Targeted variant reference panel for target variant attribution

Abstract

The present disclosure relates to a system for generating a target variant reference panel including target variant positions with target variant indices, or for using a target variant reference panel to assign genotype calls for corresponding target variants. In embodiments, the system generates an initial reference panel including various phased genomic samples of different haplotypes. The disclosed system further adds target variant positions to the initial reference panel to indicate the presence or absence of target variants, creating a target variant reference panel including target variant positions with target variant indices. Furthermore, the system can utilize the target variant reference panel to assign genotype calls indicating the presence or absence of target variants in a target genomic sample based on a comparison of the haplotypes represented in the target variant reference panel with nucleotide reads corresponding to the target genomic sample.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 377,682, entitled "A TARGET-VARIANT-REFERENCE PANEL FOR IMPUTING TARGET VARIANTS," filed September 29, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] In recent years, biotechnology companies and research institutions have improved the hardware and software for sequencing nucleotides and determining nucleic acid base calls for genomic samples. For example, some existing sequencing machines and sequencing data analysis software (collectively referred to as "existing sequencing systems") predict individual nucleotides within a sequence by using traditional Sanger sequencing or sequencing-by-synthesis (SBS) methods. When using SBS, existing sequencing systems can monitor thousands of oligonucleotides being synthesized in parallel from templates and predict nucleic acid base calls for growing nucleotide reads based on images of fluorescently tagged nucleic acid bases incorporated into the oligonucleotides. After capturing such images, some existing sequencing systems determine nucleic acid base calls for nucleotide reads corresponding to the oligonucleotides and transmit the base call data to a computing device with sequencing data analysis software. Using sequencing data analysis software, existing sequencing systems align the nucleotide reads with a reference genome. Based on the differences between the aligned nucleotide reads and the reference genome, existing systems can further utilize variant callers to identify variants in the genomic sample, such as single nucleotide polymorphisms (SNPs), repeat expansion variants, or insertions or deletions (indels).

[0003] Despite these advances, existing sequencing systems often make inaccurate variant calls for difficult-to-call genomic regions, such as regions with variable-number tandem repeat (VNTR) expansions, short tandem repeat (STR) expansions, structural variants, or other types of variants. For specific difficult-to-call genomic regions of a genomic sample, existing sequencing systems frequently use reference panels and genotype imputation models to assign nucleobase calls and phase haplotypes based on the variants detected in the genomic sample. For example, existing sequencing systems frequently use various types of hidden Markov models (HMMs) customized for genotype imputation to impute nucleobase calls for specific genomic regions, such as by using the Genotype Likelihood Imputation and Phasing Method (GLIMPSE) or IMPUTE. Based on the variants shared between the haplotypes of the reference panel and the nucleotide reads of the genomic sample, the genotype imputation model can impute variants for difficult-to-call genomic regions of the genomic sample with varying degrees of accuracy.

[0004] Variant calls for difficult-to-call genomic regions can range from insignificant to significant, depending on the gene or other genomic region. Because existing sequencing systems often use reference panels that do not adequately capture or mark repeat expansion variants (e.g., VNTR or STR) or specific pathogenic variant polymorphisms, incorrect variant calls can have significant consequences. For example, a variant call identifying a specific repeat expansion variant in the replication factor C subunit 1 (RFC1) gene may accurately or inaccurately identify the genetic indicator of a phenotype in the cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) spectrum. For example, a biallelic intronic AAGGG repeat expansion in the RFC1 gene makes such variant calling particularly challenging. As a further example, a variant call that accurately or inaccurately identifies a variant in the cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene may result in either accurately identifying the genetic indicator of neuroleptic malignant syndrome or completely missing the genetic indicator. Therefore, variant calling for such pathogenic variants in genes can be important, but appropriate reference panels with sufficient polymorphisms to support accurate variant calling are often lacking.

[0005] Despite the importance of accurately determining variant calls for repeat expansions and pathogenic variants, existing sequencing systems often fail to generate variant calls or generate inaccurate variant calls due to poor quality nucleotide read data, poor alignment of nucleotide reads, or inadequate reference panels. In fact, many existing sequencing systems either fail to generate genotype calls or generate inaccurate genotype calls because (i) the nucleotide reads corresponding to the target genomic region for the target variant provide insufficient coverage, (ii) the alignment model cannot accurately map the nucleotide reads for such genomic region on the reference genome, or (iii) the existing reference panel contains insufficient data to support accurate assignment.

