indicator set in two different intensity channels corresponding to the oligonucleotide cluster of a specific type of nucleotide base is shown. To illustrate this on / off state, Figure 4 The intensity of light at specific frequencies (e.g., frequency bands) emitted or not emitted from the oligonucleotide cluster 402 in the cropped images are depicted, and these cropped images are shown in rows next to the nucleobase calls adenine (A) 408, cytosine (C) 410, thymine (T) 412, and guanine (G) 414.

[0074] For example, Figure 4As shown, when a nucleobase call of adenine (A) 408 is made for the oligonucleotide cluster 402, the crosstalk-aware base calling system 106 determines that the first set of illumination indicators indicating the oligonucleotide cluster 402 is "on" (e.g., illuminated by a light intensity of a particular frequency, or emitting a light intensity of a particular frequency) in both the first channel captured by the first channel image 404 and the second channel captured by the second channel image 406. In contrast, when a nucleobase call of cytosine (C) 410 is made for the oligonucleotide cluster 402, the crosstalk-aware base calling system 106 determines that the second set of illumination indicators indicating the oligonucleotide cluster 402 is "on" in the first channel captured by the first channel image 404, and is "off" (e.g., not illuminated by a light intensity of a particular frequency, or not emitting a light intensity of a particular frequency) in the second channel captured by the second channel image 406. When a nucleobase call of thymine (T) 412 is made for the oligonucleotide cluster 402, the crosstalk-aware base calling system 106 determines that a third set of illumination indicators indicative of the oligonucleotide cluster 402 are "off" in the first channel captured by the first channel image 404, and "on" in the second channel captured by the second channel image 406. Finally, when a nucleobase call of guanine (G) 414 is made for the oligonucleotide cluster 402, the crosstalk-aware base calling system 106 determines that a fourth set of illumination indicators indicative of the oligonucleotide cluster 402 are "off" in both the first channel captured by the first channel image and the second channel captured by the second channel image 406.

[0075] As previously discussed, the illumination state of the illumination indicator (e.g., an on / activated state, or an off / inactivated state) can be in a paired form or in a continuous form. For example, if the illumination indicator is "on" in the intensity channel (and emits a light intensity of a specific frequency) during sequencing, the "on" state can be represented by 1. Conversely, if the illumination indicator is "off" in the intensity channel (and does not emit a light intensity of a specific frequency) during sequencing, the "off" state can be represented by 0.

[0076] Therefore, the illumination state of an oligonucleotide cluster in more than one channel can be represented by a set of illumination indicators. For example, a set of illumination indicators represented by [1,1] can indicate that the illumination indicator of the oligonucleotide cluster is "on" in the first intensity channel and the second intensity channel. In addition, the crosstalk-aware base detection system 106 can decode the set of illumination indicators based on the detection of the nuclear bases. For example, a set of illumination indicators of an oligonucleotide cluster having an adenine (A) nucleotide base can be represented by [1,1]; a set of illumination indicators of an oligonucleotide cluster having a cytosine (C) nucleotide base can be represented by [1,0]; a set of illumination indicators of a thymine (T) nucleotide base can be represented by [0,1]; a set of illumination indicators of a guanine (G) nucleotide base can be represented by [0,0].

[0077] As previously mentioned, the illumination state of the illumination indicator mark can be continuous. More specifically, a given illumination indicator mark can indicate the degree to which the oligonucleotide cluster is illuminated by the light intensity of a specific frequency (e.g., frequency band). For example, based on the possibility that the oligonucleotide cluster falls within the intensity value boundary defined by the Gaussian mixture model, the crosstalk-aware base detection system 106 can determine the degree to which the illumination indicator mark is illuminated in a given intensity channel. In addition, the crosstalk-aware base detection system 106 can determine the degree to which the continuous illumination indicator mark is illuminated based on the intensity value of the oligonucleotide cluster.

[0078] Taking into account the relationship between lighting indicators and nuclear base calls, in some embodiments, the crosstalk-aware base calling system 106 can update or adjust the lighting indicator set based on the modified signal of the target cluster. For example, the crosstalk-aware base calling system 106 can generate a modified (and more accurate) intensity value for the target cluster by removing the inter-cluster interference measure from the initial intensity value of the target cluster. Based on the modified intensity value of the target cluster, the crosstalk-aware base calling system 106 can make different nuclear base calls for the target cluster. Based on the different and more accurate nuclear base calls, the crosstalk-aware base calling system 106 can adjust the lighting indicator set to more accurately represent the "on" or "off" state of the lighting indicator of the target cluster within 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 that the nuclear base call is A, and the lighting indicator set of the target cluster is [1,1]. However, based on the modified intensity values ​​of the target cluster and the corresponding nucleobase calls T, the crosstalk-aware base calling system 106 determines the adjusted set of illumination indication signatures to be [0, 1].

[0079] As discussed above, in one or more embodiments, the crosstalk-aware base calling system 106 can utilize the inter-cluster interference metric to remove crosstalk from neighboring clusters to the target cluster. According to one or more embodiments, Figure 5A and Figure 5B The crosstalk-aware base calling system 106 is shown utilizing an equalizer system, determining an inter-cluster interference metric representing optical interference of neighboring clusters to a target cluster, and generating a modified intensity value for the target cluster based on the inter-cluster interference metric.

[0080] 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 the intensity value of the target cluster by processing the received image. Generally speaking, a linear equalizer is a linear filter that can be designed or optimized to filter out noise. In some embodiments, the equalizer can convert the scattered intensity energy on the received pixels into received intensity values ​​for the target cluster and adjacent 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. Specifically, Figure 5A A model of an equalizer system is described.

[0081] In some embodiments, the crosstalk-aware base calling system 106, when implemented on a sequencing device, can utilize a linear equalizer to calculate a weighted sum of pixel intensity values ​​depicting intensity emissions from a target cluster and one or more neighboring clusters. The equalizer can be trained to produce equalizer coefficients that are configured to mix / combine pixel intensity values ​​depicting emission intensities from the target cluster and neighboring clusters in a manner that maximizes, for example, the signal-to-noise ratio.

[0082] like Figure 5A As shown, the crosstalk-aware base calling system 106 can receive an input image 503 of a nucleotide sample slide segment. The input image can include pixels depicting intensity values ​​of a target cluster and nearby neighboring clusters. Based on the received input image, the equalizer can collect light energy from the pixels and convert the energy into an intensity value (y) of the target cluster (i) in channel (j) during cycle (c). i,c,j ). The system model of the equalizer 505 can be modeled as i,c,j =a i,c,j v i,c,j +d i,c,j +n i,c,j Amplification coefficient a i,c,j Considering the scale variation between clusters on the nucleotide sample slide for cycles (c), channels (j), and clusters (i). The clean intensity signal (v i,c,j ) takes into account the unsealed and unshifted signals for cycles (c), channels (j) and clusters (i). The DC offset (d i,c,j ) takes into account random noise caused by different cluster sizes, different background intensities, varying stimulus responses, varying focus, varying sensor sensitivity, and varying lens aberrations for cycles (c), channels (j), and clusters (i). The variable n( i,c,j ) represents the additive noise of cycle (c), channel (j) and cluster (i).

[0083] When processing the input using the system model of the equalizer 505, the crosstalk-aware base calling system 106 can determine the intensity value 507P[x,y,c,j] of the pixel at cycle (c), position (x,y), and channel (j). As described below in Figure 5B As discussed in, the crosstalk-aware base calling system 106 can use pixel intensity to determine modified intensity values ​​for target clusters. Although the described embodiment uses a linear equalizer to determine the pixel intensity depicting the target cluster, other embodiments can use the described method in conjunction with an intensity detection system and / or an intensity extraction system. In some embodiments, the crosstalk-aware base calling system 106 uses an equalizer as described in U.S. Patent No. 11,188,778, entitled “Equalization-Based Image Processing and Spatial Crosstalk Attenuator,” filed by Eric Ojard et al., and U.S. Patent Application No. 18 / 059,326, entitled “Generating Cluster-Specific-Signal Corrections for Determining Nucleotide-Base Calls,” filed by Eric Ojard et al., each of which is incorporated herein by reference in its entirety.

[0084] like Figure 5B As shown, and as discussed above, the crosstalk-aware base calling system 106 can perform action 502 of determining nucleobase calls of neighboring clusters and illuminating an indication mark. As previously mentioned, the crosstalk-aware base calling system 106 can detect and / or measure light emitted by neighboring clusters in a given channel during a sequencing cycle and determine an intensity value of the emitted light. In some cases, based on the intensity values ​​of the neighboring clusters, the crosstalk-aware base calling system 106 determines the nucleobase calls of the neighboring clusters.

[0085] To illustrate this intensity value-based base calling, in some embodiments, the crosstalk-aware base calling system 106 can apply an 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 the oligonucleotide cluster in a given sequencing cycle, the crosstalk-aware base calling system 106 can determine the probability that the intensity value of the oligonucleotide cluster falls within one of the four intensity value boundaries corresponding to each type of nucleobase. The crosstalk-aware base calling system 106 can then call the nucleobase of the oligonucleotide cluster by selecting the nucleobase with the highest probability based on the intensity value boundaries.

[0086] As discussed above, in some embodiments, based on the nuclear base detection, the crosstalk-aware base detection system 106 can determine a set of lighting indicators for the cluster. For example, the crosstalk-aware base detection system 106 can determine the "on" and / or "off" state of the lighting indicators of adjacent clusters in one or more intensity channels. As discussed above, in some cases, the crosstalk-aware base detection system 106 can represent the lighting state of the lighting indicators in a paired format. For example, if the crosstalk-aware base detection system 106 makes a nuclear base detection of adenine (A) for an adjacent cluster, the crosstalk-aware base detection system 106 determines that the lighting indicator of the oligonucleotide cluster is "on" in both the first intensity channel and the second intensity channel. Based on this determination, the crosstalk-aware base detection system 106 can represent the on state of the oligonucleotide cluster in these two channels as a lighting indicator set [1,1]. As discussed in more detail below, the crosstalk-aware base detection system 106 can use the data in the lighting indicator set to determine an inter-cluster interference measure.