[0006] To illustrate the technical problems of (i) and (ii), some existing sequencing systems align nucleotide reads corresponding to repeat expansions with target genomic regions only to leave read coverage holes in the center of the target genomic regions. Because the target genomic regions of repeat expansions or pathogenic variants may exhibit such read coverage holes, existing sequencing systems either do not generate genotype calls or generate inaccurate genotype calls. In fact, without direct evidence from a reference panel with nucleotide reads for the genomic regions corresponding to the repeat expansions or appropriate data for such repeat expansions, existing sequencing systems cannot accurately genotype repeat expansions, such as those in RFC1 and CYP21A2, or other important pathogenic variants.

[0007] These are among the additional problems and challenges with existing sequencing systems. Summary of the Invention

[0008] The present disclosure describes one or more embodiments of a system, method, and non-transitory computer-readable storage medium that solve one or more of the above problems or provide other advantages over the art. For example, the disclosed system can generate a target variant reference panel including target variant positions with target variant indices, or use the target variant reference panel to impute genotype calls for corresponding target variants. More specifically, in one or more embodiments, the disclosed system generates an initial reference panel including various phased genomic samples of different haplotypes. The disclosed system further adds target variant positions to the initial reference panel to indicate the presence or absence of target variants, creating a target variant reference panel including target variant positions with target variant indices. Additionally or alternatively, the disclosed system can utilize the target variant reference panel to impute genotype calls indicating the presence or absence of target variants in a target genomic sample based on a comparison of (i) haplotypes represented in the target variant reference panel and (ii) nucleotide reads corresponding to the target genomic sample.

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

[0010] The detailed description provides additional specificity and detail in one or more embodiments through the use of the accompanying drawings, as briefly described below. [Figure 1] 1 shows a schematic diagram of a computing system in which a customized genotype assignment system can operate in accordance with one or more embodiments. [Figure 2A] 1 illustrates a customized genotype imputation system for generating a targeted variant reference panel according to one or more embodiments. [Figure 2B]1 illustrates a customized genotype imputation system that utilizes a targeted variant reference panel to impute genotype calls, according to one or more embodiments. [Figure 3] 1 shows nucleotide reads of a genomic sample misaligned to a genomic region containing a repeat expansion, according to one or more embodiments. [Figure 4] 1 shows a clustering pattern of genomic samples containing target variants according to one or more embodiments. [Figure 5] 1 illustrates a customized genotype assignment system that generates a target variant reference panel that includes target variant positions, according to one or more embodiments. [Figure 6] 1 shows an exemplary output file containing a target variant reference panel according to one or more embodiments. [Figure 7] 1 shows a graph depicting non-reference genotype concordance rate for a customized genotype assignment system using a target variant-specific target variant reference panel versus allele frequency according to one or more embodiments. [Figure 8] 1 illustrates a customized genotype assignment system that utilizes a targeted variant reference panel to assign genotype calls to targeted variants in a genomic sample, according to one or more embodiments. [Figure 9] 1 illustrates a graphical user interface for providing information about the imputed genotype calls for target variants, according to one or more embodiments. [Figure 10] 1 shows a flowchart of a series of actions for generating a target variant reference panel according to one or more embodiments. [Figure 11] 1 shows a flowchart of a series of operations for utilizing a targeted variant reference panel to impute genotype calls according to one or more embodiments. [Figure 12] FIG. 1 illustrates a block diagram of an exemplary computing device for implementing one or more embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure describes one or more embodiments of a customized genotype assignment system that generates a target variant reference panel including target variant positions for target variant indicators or utilizes a target variant reference panel to assign genotype calls for corresponding target variants. Illustratively, in one or more embodiments, the customized genotype assignment system creates an initial reference panel including a genomic sample of genetically diverse haplotypes. The customized genotype assignment system further adds the target variant positions to the initial reference panel and phases the alleles of the genomic sample to determine the presence or absence of the target variants in the corresponding alleles present on the maternal and paternal haplotypes. By adding such target variant positions, the customized genotype assignment system generates a target variant reference panel including target variant indicators within the target variant positions for the phased alleles of the genomic sample. After generating or accessing such a target variant reference panel, in one or more embodiments, the customized genotype assignment system utilizes the target variant reference panel to determine a genotype call indicating the presence or absence of the target variant in the target genomic sample.

[0012] As described above, in one or more embodiments, the customized genotype assignment system generates a targeted variant reference panel. To generate the targeted variant reference panel, in one or more embodiments, the customized genotype assignment system generates an initial reference panel that includes genomic samples with genetically diverse haplotypes. Illustratively, in one or more embodiments, the customized genotype assignment system generates an initial reference panel that includes genomic samples from various populations, ancestries, continents, and / or countries. In some embodiments, the haplotypes in the initial reference panel include one or more marker variants (e.g., single nucleotide polymers (SNPs) or small insertions and / or deletions).

[0013] Based on the initial reference panel, in some implementations, the customized genotype assignment system generates a target variant reference panel by adding target variant positions to the initial reference panel. For example, in some embodiments, the customized genotype assignment system adds data fields as placeholders for indicators of target variants present in the alleles of various haplotypes represented in the initial reference panel. In one or more embodiments, the customized genotype assignment system inserts target variant indicators into such data fields (or into other target variant positions) to indicate whether a given genomic sample contains the target variant. In contrast to conventional reference panels that do not include such target variant positions, the customized genotype assignment system can utilize the target variant positions of the targeted variant reference panel to more accurately identify the target variant.

[0014] In addition to adding target variant locations, in some cases, the customized genotype assignment system phases alleles of a genomic sample represented by a target variant reference panel based on SNPs or other marker variants represented by alleles of various haplotypes. Illustratively, in some embodiments, the customized genotype assignment system utilizes a haplotype phasing model to phase alleles of a genomic sample based on known haplotypes and other inheritance patterns. More specifically, in one or more embodiments, the customized genotype assignment system (i) identifies one or more genomic coordinates corresponding to the target variant and (ii) phases the alleles from the haplotypes corresponding to those genomic coordinates based on the marker variants represented by the alleles. By phasing the alleles of a genomic sample with indices of the target variant locations, the customized genotype assignment system can include target variant indices for target variants specific to the phased alleles of various haplotypes in the target variant reference panel. As described below, the customized genotype assignment system can utilize various other phasing models to phase alleles of a genomic sample represented by a target variant reference panel.

[0015] In addition to or as an alternative to generating a target variant reference panel, in one or more embodiments, the customized genotype assignment system utilizes the target variant reference panel to assign one or more genotype calls for target variants in the target genomic sample. Illustratively, in one or more embodiments, the customized genotype assignment system receives and / or identifies nucleotide reads corresponding to the target genomic sample. The customized genotype assignment system further accesses a target variant reference panel that includes target variant indices within target variant positions for phased alleles of the genomic sample of different haplotypes. Based on comparing the alleles of the haplotypes represented by the target variant reference panel with the nucleotide reads corresponding to the target genomic sample, in some embodiments, the customized genotype assignment system assigns genotype calls for the target variants in the target genomic sample.

[0016] For example, in one or more embodiments, the sequencing device receives a nucleotide-sample slide (e.g., a flow cell) containing oligonucleotides extracted from a target genomic sample and determines nucleotide reads corresponding to the oligonucleotides in the target genomic sample. Additionally or alternatively, the customized genotype assignment system can receive data representing nucleotide reads for the target genomic sample. In some cases, the customized genotype assignment system receives nucleotide reads for the target genomic sample from a third-party sequencing system.

[0017] As described above, in one or more embodiments, the customized genotype assignment system compares the reads of the target genomic sample with the alleles of the genomic sample included in the target variant reference panel. For example, the customized genotype assignment system can identify marker variants in the target sample surrounding one or more genomic coordinates corresponding to the target variant. The customized genotype assignment system further compares the marker variants indicated by the nucleotide reads of the target genomic sample with corresponding marker variants in the alleles of haplotypes in the target variant reference panel. In some cases, the customized genotype assignment system phases the nucleotide reads of the target genomic sample to identify corresponding alleles in the maternal haplotype and paternal haplotype in the target variant reference panel.