[0087] like Figure 5B As further shown, in some embodiments, the crosstalk-aware base calling system 106 utilizes a signal model 504 of a target cluster. More specifically, the crosstalk-aware base calling system 106 can utilize a function to determine an initial intensity value (P[x,y,c,j]) of a pixel (P) representing an oligonucleotide target cluster at position [x,y], where (x) represents the horizontal coordinate of the pixel and (y) represents the vertical coordinate of the pixel. As indicated by the signal model, 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 neighboring clusters.

[0088] like Figure 5B As shown, for example, the intensity value of the target cluster (P[x,y,c,j]) can be modeled as:

[0089]

[0090] Background intensity Estimate the background intensity value at position (x, y) in the captured image within the channel ( / ) during the sequencing cycle (c). In some cases, the estimated background intensity value may include noise or bias inherent in the genomic sample or sequencing equipment. For example, in some embodiments, the background intensity may increase the intensity value of the target cluster. In addition, the function The sum of the intensity values ​​from the target cluster and the crosstalk from neighboring clusters is estimated.

[0091] like Figure 5BAs further shown, the sum of the target cluster intensity values ​​may include the magnitude of the target cluster within intensity channel (j) and the neighboring cluster with cluster index (i) during sequencing cycle (c). Specifically, based on the intensity value of the target cluster, the crosstalk-aware base calling system 106 can estimate the amplitude of the target cluster within the intensity channel, as well as the amplitudes of one or more adjacent clusters. Figure 5B As indicated, the crosstalk-aware base calling system 106 can determine the illumination indicator signature encoded for the target cluster in the intensity channel (j) during the sequencing cycle (c). and corresponding lighting indication identifiers of one or more adjacent clusters having cluster index (i).

[0092] As discussed above, in some cases, the paired format encoded in the target cluster can be represented by a set of illumination indication identifiers for the target cluster (e.g., [1,1], [1,0], [0,1], or [0,0]). However, as discussed above, crosstalk from neighboring clusters with high intensity values ​​may amplify the intensity value of the target cluster. The increased intensity value of the target cluster may result in an erroneous indication that the target cluster is turned on in the first intensity channel or the second intensity channel during sequencing.

[0093] Figure 5B As further illustrated, the signal model 504 of the target cluster may include an estimate of a point spread function (PSF) covering the PSF response relative to the center position (x i ,y i ). For example, the crosstalk-aware base calling system 106 may respond to each position (x, y) closer to the PSF center position (x i ,y i ) for the first position to estimate a higher PSF response, or for the position farther from the center of the PSF response (x i ,y i ) is estimated at a second location of the target cluster. As mentioned above, the estimated PSF can illustrate how crosstalk diffused from neighboring clusters interferes with the intensity values ​​of the target cluster. More specifically, the estimated PSF can estimate the PSF response of the location of the target cluster relative to the center of the PSF responses from neighboring clusters.

[0094] like Figure 5B As further shown, the crosstalk-aware base calling system 106 can determine an inter-cluster interference metric 506. As mentioned above, the inter-cluster interference metric (I i0 _ i1 ) can represent the optical interference of one cluster (denoted as i1) to another cluster (denoted as i0). For example, the inter-cluster interference metric (I i0 _ i1 ) can represent the optical interference of adjacent clusters to the target cluster.

[0095] like Figure 5B Function I i0 _ i1 As indicated, the crosstalk-aware base calling system 106 can be configured to generate a base call by dividing the magnitudes of adjacent clusters by Illuminated indicators of adjacent clusters The inter-cluster interference measure for a given sequencing cycle (c) is estimated by multiplying the PSF corresponding to the target cluster position. Specifically, the crosstalk-aware base calling system 106 can estimate the inter-cluster interference measure for a given sequencing cycle (c) by, in part, estimating the amplitude of the neighboring cluster (i1) in the channel (j) during the sequencing cycle (c). To determine the inter-cluster interference measure (I i0 _ i1 As mentioned previously, the crosstalk-aware base calling system 106 can estimate the magnitude of the neighboring cluster (i1) based on the intensity value of the neighboring cluster (i2).

[0096] The crosstalk-aware base calling system 106 can further estimate the illumination indicator of the adjacent cluster (i1) based on the intensity value of the adjacent cluster (i1). For example, based on the high intensity value of the adjacent cluster (i1), the crosstalk-aware base calling system 106 determines the nuclear base call (e.g., A) of the adjacent cluster in the first intensity channel and the second intensity channel, and the corresponding illumination indication label (e.g., [1,1]) of the adjacent cluster (i1).

[0097] In addition, if Figure 5B As shown, the crosstalk-aware base calling system 106 can be used to determine the position of the adjacent cluster (i1) relative to the center position (x i1 ,y i1 ) (or region) to estimate the location (x) of the target cluster (i0) i0 ,y i0 ). As mentioned above, the estimated PSF corresponding to the location of the target cluster can describe how the intensity values ​​of the neighboring clusters affect the intensity value of the target cluster based on the locations of the neighboring clusters and the target cluster.

[0098] like Figure 5B As further indicated by the corresponding functions, the crosstalk-aware base calling system 106 can deduct the inter-cluster metric 508 from the sum of the intensity values ​​of the target cluster. Specifically, the crosstalk-aware base calling system 106 can remove the inter-cluster interference metric of the neighboring cluster (i1) from the sum of the intensity values ​​of the target cluster (i0). Similarly, Figure 5BFurther depicted, the crosstalk-aware base calling system 106 can determine the inter-cluster interference measure for the neighboring cluster (i2) up to the neighboring cluster (in), and then remove it from the sum of the intensity values ​​of the target cluster (i0). Thus, the crosstalk-aware base calling system 106 can determine the inter-cluster interference measure for multiple neighboring clusters, and then remove it from the intensity value of a single target cluster. For example, in some embodiments, the crosstalk-aware base calling system 106 can estimate the inter-cluster interference measure for the neighboring cluster with the highest intensity that is closest to the target cluster and remove it. In addition, the crosstalk-aware base calling system 106 can subtract the crosstalk originating from the neighboring cluster (i1) from any other cluster position on the flow cell.

[0099] As mentioned above, the crosstalk-aware base calling system 106 can repeatedly determine the inter-cluster interference measure of the crosstalk of the neighboring cluster subsets to the corresponding subset of the target cluster, and then remove the inter-cluster interference measure of the neighboring cluster subsets from the corresponding subset of the target cluster based on the intensity value range of the neighboring cluster subsets. For example, the crosstalk-aware base calling system 106 can determine the nucleobase calls of the first subset of neighboring clusters of oligonucleotides that emit the brightest signals within the highest intensity value range (e.g., the brightest top 10%). In some cases, the crosstalk-aware base calling system 106 calls the nucleobases of the brightest clusters because they have the highest probability of falling within the intensity value boundary associated with one of the nucleobases (e.g., A). Based on the nucleobase calls of the first subset of oligonucleotide neighbor clusters, the crosstalk-aware base calling system 106 determines (i) an illumination indicator signature of a corresponding cluster from the first subset of oligonucleotide neighbor clusters, and (ii) an inter-cluster interference measure of each neighbor cluster from the first subset of oligonucleotide neighbor clusters relative to each target cluster from the subset of oligonucleotide target clusters. The crosstalk-aware base calling system 106 further removes the inter-cluster interference measure of the first subset of oligonucleotide neighbor clusters from the sum of the intensity values ​​of each target cluster.

[0100] After such removal, the crosstalk-aware base calling system 106 can determine the nucleobase calls, illumination indicators, and inter-cluster interference measures for a second subset of neighboring clusters within a second range of intensity values ​​(e.g., the brightest top 20% to 30%). The crosstalk-aware base calling system 106 can further remove the inter-cluster interference measures for each neighboring cluster of the second subset of neighboring clusters from the sum of the intensity values ​​for each target cluster from the second subset of oligonucleotide target clusters.

[0101] like Figure 5B As further shown, after the crosstalk-aware base calling system 106 removes the inter-cluster interference measure, the crosstalk-aware base calling system 106 can generate a modified intensity value 510 for the pixel depicting the target cluster. Figure 5B Indicated, for example, the modified intensity values ​​of the pixels depicting the target cluster Equal to the sum of the following: (i) background intensity and (ii) the magnitudes of the neighboring clusters and the target cluster With lighting indicator The sum is multiplied by the PSF. As indicated previously, the modified intensity value Indicating a more accurate intensity value and / or a purer signal for a target cluster during a sequencing cycle. Based on the modified intensity value, the crosstalk-aware base calling system 106 can make more accurate nucleobase calls with minimal or no crosstalk interference from one or more neighboring clusters.

[0102] For example, as previously mentioned, the crosstalk-aware base calling system 106 can calculate the probability of a signal falling within the intensity value boundaries of a particular nucleobase (A, C, G, or T) based on a Gaussian probability distribution and expectation maximization. By removing the inter-cluster interference measure, the crosstalk-aware base calling system 106 can determine a more accurate probability that the signal falls within the intensity value boundaries 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 detection or prediction of the nucleobase incorporated into the cluster. In other embodiments, the updated probability may not change the detection or prediction of the nucleobase incorporated into the cluster, but may provide a higher base call quality measure (e.g., QUAL score) that the signal from the cluster falls within the intensity value boundaries of the initially detected or predicted nucleobase.

[0103] As just indicated, in some cases, the crosstalk-aware base calling system 106 estimates the PSF response of a segment of a nucleotide sample slide that includes a target cluster and an adjacent cluster. According to one or more embodiments, Figure 6 The point spread function of the estimated cluster intensity values ​​by the crosstalk-aware base calling system 106 is shown.

[0104] like Figure 6 As shown, the estimated PSF can describe the response at a specific location or region relative to the central PSF response of a point source (e.g., an oligonucleotide cluster). More specifically, Figure 6 The mathematically modeled PSF response 602 of the intensity values ​​from the oligonucleotide cluster is shown. Figure 6 As shown, the estimated PSF of the oligonucleotide cluster intensity values ​​is most concentrated (e.g., brightest) at the central location or region and decreases as the signal from the oligonucleotide cluster moves away from the central location or region of the oligonucleotide cluster. The crosstalk-aware base calling system 106 can use the estimated PSF response to estimate the degree of crosstalk caused by neighboring clusters to the target cluster.

[0105] 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) method, the detector receives a signal (y) to determine a PSF estimate The received signal (y) can be expressed as y=Mh+n, where h is the complex channel impulse response (e.g., PSF response), M is the cyclic training sequence matrix, and n is the noise. After generating the training sequence matrix (M) and minimizing After the squared error, the estimated least squares channel impulse response It can be expressed as in() H and() -1 denote the Hermitian matrix and the inverse matrix respectively. Finally, given is the best linear unbiased estimate of the channel coefficient, and the equation mentioned above can be simplified to Where P represents the length of the training sequence. Therefore, based on the equation To estimate the PSF. In some implementations, the crosstalk-aware base calling system 106 determines a PSF estimate as described in Jinho Choi, Adaptive and Iterative Signal Processing in Communications (Cambridge Univ. Press 2006), or Markku Pukkila, Channel Estimation Modeling (2000), available at http: / / www.comlab.hut.fi / opetus / 333 / presentations_2000 / chan_est.pdf, both of which are incorporated herein by reference in their entirety.

[0106] According to one or more embodiments, 7A to 7C The crosstalk between oligonucleotide clusters and the effect of removing the light interference of certain oligonucleotide clusters are demonstrated. Specifically, for the purpose of illustration, 7A to 7C Provides a simulated image of oligonucleotide clusters on a nucleotide sample slide and the crosstalk between oligonucleotide clusters. 7A to 7C The image in shows the clusters as a grid of evenly spaced squares, but the actual oligonucleotide clusters are not evenly dispersed on the nucleotide sample slide. 7A to 7C The clusters at the center of each pixel within a square grid are depicted to more clearly demonstrate the impact of crosstalk. 7A to 7C A nucleotide sample slide utilizing a square grid is shown, but other embodiments of nucleotide sample slides may utilize various shapes (eg, diamonds, hexagons, etc.).

[0107] As a schematic diagram, Fig. 7AAn image 700 is depicted that maps the intensity values ​​of oligonucleotide clusters that respond to light excitation in an intensity channel. Image 700 may represent a section of a nucleotide sample slide (e.g., a flow cell) on which oligonucleotide clusters have been seeded. Fig. 7A Shown, the image 700 of intensity channel comprises several oligonucleotide clusters, and uses the intensity value corresponding to pixel mapping.Specifically, the image 700 of intensity channel uses pixel to represent the intensity value at a given position in the flow cell.The intensity value described by each pixel is the intensity value sum of oligonucleotide clusters, noise and the crosstalk from adjacent clusters.

[0108] Fig. 7A Oligonucleotide clusters adjacent to other clusters on the nucleotide sample slide section are also depicted. Specifically, Fig. 7A The oligonucleotide cluster of the target cluster is first adjacent, second adjacent or third adjacent. When the adjacent cluster is positioned to leave a cluster of the target cluster, or when it is adjacent to the target cluster relative to other clusters, this type of adjacent cluster is first adjacent to the target cluster. For example, eight adjacent clusters in the first adjacent boundary 712 are first adjacent to the oligonucleotide "disconnection" cluster 702a, because relative to other clusters, these eight adjacent clusters are next to (and closer to) the oligonucleotide "disconnection" cluster 702a. Relatedly, when the adjacent cluster is positioned to leave two clusters of the target cluster, or when it is positioned at the position immediately after the target cluster, this type of adjacent cluster is second adjacent to the target cluster. For example, as Fig. 7A As shown, the 16 adjacent clusters within the second adjacent boundary 714 (and outside the first adjacent boundary 712) are second adjacent to the oligonucleotide "break" cluster 702a because these 16 adjacent clusters are located immediately after the oligonucleotide "break" cluster 702a. Similarly, when the adjacent cluster is located three clusters away from the target cluster, or is located immediately after the target cluster, such adjacent cluster is third adjacent to the target cluster. Figure 3 As shown, the 24 neighboring clusters within the third neighboring boundary 716 (and outside the second neighboring boundary 714) are third neighboring with respect to the oligonucleotide "break" cluster 702a because the 24 neighboring clusters are located three clusters away from the oligonucleotide "break" cluster 702a (or located immediately after and then after the oligonucleotide "break" cluster). As noted above, in some embodiments, the crosstalk-aware base calling system 106 determines the inter-cluster interference measure for clusters that are first neighboring, second neighboring, and / or third neighboring to the target cluster.

[0109] To represent the different types of light emitted by each cluster, Fig. 7ADepict different patterns representing the changing intensity values ​​of different clusters and different circle types representing the cluster illumination indicator logo in image 700. As indicated by denser or darker patterns and sparser or brighter patterns, darker and / or darker pixels represent oligonucleotide positions and / or oligonucleotide clusters with lower intensity values, while brighter and / or brighter pixels represent oligonucleotide positions and / or oligonucleotide clusters with higher intensity values. In addition, the pixels comprising black circles with white borders represent that for a given channel, the oligonucleotide "disconnects" clusters that do not (or have not yet been detected) emit the light intensity of a specific frequency (e.g., frequency band), while the pixels comprising white circles with black borders represent that for a given channel, the oligonucleotide "connects" clusters that emit (or have been detected to emit) the light intensity of a specific frequency (e.g., frequency band) are emitted.

[0110] As discussed previously, it may be difficult to make nucleobase calls for oligonucleotide "off" clusters that are adjacent to or surrounded by oligonucleotide "on" clusters with high intensity values ​​because crosstalk from oligonucleotide "on" clusters with high intensity values ​​can distort the intensity values ​​of the oligonucleotide "off" clusters. Specifically, Fig. 7A shows how crosstalk emitted from an oligonucleotide "on" cluster with a high intensity value can increase the intensity value of an adjacent or neighboring oligonucleotide "off" cluster. Fig. 7A In the example, oligonucleotide "off" cluster 702a appears to be an oligonucleotide "on" cluster because crosstalk from bright oligonucleotide neighboring clusters 706a and 706b makes the pixel containing oligonucleotide "off" cluster 702a appear brighter (e.g., increases the intensity value of the pixel). By making the pixel containing oligonucleotide "off" cluster 702a appear brighter, the probability of making an incorrect nucleobase call for oligonucleotide "off" cluster 702a increases. In addition, Fig. 7A shows how the lower intensity values ​​of dim "on" clusters of oligonucleotides make it difficult to determine the nucleobase call of dim "on" clusters of oligonucleotides because they appear to have similar intensity values ​​to the intensity values ​​of adjacent oligonucleotide "off" clusters on the cluster. More details on dim "on" clusters of oligonucleotides are in Fig. 8A discussed in.

[0111] According to one or more embodiments, Figure 7BThe crosstalk-aware base calling system 106 is shown initially determining the nucleobase calls and lighting indicators for a subset of clusters as part of an orderly method for removing crosstalk. For example, the subset of oligonucleotide clusters highlighted by selection boundaries 708a, 708b, 708c, 708d, 708e, 708f, 708g, 708h, 708i, 708j, and 708k represent the oligonucleotide clusters that emit the highest intensity values ​​within a range of highest intensity values ​​(e.g., top 10% or top 15%). As previously discussed, the high intensity values ​​of such a subset of oligonucleotide clusters allow the crosstalk-aware base calling system 106 to more confidently determine the nucleobase calls. Based on the nucleobase calls for the subset of oligonucleotide clusters highlighted by selection boundaries 708a-708k, the crosstalk-aware base calling system 106 can more accurately determine that the oligonucleotide cluster is "on" within the intensity channel.

[0112] Although nucleobase calling is easier for the subset of oligonucleotide clusters highlighted by selection boundaries 708a-708k, in some cases these oligonucleotide clusters generate the most crosstalk (e.g., light interference) and affect neighboring oligonucleotide clusters with lower intensity values. Figure 7B As shown, oligonucleotide "disconnection" cluster 702a is surrounded by a subset of oligonucleotide clusters highlighted by selection boundaries 708d, 708g, and 708h, which emits the highest intensity value and the highest level of crosstalk. The crosstalk from the subset of oligonucleotide clusters highlighted by selection boundaries 708d, 708g, and 708h increases the intensity value of oligonucleotide "disconnection" cluster 702a. Due to the increase in the intensity value of oligonucleotide "disconnection" cluster 702a, it is more likely that oligonucleotide "disconnection" cluster 702a is given an inaccurate nucleobase call-there is no effective way to remove the crosstalk.

[0113] Although FIG. 7A to FIG. 7B shows crosstalk effects between oligonucleotide clusters, but Figure 7C The effect of removing crosstalk from certain oligonucleotide clusters is shown. Figure 7C As shown, the crosstalk-aware base calling system 106 removes crosstalk from the intensity values ​​of the various target clusters from the subset of oligonucleotide clusters highlighted by the selection boundaries 708a-708k. To illustrate the intensity values ​​of the light emitted by the target cluster (without the light emitted by the subset of oligonucleotide clusters highlighted by the selection boundaries 708a-708k), Figure 7CAn image 700 is depicted having a pattern indicating that light emitted by such a cluster subset has been removed. Prior to removing crosstalk, and as discussed above, the crosstalk-aware base calling system 106 can (i) determine nucleobase calls and determine a set of illumination indicative signatures for the oligonucleotide cluster subset highlighted by the selection boundaries 708a-708k, (ii) determine an inter-cluster interference metric for each cluster in the oligonucleotide cluster subset highlighted by the selection boundaries 708a-708k, and (iii) remove the inter-cluster interference metric for the oligonucleotide cluster subset from other oligonucleotide neighboring clusters having darker intensity values.