[0018] Based on comparing the alleles of the haplotypes represented by the target variant reference panel with the nucleotide reads corresponding to the target genome sample, the customized genotype assignment system generates a prediction of whether the target genome sample carries the target variant. For example, in some cases, the customized genotype assignment system determines a phased genotype call indicating the presence or absence of the target variant at the allele corresponding to the maternal haplotype or the paternal haplotype. Thus, the customized genotype assignment system can determine whether the target genome sample is a carrier of the target variant at a particular allele, a case of the target variant at both alleles, or unaffected by the target variant at either allele. Thus, in one or more embodiments, the customized genotype assignment system can generate and provide notifications or graphics showing the phased genotype call in a graphical user interface via a computing device.

[0019] As alluded to above, customized genotype assignment systems offer several technical advantages and benefits over existing sequencing systems and methods. For example, customized genotype assignment systems improve the accuracy of genotype calls for target variants. By generating or utilizing a target variant reference panel to assign genotype calls for target variants corresponding to haplotypes of a genomic sample, customized genotype assignment systems improve the accuracy of assignment for target variants, particularly for difficult-to-call genomic regions that exhibit repeat expansions or other variant types. For example, by utilizing a target variant reference panel that includes target variant locations, customized genotype assignment systems can generate accurate and phased genotype calls for target variants in genomic regions of a reference genome that are difficult to align nucleotide reads, including genomic regions where many existing sequencing systems are unable to generate any genotype calls or are unable to generate accurate genotype calls. For example, a customized genotype assignment system can generate accurate genotype calls for repeat expansions in the RFC1 gene, the CYP2D6 gene, or various other genes referenced below, in part by generating or using a target variant reference panel that includes both marker variants and target variant positions with target variant indices for a particular genomic sample.

[0020] A customized genotype assignment system improves genotype calling by utilizing a first-of-its-kind reference panel. More specifically, the customized genotype assignment system generates or utilizes a customized target variant reference panel with target variant positions specific to one or more target variants. Existing reference panels do not include target variant positions with target variant indicators of the presence or absence of the target variant on maternal and paternal haplotypes. The disclosed target variant reference panel enables the customized genotype assignment system to compare nearby marker variants within nucleotide reads of alleles of a target genome sample and haplotypes represented by the target variant reference panel with corresponding target variant indicators, facilitating more accurate genotype calling, including more accurate phased genotype calling for repeat expansions and other pathogenic variants.

[0021] In addition to improved genotype calling for target variants, in one or more embodiments, the customized genotype assignment system improves computational efficiency and uses less memory than existing reference panels by generating a target variant reference panel that includes data for one or more target genomic regions (or genomic regions of interest) corresponding to the target variant. For example, in some embodiments, the customized genotype assignment system restricts the target variant reference panel to include data representing haplotypes of a genomic sample corresponding to one or more target genomic regions corresponding to the target variant, but does not include data representing haplotypes outside the one or more target genomic regions. This improves efficiency and conserves computing resources by reducing or eliminating excessive analysis of other genomic coordinates performed by conventional systems. Because some existing reference panels can include a haplotype matrix with 50,000,000 cells representing different marker variants and haplotypes, and existing sequencing systems can determine 40,000 genotype calls based on 40,000 haplotype matrices in the reference panel, a relatively small reduction in the size of the target variant reference panel can result in significant memory and computational savings. By reducing or eliminating unnecessary genomic regions and using a targeted variant reference panel that contains data limited to one or more target genomic regions, the customized genotype imputation system uses less memory and speeds up the computer processing time for imputing genotype calls for target variants.

[0022] As indicated by the preceding discussion, the present disclosure utilizes various terms to describe the features and advantages of the customized genotype assignment system. Further details regarding the meaning of such terms are now provided. For example, as used herein, the term "nucleotide read" or simply "read" refers to the estimated sequence of one or more nucleotide bases (or nucleobase pairs) from all or a portion of a sample nucleotide sequence. In particular, a nucleotide read includes the sequence of a determined or predicted nucleobase call for a nucleotide fragment (or group of monoclonal nucleotide fragments) from a sequencing library corresponding to a genomic sample. For example, in some cases, a sequencing device determines a nucleotide read by generating nucleobase calls for nucleobases that have passed through nanopores in a nucleotide-sample slide, determined via fluorescent tagging, or determined from wells in a flow cell.