[0114] After removing the inter-cluster interference measure, image 700 depicts more accurate intensity values ​​for the darker oligonucleotide "on" and "off" clusters. For example, the crosstalk-aware base calling system 106 eliminates or removes the crosstalk emitted by oligonucleotide clusters 710a and 710b that interferes with the relatively low intensity value of the oligonucleotide target cluster. Figure 7C As proposed, the intensity value of oligonucleotide "broken" cluster 702a more clearly shows that oligonucleotide cluster 702a is "broken" in a particular channel. Therefore, the intensity value of oligonucleotide cluster 702a is more closely similar to the intensity value of oligonucleotide cluster 702b - both do not emit light intensity of a particular frequency (e.g., frequency band) in the channel captured by image 700.

[0115] like Figure 7C As further shown, by removing the crosstalk from the neighboring cluster of oligonucleotides having the highest intensity value to the target cluster, the crosstalk-aware base calling system 106 determines a modified intensity value for the oligonucleotide cluster 704, thereby clarifying that the oligonucleotide cluster 704 is "on" in the intensity channel during sequencing, or emits light intensities of a particular frequency (e.g., frequency band). Thus, the crosstalk-aware base calling system 106 can (i) make more accurate nucleobase calls based on the more accurate modified intensity values ​​for the oligonucleotide target cluster, and (ii) more confidently determine that a given cluster of oligonucleotides is "on" in a given channel during sequencing cycles, or emits light intensities of a particular frequency (e.g., frequency band).

[0116] As noted above, the crosstalk-aware base calling system 106 improves the accuracy with which illumination indications (and corresponding nucleobase calls) can be determined by determining and removing inter-cluster interference metrics. FIG. 8A to FIG. 8B Depicted are histograms of intensity values ​​for oligonucleotide clusters with and without crosstalk from neighboring clusters.

[0117] For example, Fig. 8AAs shown, the oligonucleotide clusters with higher intensity values ​​806 depicted by the black values ​​in the bar graph represent clusters with the following intensity values: for these intensity values, based on the lighting indicator that is clearly "on", accurate nuclear base detection can be more easily determined. Relatively high (or brightest) intensity values ​​reduce the possibility of inaccurate nuclear base detection by the crosstalk-aware base calling system 106 due to crosstalk from adjacent clusters. Therefore, the crosstalk-aware base calling system 106 can more easily determine whether the oligonucleotide clusters with relatively high intensity values ​​are "on". In contrast, the oligonucleotide clusters with lower intensity values ​​802 depicted by the white values ​​in the bar graph represent clusters with the following intensity values: for these intensity values, based on the lighting indicator that is clearly "off", accurate nuclear base detection can be more easily determined. Therefore, in the embodiment shown, the crosstalk-aware base calling system 106 can more easily determine that the oligonucleotide clusters with lower intensity values ​​802 are "off".

[0118] However, if Fig. 8A As further shown, this histogram includes overlapping intensity values ​​804 regions described by black and white stripe values, and it is difficult to determine core base detection and illumination indication mark for this region.For example, when the increased intensity value of oligonucleotide " disconnection " cluster overlaps with the intensity value of oligonucleotide " connection " cluster, the cluster with overlapping intensity value 804 can prove to be difficult to determine accurate core base detection and illumination indication mark.As discussed above, in some cases, the crosstalk from bright oligonucleotide adjacent clusters increases the intensity value of dim oligonucleotide " disconnection " cluster, and makes oligonucleotide " disconnection " cluster show as " connection ", or show the intensity value that may be " connection " or may not be " connection ".In addition, and as mentioned previously, some " connection " clusters of oligonucleotide do not actually emit the light intensity of specific frequency (for example, frequency band) in the intensity channel with high intensity value, and may show as " disconnection " in this intensity channel.

[0119] In some cases, existing sequencing systems identify an intensity value threshold for determining whether the intensity value of a given oligonucleotide cluster indicates that the given cluster emits a particular frequency (e.g., frequency band) of light intensity in an intensity channel. Fig. 8A As shown, the intensity value threshold may play little role in accurately determining whether an oligonucleotide cluster exhibiting overlapping intensity values ​​804 should have an "on" or "off" illumination indicator for a given intensity channel. Fig. 8A The depicted bar graph demonstrates that existing sequencing systems that use intensity value thresholds without an effective method to remove or subtract crosstalk are unable to accurately resolve the illumination indication signatures and corresponding nucleobase calls of oligonucleotide clusters that exhibit overlapping intensity values ​​804.

[0120] According to one or more embodiments, Figure 8BAs shown, by determining and removing an inter-cluster interference measure representing crosstalk emanating from adjacent clusters of oligonucleotides, the crosstalk-aware base calling system 106 determines a more accurate modified intensity value (and corresponding nucleobase call) for a target cluster. For example, Figure 8B Modified (or more accurate) intensity values ​​for oligonucleotide clusters are shown. Figure 8B As shown, the modified intensity values ​​808 for the oligonucleotide "off" clusters represented as white values ​​do not overlap with the modified intensity values ​​810 for the oligonucleotide "on" clusters represented as black values. By clearly separating the modified intensity values ​​808 for the oligonucleotide "off" clusters and the modified intensity values ​​810 for the oligonucleotide "on" clusters, the crosstalk-aware base calling system 106 can apply a range of intensity values ​​to clearly distinguish between the oligonucleotide "on" clusters and the oligonucleotide "off" clusters and more accurately determine the nucleobase calls of such oligonucleotide clusters.

[0121] Figures 1 to 8B , corresponding text and examples provide many different methods, systems, devices and non-transitory computer-readable media of the crosstalk-aware base calling system 106. In addition to the foregoing, one or more embodiments may also be described in terms of a flowchart including actions for achieving a particular result, such as Fig. 9 shown. Fig. 9 Can be performed with more or less actions.In addition, these actions can be performed in different orders.Additionally, the actions described herein can be repeated or performed in parallel with each other or in parallel with different instances of the same or similar actions.

[0122] Fig. 9 A flow chart showing a series of acts 900 for generating a quality metric for nucleobase calling using an inter-cluster interference metric according to one or more embodiments. Fig. 9 Actions according to one embodiment are shown, but alternative embodiments may omit, add, reorder, and / or modify Fig. 9 In some implementations, Fig. 9 In some cases, a non-transitory computer-readable medium stores thereon instructions that, when executed by at least one processor, cause a computing device to perform Fig. 9 In some implementations, the system performs Fig. 9 For example, in one or more cases, the system includes at least one processor and a non-transitory computer-readable medium containing instructions that, when executed by the at least one processor, cause the system to perform Fig. 9 action.

[0123] The series of acts 900 includes an act 902 for detecting a set of intensity values ​​from a first cluster and a second cluster. For example, act 902 may involve detecting intensity values ​​from a first signal from the first cluster and a second signal from the second cluster.

[0124] Additionally, the series of acts 900 includes an act of determining a set of illumination indicators for the first cluster 904. For example, act 904 can involve determining nucleobase calls for the first cluster, and determining the set of illumination indicators for the first cluster based on the nucleobase calls.

[0125] Additionally, sequence of acts 900 includes an act of determining an inter-cluster interference measure 906. For example, act 906 may involve estimating a degree of crosstalk from the first cluster to the second cluster by multiplying an estimated magnitude of the first cluster, a set of lighting indicators of the first cluster, and a point spread function response.

[0126] The series of actions 900 also includes an action 908 of generating a modified intensity value for a second cluster by removing the inter-cluster interference measure. Specifically, action 908 can involve, for a sequencing cycle, generating a second set of modified intensity values ​​for a second signal from the second cluster by removing the inter-cluster interference measure from the second set of intensity values, subtracting the inter-cluster interference measure from the sum of the intensity values ​​of the second cluster of oligonucleotides.

[0127] In some cases, the series of actions includes the following additional actions: determining the set of illumination indication signatures further based on the magnitudes of the first set of intensity values ​​and an estimated point spread function of the nucleotide sample slide segment including the first cluster of oligonucleotides; and then determining the inter-cluster interference measure further based on the estimated point spread function.

[0128] In one or more embodiments, the series of acts 900 further includes the additional act of estimating a point spread function using the locations of the second cluster of oligonucleotides or a different cluster of oligonucleotides as points and including a region containing the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

[0129] In some cases, the series of acts 900 includes the additional act of firstly adjacent, secondly adjacent, or thirdly adjacent a first position of a first cluster of oligonucleotides within the nucleotide sample slide to a second position of a second cluster of oligonucleotides within the nucleotide sample slide.

[0130] In addition, in one or more embodiments, this series of actions 900 includes the following additional actions: for a sequencing cycle, determining the nucleobase calls of a first cluster of oligonucleotides based on a first set of nucleobase intensity values ​​and an intensity value boundary; and further determining a lighting indication mark set based on the nucleobase calls of the first cluster of oligonucleotides.

[0131] In some embodiments, the series of actions 900 also includes the following additional actions: determining the nucleobase calls of 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 the value of the inter-cluster interference metric from the intensity values ​​from the second set of intensity values ​​corresponding to the first channel or from the intensity values ​​from the second set of intensity values ​​corresponding to the second channel. In some cases, the series of actions 900 includes generating a modified second set of intensity values ​​by subtracting the value of the inter-cluster interference metric from the intensity values ​​from the second set of intensity values ​​corresponding to the first channel or from the intensity values ​​from the second set of intensity values ​​corresponding to the second channel. Therefore, in some embodiments, the inter-cluster interference metric can be removed or eliminated from the intensity values ​​of both the first channel and the second channel.

[0132] In addition, in other embodiments, this series of actions 900 may include the following additional actions: determining a first illumination indicator that indicates whether the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second illumination indicator that indicates whether the second cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle; or determining a first continuous illumination indicator that indicates the extent to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicator that indicates the extent to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

[0133] In one or more cases, the series of actions 900 includes the following additional actions: determining an adjusted set of illumination indicator markers for the sequencing cycle and based on the modified second set of intensity values, the adjusted set of illumination indicator markers indicating whether the second cluster of oligonucleotides is illuminated during the sequencing cycle and being different from the initial set of illumination indicator markers corresponding to the second set of intensity values.

[0134] In some implementations, the series of acts 900 further includes the additional act of determining, for the sequencing cycle and based on the modified second set of intensity values, that the nucleobase calls of the second cluster of oligonucleotides are different from the nucleobases corresponding to the second set of intensity values.

[0135] In another embodiment, the series of actions 900 includes the following additional actions: for a sequencing cycle, detecting a third set of intensity values ​​for a third signal from a third cluster of oligonucleotides; determining an additional set of illumination indicator markers based on the third set of intensity values, the additional set of illumination indicator markers indicating whether the third cluster of oligonucleotides is illuminated during the sequencing cycle; determining an additional inter-cluster interference metric based on the additional set of illumination indicator markers, the additional inter-cluster interference metric estimating optical interference of the third cluster of oligonucleotides on the second cluster of oligonucleotides; and for the sequencing cycle, generating a second set of modified intensity values ​​for a 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.

[0136] Furthermore, in one or more embodiments, the series of actions 900 further include the following additional actions: determining that a first set of intensity values ​​for a first signal from a first cluster of oligonucleotides is within a range of intensity values; determining that a second set of intensity values ​​for a second signal from a second cluster of oligonucleotides is not within the range of intensity values; generating a modified second set of intensity values ​​by removing from the second set of intensity values ​​an inter-cluster interference measure that estimates optical interference of the first cluster of oligonucleotides with the second cluster of oligonucleotides based on the first set of intensity values ​​being within the range of intensity values ​​and the second set of intensity values ​​being not within the range of intensity values. Alternatively, the series of actions 900 includes: generating a modified second set of intensity values ​​by removing from the second set of intensity values ​​an inter-cluster interference measure that estimates optical interference of one or more pixels depicting the first cluster of oligonucleotides with one or more pixels depicting the second cluster of oligonucleotides based on the first set of intensity values ​​being within the range of intensity values ​​and the second set of intensity values ​​being not within the range of intensity values.

[0137] In some cases, this series of actions 900 includes the following additional actions: determining a first nucleobase call of a first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values ​​within a range of intensity values ​​based on the first set of intensity values; and determining a second nucleobase call of a second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values ​​not within the range of intensity values ​​based on a modified second set of intensity values.

[0138] 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 of 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 of a second signal from a second cluster of oligonucleotides in a single channel; and determines a set of illumination indicator markers by determining a single illumination indicator marker that indicates whether the first cluster of oligonucleotides is illuminated in the single channel during a sequencing cycle.

[0139] Methods described herein can be used in combination with multiple nucleic acid sequencing techniques. Particularly suitable techniques are those in which nucleic acids are attached to fixed positions in an array so that their relative positions do not change and in which the array is repeatedly imaged. Embodiments in which images are obtained in different color channels (e.g., matching different markers for distinguishing a nucleotide base type from another nucleotide base type) are particularly suitable. In some embodiments, the process of determining the nucleotide sequence of a target nucleic acid (i.e., nucleic acid polymer) can be an automated process. Preferred embodiments include sequencing by synthesis (SBS) technology.

[0140] SBS techniques generally include enzymatic extension of nascent nucleic acid chains by repeated addition of nucleotides to the template strand. In traditional SBS methods, a single nucleotide monomer can be provided to the target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to the target nucleic acid in the presence of a polymerase in the delivery.

[0141] The SBS technology described below can utilize single-end sequencing or double-end sequencing. In single-end sequencing, the sequencing equipment reads the fragment from one end to the other to generate a sequence of base pairs. In contrast, during double-end sequencing, the sequencing equipment starts with a read, completes a read of a specific read length in the same direction, and then starts another read from the opposite end of the fragment.

[0142] SBS can utilize nucleotide monomers with terminator parts or lack nucleotide monomers of any terminator parts.The method using the nucleotide monomer lacking terminator includes for example pyrophosphate sequencing and the sequencing of nucleotides using gamma-phosphate labeling, as described in further detail below.In the method using the nucleotide monomer lacking terminator, the number of nucleotides added in each cycle is usually variable, and the number depends on the mode of template sequence and nucleotide delivery.For the SBS technology utilizing the nucleotide monomer with terminator part, terminator can be effectively irreversible under the sequencing conditions used, such as the situation of traditional Sanger sequencing utilizing dideoxynucleotides, or terminator can be reversible, such as the situation of the sequencing method developed by Solexa (now Illumina, Inc.).

[0143] The SBS technique can utilize nucleotide monomers with a labeling portion or nucleotide monomers lacking a labeling portion. Thus, incorporation events can be detected based on the following items: properties of the label, such as the fluorescence of the label; properties of the nucleotide monomer, such as molecular weight or charge; byproducts of the incorporated nucleotide, such as the release of pyrophosphate; and the like. In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or alternatively, the two or more different labels may be indistinguishable under the detection technology used. For example, different nucleotides present in the sequencing reagent may have different labels, and they may be distinguished using appropriate optical devices, as exemplified by the sequencing method developed by Solexa (now Illumina, Inc.).

[0144] Preferred embodiments include pyrophosphate sequencing technology. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent chain (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. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568 and U.S. Pat. No. 6,274,320, the disclosures of which are incorporated herein by reference in their entirety). In pyrophosphate sequencing, the released PPi can be detected by being immediately converted into ATP by adenosine triphosphate (ATP) sulfurylase, and the level of ATP generated is detected by photons generated by luciferase. The nucleic acid to be sequenced can be attached to a feature in an array, and the array can be imaged to capture the chemiluminescent signal generated by the incorporation of nucleotides at the feature of the array. The image can be obtained after the array is treated with a specific nucleotide type (e.g., A, T, C or G). The images obtained after adding each nucleotide type will differ in which features in the array are detected. These differences in the image reflect the different sequence contents of the features on the array. However, the relative position of each feature will remain unchanged in the image. The 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 a reversible terminator-based sequencing method.

[0145] In another exemplary type of SBS, cycle sequencing is accomplished by the stepwise addition of reversible terminator nucleotides comprising, for example, cleavable or photobleachable dye labels, as described in, for example, WO 04 / 018497 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated herein by reference. The method is commercialized by Solexa (now Illumina Inc.) and is also described in WO 91 / 06678 and WO 07 / 123,744, the disclosures of each of which are incorporated herein by reference. The availability of fluorescently labeled terminators (wherein termination can be reversed and the fluorescent label can be cleaved) facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate these modified nucleotides and extend from these modified nucleotides.

[0146] Preferably, in the sequencing embodiment based on reversible terminator, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label can be removable, such as removed by cleavage or degradation. Images can be captured after the label is incorporated into the arrayed nucleic acid feature. In a specific embodiment, each cycle involves four different nucleotide types being delivered to the array simultaneously, and each nucleotide type has spectrally different labels. Four images can then be obtained, each image using a detection channel that is selective to one label in the four different labels. Alternatively, different nucleotide types can be added sequentially, and the image of the array can be obtained between each addition step. In such embodiments, each image will show the nucleic acid feature of the nucleotides that have been incorporated with a specific type. Due to the different sequence content of each feature, different feature will be present or not present in different images. However, the relative position of the feature will remain unchanged in the image. The image obtained by such reversible terminator-SBS method can be stored, processed and analyzed as described herein. After the image capture step, the label can be removed and the reversible terminator part can be removed for subsequent cycles of nucleotide addition and detection. Removing labels after they have been detected in a particular cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are set forth below.

[0147] In a specific embodiment, some or all of the nucleotide monomers may include a reversible terminator. In such embodiments, the reversible terminator / cleavable fluorophore may include a fluorophore (Metzker, Genome Res. 15: 1767-1776 (2005), which is incorporated herein by reference) connected to a ribose moiety via a 3' ester bond. Other methods have separated terminator chemistry from the cleavage of a fluorescent label (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety). Ruparel et al. describe the development of reversible terminators that use small 3' allyl groups to block extension, but can be easily deblocked by a short treatment with a palladium catalyst. The fluorophore is attached to the base via a photocleavable linker that can be easily cleaved by exposure to long wavelength ultraviolet light for 30 seconds. Therefore, disulfide reduction or photocleavage can be used as a cleavable linker. Another method of reversible termination is to use natural termination, which occurs after a bulky dye is placed on a dNTP. The presence of a charged bulky dye on a dNTP can act as an efficient terminator by steric hindrance and / or electrostatic hindrance. Unless the dye is removed, the presence of an incorporation event prevents further incorporation. The cracking of the dye removes the fluorophore and effectively reverses termination. The example of modified nucleotides is also described in U.S. Patent No. 7,427,673 and U.S. Patent No. 7,057,026, the disclosure of which is incorporated herein by reference in its entirety.

[0148] Additional exemplary SBS systems and methods that may 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, PCT Publication No. WO05 / 065814, U.S. Patent Application Publication No. 2005 / 0100900, PCT Publication No. WO 06 / 064199, PCT Publication No. WO07 / 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.

[0149] Some embodiments can use the detection of four different nucleotides using less than four different marks.For example, the method and system described in the material of the U.S. Patent Application Publication No. 2013 / 0079232 incorporated can be used to perform SBS.As a first example, a pair of nucleotide types can be detected at the same wavelength, but based on the intensity difference of a member in the pair relative to another member, or based on a member in the pair causing a change (for example, by chemical modification, photochemical modification or physical modification) that a signal that is obvious compared with the signal of another member of the pair detected appears or disappears.As a second example, three of the four different nucleotide types can be detected under specific conditions, and the fourth nucleotide type lacks a mark (for example, the minimum detection caused by background fluorescence, etc.) that can be detected under those conditions or detected minimally under those conditions.The first three nucleotide types can be determined to be incorporated into nucleic acid based on the existence of its corresponding signal, and the fourth nucleotide type can be determined to be incorporated into nucleic acid based on the absence of any signal or the minimum detection of any signal.As a third example, a nucleotide type can include a mark detected in two different channels, and other nucleotide types are detected in no more than one channel. The three exemplary configurations described above are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type detected in a first channel (e.g., dATP with a label detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP with a label detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first channel and the second channel (e.g., dTTP with at least one label detected in both channels when excited by the first excitation wavelength and / or the second excitation wavelength), and a fourth nucleotide type that lacks a label detected or minimally detected in either channel (e.g., dGTP without a label).