[0023] Furthermore, as used herein, the term "nucleobase calling" (or sometimes simply "base calling") refers to the determination or prediction of a specific nucleotide base (or nucleotide pair) for a genomic coordinate of a sample genome, or for an oligonucleotide in a sequencing cycle, or for a genomic coordinate of a sample genome. In particular, nucleobase calling can refer to (i) the determination or prediction of the type of nucleobase incorporated within an oligonucleotide on a nucleotide-sample slide (e.g., read-based nucleobase calling), or (ii) the determination or prediction of the type of nucleobase present at a genomic coordinate or region within a genome, including variant or non-variant calls in a digital output file. In some cases, for a nucleotide read, nucleobase calling involves the determination or prediction of a nucleobase based on intensity values ​​obtained from fluorescently tagged nucleotides attached to oligonucleotides on a nucleotide-sample slide (e.g., in a cluster of a flow cell). Alternatively, nucleobase calling involves the determination or prediction of a nucleobase from a chromatogram peak or current change resulting from a nucleotide passing through a nanopore on a nucleotide-sample slide. In contrast, a nucleobase call may also include a final prediction of a nucleobase at a genomic coordinate of a sample genome for a variant call file (VCF) or other base call output file based on the nucleotide reads corresponding to the genomic coordinate. Thus, a nucleobase call may include a base call corresponding to a genomic coordinate and a reference genome, for example, an indication of a variant or non-variant at a specific position corresponding to the reference genome. In practice, a nucleobase call may refer to a variant call, including but not limited to a single nucleotide variant (SNV), an insertion or deletion (indel), or a base call that is part of a structural variant. As suggested above, a single nucleobase call may be an adenine (A) call, a cytosine (C) call, a guanine (G) call, or a thymine (T) call.

[0024] Furthermore, as used herein, the term "variant" refers to one or more nucleobase calls that differ or vary from a reference base (or multiple reference bases) of a reference genome. Illustratively, a variant nucleobase call may include (or be part of) various structural variants that differ from one or more reference bases of a reference genome. Illustratively, a variant may include a SNP, a deletion, an insertion, a duplication, an inversion, a translocation, or a copy number variation (CNV). In one or more embodiments, a variant includes a mutation, such as a natural or synthetically introduced mutation, such as a CRISPR-induced mutation.

[0025] Relatedly, as used herein, the term "target variant" refers to a variant selected or identified for detection or assignment. In some cases, the target variant includes a variant identified for detection by a variant caller, variant calling model, or other caller. For example, the target variant may be identified by a repeat expansion detection model, structural variant caller, CYP2D6 caller, CNV caller, small variant caller, or other caller for detection. As described below, the target variant can be a variant of a specific gene, including, but not limited to, the replication factor C subunit 1 (RFC1) gene, the cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene, the cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene, the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, the survival motor neuron 1 (SMN1) gene, the survival motor neuron 2 (SMN2) gene, the glucosylceramide beta (GBA) gene, the blood group Rh(CE) (RHCE) gene, the lipoprotein (A) (LPA) gene, the fragile X mental retardation 1 (FMR1) gene, the hexosaminidase subunit alpha (HEXA) gene, the hemoglobin subunit alpha 1 (HBA1) gene, the hemoglobin subunit alpha 2 (HBA2) gene, or the hemoglobin subunit beta (HBB) gene.