[0150] In addition, as described in the materials of incorporated U.S. Patent Application Publication No. 2013 / 0079232, a single channel can be used to obtain sequencing data. In such so-called single dye sequencing methods, a first nucleotide type is marked, but the mark is removed after the first image is generated, and a second nucleotide type is marked only after the first image is generated. A third nucleotide type retains its mark in both the first image and the second image, and a fourth nucleotide type remains unmarked in both images.

[0151] Some embodiments can utilize sequencing technology while connecting. Such technology utilizes DNA ligase to incorporate oligonucleotides and recognize the incorporation of such oligonucleotides. Oligonucleotides generally have different labels related to the identity of specific nucleotides in the sequence of oligonucleotide hybridization. As with other SBS methods, images can be obtained after the array of nucleic acid feature parts is treated with labeled sequencing reagents. Each image will show the nucleic acid feature parts of the labeling that has been incorporated into a specific type. Due to the different sequence content of each feature part, different feature parts will be present or absent in different images, but the relative position of the feature parts will remain unchanged in the image. The images obtained by the sequencing method based on connection can be stored, processed and analyzed as described herein. Exemplary SBS systems and methods that can be used with the methods and systems described herein are described in U.S. Patent No. 6,969,488, U.S. Patent No. 6,172,218 and U.S. Patent No. 6,306,597, and the disclosures of these patents are incorporated herein by reference in their entirety.

[0152] Some embodiments may utilize nanopore sequencing (Deamer, DW and 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 JA 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 entirety). In such embodiments, the target nucleic acid passes through the nanopore. The nanopore may 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 conductivity of the pore. (U.S. Pat. No. 7,001,792; Soni, GV and 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. and 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 entirety). The data obtained from nanopore sequencing can be stored, processed and analyzed as described herein. In particular, according to the exemplary processing of optical images and other images described herein, the data can be processed like an image.

[0153] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide, as described, for example, in U.S. Pat. No. 7,329,492 and U.S. Pat. No. 7,211,414 (each of which is incorporated herein by reference), or may be detected with a zero-mode waveguide, as described, for example, in U.S. Pat. No. 7,315,019 (which is incorporated herein by reference), and may be detected with fluorescent nucleotide analogs and engineered polymerases, as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Patent Application Publication No. 2008 / 0108082 (each of which is incorporated herein by reference). Illumination can be limited to a volume of magnitude 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., "Parallelconfocal 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 entirety). Images obtained by such methods can be stored, processed, and analyzed as described herein.

[0154] Some SBS embodiments include detecting the proton released when nucleotides are incorporated into extension products. For example, sequencing based on the detection of releasing protons can use the commercially available electrical detectors and related technologies available from Ion Torrent company (Guilford, CT, it is a Life Technologies subsidiary) or the sequencing methods and systems described in US 2009 / 0026082A1, US2009 / 0127589 A1, US2010 / 0137143 A1 or US 2010 / 0282617A1, each of which is incorporated herein by reference. The method for amplifying target nucleic acids using kinetic exclusion set forth herein can be easily applied to substrates for detecting protons. More specifically, the method set forth herein can be used to produce amplicon clone colonies for detecting protons.

[0155] The above-mentioned SBS method can be advantageously carried out in a variety of formats so that a plurality of different target nucleic acids are manipulated simultaneously. In a specific embodiment, different target nucleic acids can be processed in a common reaction vessel or on the surface of a specific substrate. This allows to conveniently deliver sequencing reagents, remove unreacted reagents and detect incorporation events in a variety of ways. In the embodiment using the target nucleic acid of surface binding, the target nucleic acid can be an array format. In the array format, the target nucleic acid can be attached to the surface in a spatially distinguishable manner generally. The target nucleic acid can be attached by direct covalent attachment, attached to beads or other particles or attached to a polymerase or other molecules attached to the surface to combine. The array can include a single copy of the target nucleic acid at each site (also referred to as a feature portion), or multiple copies with the same sequence can be present at each site or feature portion. Multiple copies can be produced by an amplification method (such as bridge amplification or emulsion PCR as described in further detail below).

[0156] The methods described herein may use arrays having features at any of a variety of densities, including, 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 or higher.

[0157] The advantage of the method described herein is that they provide rapid and effective detection of multiple target nucleic acids in parallel. Therefore, the present disclosure provides an integrated system that can use technology known in the art (such as those illustrated above) to prepare and detect nucleic acids. Therefore, the integrated system of the present disclosure may include a fluid assembly that can deliver amplification reagents and / or sequencing reagents to one or more fixed DNA fragments, and the system includes components such as pumps, valves, reservoirs, fluid pipelines, etc. The circulation cell can be configured for and / or for detecting target nucleic acids in the integrated system. Exemplary circulation cells are described in, for example, US 2010 / 0111768A1 and U.S. Serial No. 13 / 273,666, each of which is incorporated herein by reference. As illustrated for the circulation cell, one or more fluid assemblies of the integrated system can be used for amplification method and detection method. Taking nucleic acid sequencing embodiment as an example, one or more fluid assemblies of the integrated system can be used for the amplification method described herein and for delivering sequencing reagents in sequencing method (such as those illustrated above). Alternatively, the integrated system may include a separate fluid system to perform amplification method and perform detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and also determine nucleic acid sequences include, but are not limited to, MiSeq TM The ELISA platform (Illumina, Inc., San Diego, CA) and the apparatus described in U.S. Ser. No. 13 / 273,666, which is incorporated herein by reference.

[0158] The above-mentioned sequencing system sequences the nucleic acid polymers present in the sample received by the sequencing device. As defined herein, "sample" and its derivatives are used in their broadest sense, including any specimens, cultures, etc. suspected of containing a target. In some embodiments, the sample includes DNA, RNA, PNA, LNA, chimeric or hybridized nucleic acids. The sample may include any biological, clinical, surgical, agricultural, atmospheric or aquatic animal and plant specimens 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 specimens. It is also envisioned that the sample may come from: a single individual, a collection of nucleic acid samples from genetically related members, a nucleic acid sample from a genetically unrelated member, a nucleic acid sample (matched therewith) from a single individual (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 DNA and fetal DNA obtained from a maternal subject), or there is contaminating bacterial DNA in a sample containing plant or animal DNA. In some embodiments, the source of nucleic acid material may include nucleic acids obtained from newborns, such as nucleic acids commonly used for newborn screening.

[0159] Nucleic acid samples may include high molecular weight substances, such as genomic DNA (gDNA). Samples may include low molecular weight substances, such as nucleic acid molecules obtained from FFPE samples or archived DNA samples. In another embodiment, low molecular weight substances include DNA of enzymatic fragmentation or mechanical fragmentation. Samples may include cell-free circulating DNA. In some embodiments, samples may include nucleic acid molecules obtained from biopsy tissue, tumor, scrapings, swabs, blood, mucus, urine, blood plasma, semen, hair, laser capture microdissection, surgical resection and other clinical or laboratory samples. In some embodiments, samples may be epidemiological samples, agricultural samples, forensic samples or pathogenic samples. In some embodiments, samples may include nucleic acid molecules obtained from animals (such as humans or mammalian sources). In another embodiment, samples may include nucleic acid molecules obtained from non-mammalian sources (such as plants, bacteria, viruses or fungi). In some embodiments, the source of nucleic acid molecules may be archived or extinct samples or species.

[0160] In addition, the methods and compositions disclosed herein can be used to amplify nucleic acid samples with low-quality nucleic acid molecules, such as degraded and / or fragmented genomic DNA from forensic samples. In one embodiment, forensic samples may include nucleic acids obtained from crime scenes, nucleic acids obtained from missing persons DNA databases, nucleic acids obtained from laboratories associated with forensic investigations, or include forensic samples obtained by law enforcement agencies, one or more military services, or any such personnel. Nucleic acid samples can be purified samples or lysates containing crude DNA, such as from oral swabs, paper, fabrics, or other substrates that can be impregnated with saliva, blood, or other body fluids. Therefore, in some embodiments, the nucleic acid sample may include a small amount of DNA (such as genomic DNA), or a fragmented portion of DNA. In some embodiments, the target sequence may be present in one or more body fluids, wherein body fluids include but are not limited to blood, sputum, plasma, semen, urine, and serum. In some embodiments, the target sequence may be obtained from the victim's hair, skin, tissue sample, autopsy, or remains. In some embodiments, nucleic acids comprising one or more target sequences may be obtained from dead animals or humans. In some embodiments, the target sequence may include a nucleic acid obtained from non-human DNA (such as microbial, plant, or insect DNA). In some embodiments, the target sequence or amplified target sequence is directed to the purpose of human identification. In some embodiments, the disclosure as a whole relates to methods for identifying the characteristics of forensic samples. In some embodiments, the disclosure as a whole 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 standards 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 target-specific primers disclosed herein or using the primer standards outlined herein.

[0161] The components of the crosstalk-aware base detection system 106 may include software, hardware, or both. For example, the components of the crosstalk-aware base detection system 106 may 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., the user client device 108). The computer-executable instructions of the crosstalk-aware base detection system 106, when executed by one or more processors, may cause the computing device to perform the fault source identification method described herein. Alternatively, the components of the crosstalk-aware base detection system 106 may include hardware (such as a dedicated processing device) for performing certain functions or groups of functions. In addition or alternatively, the components of the crosstalk-aware base detection system 106 may include a combination of computer-executable instructions and hardware.