[0026] Furthermore, as used herein, the term "imputation" refers to statistically inferring or estimating genotypes for genomic coordinates or genomic regions. More specifically, imputing can include statistically inferring genotypes for one or more alleles corresponding to haplotypes for a genomic region of a sample genome. For example, imputation can refer to using marker variants surrounding a genomic region to determine the genotypes of alleles corresponding to haplotypes of the genomic region. In one or more embodiments, the customized genotype imputation system utilizes a reference panel from a haplotype database and a genotype imputation model (e.g., a hidden Markov-based model) to impute genotype calls. As further described herein, the customized genotype imputation system can impute genotype calls for target variants within a target genomic region based on SNPs (or other marker variants) that surround or are adjacent to the target genomic region but are part of one or more haplotypes corresponding to the target genomic region. For example, if haplotypes exhibit different sets of SNPs in a target genomic region and some genomic samples in a target variant reference panel also exhibit the target variant, the customized genotype imputation system can use the target variant indicators corresponding to such different sets of SNPs and particular haplotypes of the genomic samples to infer that the target genomic sample contains the target variant.

[0027] As used herein, the term "reference genome" refers to a digital nucleic acid sequence assembled as a representative example (or multiple representative examples) of genes and other gene sequences of an organism. Regardless of sequence length, in some cases, a reference genome represents an exemplary set of genes or a set of nucleic acid sequences in a digital nucleic acid sequence determined to be representative of an organism. For example, a linear human reference genome can be GRCh38 (or other version of the reference genome) from the Genome Reference Consortium. GRCh38 can include alternative contiguous sequences representing alternative haplotypes, such as SNPs and small indels (e.g., 10 base pairs or less, 50 base pairs or less).

[0028] Furthermore, as used herein, the term "reference panel" refers to a digital collection or database of haplotypes from genomic samples for which one or more ancestral or progenitor haplotypes have been determined. In some cases, a reference panel includes a digital database of haplotypes from genomic samples representative of (or common among) a population of organisms, for which multiple ancestral or progenitor haplotypes have been determined. A reference panel can also include data files or other organizations of data reflecting genomic sequences and various variant markers (e.g., SNPs) in those genomic sequences. By way of example, a reference panel can include data corresponding to genomic sequences and various tags or other metadata that characterize or classify the genomic sequences. In some cases, a customized genotype assignment system accesses initial reference panels developed by the Haplotype Reference Consortium (HRM), the 1000 Genomes Project, or Illumina, Inc., when generating a reference panel that includes marker variant indices for marker variants at genomic coordinates corresponding to genomic samples of different haplotypes.

[0029] Furthermore, as used herein, the term "target variant reference panel" refers to a reference panel that includes data about genomic sequences from genomic samples of different haplotypes and one or more target variant locations that include target variant indicators for one or more target variants. In particular, a target variant reference panel can include genomic sequences that include data representations for various marker variants (e.g., SNPs) and data fields for indicating the presence or absence of one or more target variants. For example, a target variant reference panel can include various genomic samples phased into maternal and paternal sequences and data fields representing target variant locations that indicate the presence or absence of the target variant for both the paternal and maternal genomic sequences.

[0030] Relatedly, as used herein, the term "target variant location" refers to a data attribute, feature, cell, or field for a target variant index. In particular, the term target variant location can include a data cell or data field into which a target variant index can be added or inserted to identify the presence or absence of a target variant in an allele, haplotype, or genomic sample. By way of example, a target variant location can include a data field in a target variant reference panel where a "0" indicates the absence of the target variant and / or a "1" indicates the presence of the target variant. In some cases, a target variant reference panel includes a target variant location for a target variant index of a biallelic target variant. Additionally or alternatively, in some embodiments, a target variant reference panel can include multiple target variant locations including multiple data entries or other target variant indexes for multiallelic target variants.

[0031] Furthermore, as used herein, the term "marker variant" refers to a variant at a polymorphic site in a population. In particular, a marker variant includes one of two or more alleles present in a population at a polymorphic genomic coordinate or genomic region at a frequency greater than a threshold frequency, for example, greater than 1% of the population. In some cases, a marker variant includes a SNP present at a polymorphic genomic coordinate among human populations represented in a reference panel. Additionally, or alternatively, a marker variant may include an insertion or deletion (indel), a structural variant, or other variant at a polymorphic site in a population. As alluded to above, alleles for a particular haplotype represented by a reference panel may include SNPs or other variant markers used for assignment.