[0162] In addition, the 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 can be implemented, for example, as part of a stand-alone application, as a module of an application, as a plug-in of an application, as a library function or function that can be called by other applications, and / or as a cloud computing model. Therefore, the components of the crosstalk-aware base calling system 106 can be implemented as part of a stand-alone application on a personal computing device or a mobile device. In addition or alternatively, the components of the crosstalk-aware base calling system 106 can 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.

[0163] As discussed in more detail below, embodiments of the present disclosure may include or utilize a special or general-purpose computer including computer hardware (such as, for example, one or more processors and system memory). 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. Specifically, one or more of the processes described herein may be implemented at least in part as instructions embodied in a non-transient computer-readable medium and capable of being executed by one or more computing devices (e.g., any of the media content access devices described herein). Generally speaking, a processor (e.g., a microprocessor) receives instructions from a non-transient computer-readable medium (e.g., a memory, etc.) and executes those instructions, thereby executing one or more processes, including one or more of the processes described herein.

[0164] Computer readable media can be any available media that can be accessed by a general or special purpose computer system. A computer readable medium that stores computer executable instructions is a non-transitory computer readable storage medium (device). A computer readable medium that carries computer executable instructions is a transmission medium. Therefore, by way of example and not limitation, embodiments of the present disclosure may include at least two distinct types of computer readable media: a non-transitory computer readable storage medium (device) and a transmission medium.

[0165] Non-transitory computer-readable storage media (devices) include RAM, ROM, EEPROM, CD-ROM, solid-state drives (SSD) (e.g., RAM-based), flash memory, phase-change memory (PCM), other types of memory, other optical disk storage devices, magnetic disk storage devices or other magnetic storage devices, or any other media that can be used to store desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general or special purpose computer.

[0166] "Network" is defined as one or more data links that enable the transmission of electronic data between computer systems and / or modules and / or other electronic devices. When information is transferred or provided to a computer via a network or another communication connection (hardwired, wireless, or a combination of hardwired or wireless), the computer appropriately regards the connection as a transmission medium. The transmission medium may include a network and / or data link, which can be used to carry the desired program code means in the form of computer executable instructions or data structures, and which can be accessed by general or special computers. The above combinations should also be included in the scope of computer-readable media.

[0167] 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 a transmission medium to a non-transitory computer readable storage medium (device) (or vice versa). For example, a computer executable instruction or data structure received over a network or data link may be buffered in RAM within a network interface module (e.g., NIC), and then ultimately transferred to the computer system RAM and / or to a less volatile computer storage medium (device) at the computer system. Thus, it should be understood that a non-transitory computer readable storage medium (device) may be included in a computer system component that also (or even primarily) utilizes a transmission medium.

[0168] Computer executable instructions include, for example, instructions and data that make a general-purpose computer, a special-purpose computer, or a special-purpose processing device perform certain functions or groups of functions when executed at a processor. In some embodiments, computer executable instructions are executed on a general-purpose computer to turn a general-purpose computer into a special-purpose computer that implements an element of the present disclosure. Computer executable instructions can be, for example, binary numbers, intermediate format instructions such as assembly language, or even source code. Although the subject matter has been described in a language specific to structural features and / or method actions, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the described features or actions. On the contrary, the described features and actions are disclosed as exemplary forms of implementing the claims.

[0169] Those skilled in the art will appreciate that the present disclosure can be practiced in a network computing environment with 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, mobile phones, PDAs, tablet computers, pagers, routers, switches, and the like. The present disclosure can also be practiced in a distributed system environment, where both local and remote computer systems linked by a network (by a hardwired data link, a wireless data link, or a combination of a hardwired and wireless data link) perform tasks. In a distributed system environment, program modules can be located in both local and remote memory storage devices.

[0170] Embodiments of the present disclosure may also be implemented in a cloud computing environment. In this specification, "cloud computing" is defined as a model for implementing on-demand network access to a shared pool of configurable computing resources. For example, cloud computing may be employed in the marketplace to provide ubiquitous and convenient on-demand access to a shared pool of configurable computing resources. A shared pool of configurable computing resources may be quickly provisioned via virtualization and released with low management effort or service provider interaction, and then scaled accordingly.

[0171] The cloud computing model may consist of various characteristics, such as, for example, on-demand self-service, broad network access, resource pooling, rapid elasticity, metered services, etc. The cloud computing model may also exhibit various service models, such as, for example, software as a service (SaaS), platform as a service (PaaS), and infrastructure as a service (IaaS). The cloud computing model may also be deployed using different deployment models, such as private cloud, community cloud, public cloud, hybrid cloud, etc. In this specification and in the claims, a "cloud computing environment" is an environment in which cloud computing is employed.

[0172] Fig.10 1 shows a block diagram of a computing device 1000 that can be configured to perform one or more of the above processes. It should be understood that one or more computing devices (such as computing device 1000) can implement the crosstalk-aware base calling system 106 and the sequencing system 104. Fig.10 As shown, 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 that can be communicatively coupled via a communication infrastructure 1012. In some embodiments, computing device 1000 may include Fig.10 Those shown may have fewer or more components. The following paragraphs describe in more detail Fig.10 Components of computing device 1000 are shown.

[0173] In one or more embodiments, the processor 1002 includes hardware for executing instructions, such as those constituting a computer program. As an example and not by way of limitation, in order to execute instructions for dynamically modifying a workflow, the processor 1002 may retrieve (or obtain) these instructions from an internal register, an internal cache, a memory 1004, or a storage device 1006, and then decode and execute them. The memory 1004 may be a volatile or non-volatile memory for storing data, metadata, and programs executed by the processor. The storage device 1006 includes a storage device, such as a hard disk, a flash drive, or other digital storage device, for storing data or instructions for performing the methods described herein.

[0174] I / O interface 1008 allows a user to provide input to computing device 1000, receive output from the computing device, and otherwise transfer data to the computing device and receive data from the computing device. I / O interface 1008 may include a mouse, a keypad or keyboard, a touch screen, a camera, an optical scanner, a network interface, a modem, other known I / O devices, or a combination of such I / O interfaces. 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 certain embodiments, I / O interface 1008 is configured to provide graphics data to the display to be presented to the user. Graphics data may represent one or more graphical user interfaces and / or may serve any other graphical content of a specific implementation.

[0175] The communication interface 1010 may include hardware, software, or both. In any case, the communication interface 1010 may provide one or more interfaces for communication (such as packet-based communication) between the computing device 1000 and one or more other computing devices or networks. As an example and not by way of limitation, the communication 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).

[0176] In addition, 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 the components of the computing device 1000 to each other. For example, the communication interface 1010 can use one or more networks and / or protocols to enable multiple computing devices connected through a specific infrastructure to communicate with each other to perform one or more aspects of the process described herein. For illustration, the sequencing process can allow multiple devices (e.g., client devices, sequencing devices, and server devices) to exchange information such as sequencing data and error notifications.

[0177] In the foregoing description, the present disclosure has been described with reference to specific exemplary embodiments thereof. Various embodiments and aspects of the present disclosure are 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 present disclosure. Many specific details are described to provide a thorough understanding of the various embodiments of the present disclosure.

[0178] The present disclosure can be embodied in other specific forms without departing from its spirit or essential characteristics. The embodiments described should be considered as being only exemplary and not restrictive in all respects. For example, the methods described herein can be performed with fewer or more steps / actions, or the steps / actions can be performed in different orders. Additionally, the steps / actions described herein can be repeated or performed in parallel with each other or in parallel with different examples of the same or similar steps / actions. Therefore, the scope of the application is indicated by the appended claims rather than the foregoing description. All changes within the equivalent meaning and scope of the claims will be included within their scope.

Claims

1. A system comprising: at least one processor; as well as a non-transitory computer readable medium comprising instructions that, when executed by the at least one processor, cause the system to: detecting, for a sequencing cycle, a first set of intensity values ​​of a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​of a second signal from a second cluster of oligonucleotides; determining a set of illumination indication marks based on the first set of intensity values, the set of illumination indication marks indicating whether the first cluster of oligonucleotides is illuminated during the sequencing cycle; determining an inter-cluster interference metric based on the set of illumination indicators, the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides on the second cluster of oligonucleotides; as well as A second set of modified intensity values ​​for the second signal from the second cluster of oligonucleotides is generated for the sequencing cycle by removing the inter-cluster interference measure from the second set of intensity values.

2. The system of claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to: determining the set of illumination indicators further based on magnitudes of the first set of intensity values ​​and an estimated point spread function of a section of the nucleotide sample slide including the first cluster of oligonucleotides; and The inter-cluster interference metric is determined further based on the estimated point spread function.

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

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

5. The system of claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to: determining, for the sequencing cycle, nucleobase calls for the first cluster of oligonucleotides based on the first set of intensity values ​​for nucleobases and intensity value boundaries; and The set of illumination indicator signatures is further determined based on the nucleobase calls of the first cluster of oligonucleotides.

6. The system of claim 5, further comprising instructions that, when executed by the at least one processor, cause the system to: determining the nucleobase calls of 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; and The modified second set of intensity values ​​is generated by subtracting the value of the inter-cluster interference metric from intensity values ​​from the second set of intensity values ​​corresponding to the first channel or from intensity values ​​from the second set of intensity values ​​corresponding to the second channel.

7. The system of claim 1, further comprising instructions, which when executed by the at least one processor, cause the system to determine the lighting indication identification set in the following manner: determining a first illumination indicator that indicates whether the first cluster of oligonucleotides is illuminated in a first channel during the sequencing cycle; as well as determining a second illumination indicator that indicates whether the second cluster of oligonucleotides is illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicative of an extent to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; as well as A second continuous illumination indicator is determined that indicates the extent to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

8. The system according to claim 1 further includes instructions that, when executed by the at least one processor, cause the system to determine an adjusted set of lighting indicator markers for the sequencing cycle and based on the modified second set of intensity values, the adjusted set of lighting indicator markers indicating whether the second cluster of oligonucleotides is illuminated during the sequencing cycle and being different from an initial set of lighting indicator markers corresponding to the second set of intensity values.

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

10. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: detecting a third set of intensity values ​​of a third signal from a third cluster of oligonucleotides for the sequencing cycle; determining an additional set of illumination indication marks based on the third set of intensity values, the additional set of illumination indication marks indicating whether the third cluster of oligonucleotides is illuminated during the sequencing cycle; determining an additional inter-cluster interference metric based on the additional set of illumination indicators, the additional inter-cluster interference metric estimating optical interference of the third cluster of oligonucleotides with the second cluster of oligonucleotides; and The modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is generated by removing the inter-cluster interference measure and the additional inter-cluster interference measure from the second set of intensity values ​​for the sequencing cycle.

11. The system of claim 1 , further comprising instructions that, when executed by the at least one processor, cause the system to: determining that the first set of intensity values ​​of the first signal from the first cluster of oligonucleotides is within a range of intensity values; determining that the second set of intensity values ​​of the second signal from the second cluster of oligonucleotides is not within the range of intensity values; and The modified second set of intensity values ​​is generated by removing the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides with the second cluster of oligonucleotides from the second set of intensity values ​​based on the first set of intensity values ​​being within the range of intensity values ​​and the second set of intensity values ​​not being within the range of intensity values.

12. The system of claim 11, further comprising instructions that, when executed by the at least one processor, cause the system to: determining, based on the first set of intensity values, a first nucleobase call of the first cluster of oligonucleotides that is part of a first subset of oligonucleotide clusters having intensity values ​​within the range of intensity values; and A second nucleobase call of the second cluster of oligonucleotides that is part of a second subset of oligonucleotide clusters having intensity values ​​that are not within the range of intensity values ​​is determined based on the second set of modified intensity values.

13. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: detecting the first set of intensity values ​​by detecting first intensity values ​​of the first signal from a first cluster of oligonucleotides in a single channel; detecting the second set of intensity values ​​by detecting second intensity values ​​of the second signal from a second cluster of oligonucleotides in the single channel; and The set of illumination indicia is determined by determining a single illumination indicia that indicates whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

14. A non-transitory computer readable medium having stored thereon instructions which, when executed by at least one processor, cause a computing device to: detecting, for a sequencing cycle, a first set of intensity values ​​of a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​of a second signal from a second cluster of oligonucleotides; determining a set of illumination indication marks based on the first set of intensity values, the set of illumination indication marks indicating whether the first cluster of oligonucleotides is illuminated during the sequencing cycle; determining an inter-cluster interference metric based on the set of illumination indicators, the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides on the second cluster of oligonucleotides; as well as A second set of modified intensity values ​​for the second signal from the second cluster of oligonucleotides is generated for the sequencing cycle by removing the inter-cluster interference measure from the second set of intensity values.

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: determining the set of illumination indicators further based on magnitudes of the first set of intensity values ​​and an estimated point spread function of a section of the nucleotide sample slide including the first cluster of oligonucleotides; and The inter-cluster interference metric is determined further based on the estimated point spread function.

16. The non-transitory computer readable medium of claim 15, 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 containing the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

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

18. 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: determining, for the sequencing cycle, nucleobase calls for the first cluster of oligonucleotides based on the first set of intensity values ​​for nucleobases and intensity value boundaries; and The set of illumination indicator signatures is further determined based on the nucleobase calls of the first cluster of oligonucleotides.

19. The non-transitory computer readable medium of claim 18, further comprising instructions that, when executed by the at least one processor, cause the computing device to: determining the nucleobase calls of 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; and The modified second set of intensity values ​​is generated by subtracting the value of the inter-cluster interference metric from intensity values ​​from the second set of intensity values ​​corresponding to the first channel or from intensity values ​​from the second set of intensity values ​​corresponding to the second channel.

20. 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 the lighting indicator set by: determining a first illumination indicator that indicates whether the first cluster of oligonucleotides is illuminated in a first channel during the sequencing cycle; as well as determining a second illumination indicator that indicates whether the second cluster of oligonucleotides is illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicative of an extent to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; as well as A second continuous illumination indicator is determined that indicates the extent to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

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

22. The non-transitory computer-readable medium of claim 14, further comprising instructions which, 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, that the nucleobase calls of the second cluster of oligonucleotides are different from the nucleobases corresponding to the second set of intensity values.

23. 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: detecting a third set of intensity values ​​of a third signal from a third cluster of oligonucleotides for the sequencing cycle; determining an additional set of illumination indication marks based on the third set of intensity values, the additional set of illumination indication marks indicating whether the third cluster of oligonucleotides is illuminated during the sequencing cycle; determining an additional inter-cluster interference metric based on the additional set of illumination indicators, the additional inter-cluster interference metric estimating optical interference of the third cluster of oligonucleotides with the second cluster of oligonucleotides; and The modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is generated by removing the inter-cluster interference measure and the additional inter-cluster interference measure from the second set of intensity values ​​for the sequencing cycle.

24. 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: determining that the first set of intensity values ​​of the first signal from the first cluster of oligonucleotides is within a range of intensity values; determining that the second set of intensity values ​​of the second signal from the second cluster of oligonucleotides is not within the range of intensity values; and The modified second set of intensity values ​​is generated by removing the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides with the second cluster of oligonucleotides from the second set of intensity values ​​based on the first set of intensity values ​​being within the range of intensity values ​​and the second set of intensity values ​​not being within the range of intensity values.

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

26. 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: detecting the first set of intensity values ​​by detecting first intensity values ​​of the first signal from a first cluster of oligonucleotides in a single channel; detecting the second set of intensity values ​​by detecting second intensity values ​​of the second signal from a second cluster of oligonucleotides in the single channel; and The set of illumination indicia is determined by determining a single illumination indicia that indicates whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

27. A method comprising: detecting, for a sequencing cycle, a first set of intensity values ​​of a first signal from a first cluster of oligonucleotides and a second set of intensity values ​​of a second signal from a second cluster of oligonucleotides; determining a set of illumination indication marks based on the first set of intensity values, the set of illumination indication marks indicating whether the first cluster of oligonucleotides is illuminated during the sequencing cycle; determining an inter-cluster interference metric based on the set of illumination indicators, the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides on the second cluster of oligonucleotides; as well as A second set of modified intensity values ​​for the second signal from the second cluster of oligonucleotides is generated for the sequencing cycle by removing the inter-cluster interference measure from the second set of intensity values.

28. The method according to claim 27, further comprising: determining the set of illumination indicators further based on magnitudes of the first set of intensity values ​​and an estimated point spread function of a section of the nucleotide sample slide including the first cluster of oligonucleotides; as well as The inter-cluster interference metric is determined further based on the estimated point spread function.

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 containing the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

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

31. The method of claim 27, further comprising: determining, for the sequencing cycle, nucleobase calls for the first cluster of oligonucleotides based on the first set of intensity values ​​for nucleobases and an intensity value boundary; as well as The set of illumination indicator signatures is further determined based on the nucleobase calls of the first oligonucleotide cluster.

32. The method of claim 31 further comprising: determining the nucleobase calls of 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; as well as The modified second set of intensity values ​​is generated by subtracting the value of the inter-cluster interference metric from intensity values ​​from the second set of intensity values ​​corresponding to the first channel or from intensity values ​​from the second set of intensity values ​​corresponding to the second channel.

33. The method of claim 27, wherein determining the lighting indicator identification set comprises: determining a first illumination indicator that indicates whether the first cluster of oligonucleotides is illuminated in a first channel during the sequencing cycle; as well as determining a second illumination indicator that indicates whether the second cluster of oligonucleotides is illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicative of an extent to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; as well as A second continuous illumination indicator is determined that indicates the extent to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

34. The method according to claim 27 also includes determining an adjusted set of lighting indicator marks for the sequencing cycle and based on the modified second set of intensity values, wherein the adjusted set of lighting indicator marks indicates whether the second cluster of oligonucleotides is illuminated during the sequencing cycle and is different from an initial set of lighting indicator marks corresponding to the second set of intensity values.

35. The method of claim 27, further comprising determining, for the sequencing cycle and based on the modified second set of intensity values, that the nucleobase calls of the second cluster of oligonucleotides are different from the nucleobases corresponding to the second set of intensity values.

36. The method of claim 27, further comprising: detecting a third set of intensity values ​​of a third signal from a third cluster of oligonucleotides for the sequencing cycle; determining an additional set of illumination indication marks based on the third set of intensity values, the additional set of illumination indication marks indicating whether the third cluster of oligonucleotides is illuminated during the sequencing cycle; determining an additional inter-cluster interference metric based on the additional set of illumination indicators, the additional inter-cluster interference metric estimating optical interference of the third cluster of oligonucleotides on the second cluster of oligonucleotides; as well as The modified second set of intensity values ​​for the second signal from the second cluster of oligonucleotides is generated by removing the inter-cluster interference measure and the additional inter-cluster interference measure from the second set of intensity values ​​for the sequencing cycle.

37. The method of claim 27, further comprising: determining that the first set of intensity values ​​of the first signal from the first cluster of oligonucleotides is within a range of intensity values; determining that the second set of intensity values ​​of the second signal from the second cluster of oligonucleotides is not within the range of intensity values; as well as The modified second set of intensity values ​​is generated by removing the inter-cluster interference metric estimating optical interference of the first cluster of oligonucleotides with the second cluster of oligonucleotides from the second set of intensity values ​​based on the first set of intensity values ​​being within the range of intensity values ​​and the second set of intensity values ​​not being within the range of intensity values.

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

39. The method of claim 27, wherein: Detecting the first set of intensity values ​​includes detecting, in a single channel, first intensity values ​​of the first signal from a first cluster of oligonucleotides; Detecting the second set of intensity values ​​comprises detecting, in the single channel, second intensity values ​​of the second signal from a second cluster of oligonucleotides; and Determining the set of illumination indicators includes determining a single illumination indicator that indicates whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

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