[0032] Relatedly, as used herein, the term "marker variant index" refers to a data index of a marker variant. Similarly, as used herein, the term "target variant index" refers to a data index of a target variant. In particular, the terms marker variant index or marker variant index may include a "1" in a file (e.g., a VCF) indicating the presence of a variant at a particular genomic coordinate, or a "0" in a file reflecting the absence of a variant at a particular genomic coordinate. However, it will be understood that the marker variant index and / or target variant index may include another data index reflecting the presence or absence of a variant, such as a single-letter code, an alphanumeric code, or other symbol.

[0033] Furthermore, as used herein, the term "genomic coordinate" refers to a specific location or position of a nucleotide base within a genome (e.g., the genome of an organism or a reference genome). In some cases, a genomic coordinate includes an identifier for a specific chromosome of a genome and an identifier for the position of a nucleotide base within the specific chromosome. For example, a genomic coordinate(s) may include a chromosome (e.g., chr1 or chrX) number, name, or other identifier, and a specific location(s), such as a numbered position following the identifier of the chromosome (e.g., chr1:1234570 or chr1:1234570-1234870). Furthermore, in certain implementations, a genomic coordinate refers to the source of a reference genome (e.g., mt:16568 for a mitochondrial DNA reference genome, or SARS-CoV-2:29001 for a reference genome for the SARS-CoV-2 virus) and the position of a nucleotide base within the source for the reference genome (e.g., mt:16568 or SARS-CoV-2:29001). In contrast, in certain cases, a genomic coordinate refers to the location of a nucleotide base within a reference genome, without reference to a chromosome or source (e.g., 29727).

[0034] Additionally, as used herein, the term "genomic region" refers to a range of genomic coordinates. Similar to genomic coordinates, in certain embodiments, a genomic region can be identified by a chromosomal identifier and a specific location(s), such as a chromosomal identifier followed by a numbered location (e.g., chr1:1234570-1234870).

[0035] In this context, the term "target genomic region" refers to a genomic region that includes a target variant and nucleic acid bases surrounding or adjacent to the target variant. In particular, the target genomic region may include the genomic coordinates of the target variant and at least the genomic coordinates of a marker variant within a threshold number of nucleic acid bases (e.g., 50 base pairs, 200 base pairs, 500 base pairs, 1,000 base pairs) upstream of the target genomic region and / or within a threshold number of nucleic acid bases (e.g., 50 base pairs, 200 base pairs, 500 base pairs, 1,000 base pairs) downstream of the target genomic region.

[0036] Also as used herein, the term "haplotype" refers to a nucleotide sequence present in an organism (or present in organisms from a population) that is inherited from one or more ancestors. In particular, a haplotype can include alleles or other nucleotide sequences present in organisms of a population that are inherited together by such organisms, each from a single parent. In one or more embodiments, a haplotype includes a set of SNPs on the same chromosome that tend to be inherited together. In some cases, data representing haplotypes or sets of different haplotypes is stored in or otherwise accessible to a haplotype database.

[0037] Furthermore, as used herein, the term "genomic sample" refers to a target genome or portion of a genome to be sequenced. For example, a sample genome includes a sequence of nucleotides isolated or extracted from a sample organism (or a copy of such an isolated or extracted sequence). In particular, a sample genome includes an entire genome isolated or extracted (in whole or in part) from a sample organism and composed of nitrogenous heterocyclic bases. For example, a nucleic acid polymer can include segments of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or other polymeric forms of nucleic acid or chimeric or hybrid forms of nucleic acids described below. In some cases, a sample genome is one found in a sample prepared or isolated by a kit and received by a sequencing instrument.

[0038] In this context, the term "allele" refers to a version of a nucleic acid base or nucleotide sequence at a genomic coordinate or genomic region that corresponds to a haplotype, such as a haplotype for a genomic region that encodes a gene or non-coding region. In particular, an allele comprises one of two or more versions of a nucleic acid base or nucleotide sequence at a genomic coordinate or region that tend to be inherited together in combination as part of a haplotype. As part of a haplotype, in some cases, a combination of alleles can be inherited by an organism as part of a single gene or across multiple genes.

[0039] Furthermore, as used herein, the term "genetic diversity" refers to the range of different inherited variants within a population. In particular, genetic diversity includes the range of genetic variants represented by different haplotypes representing different ancestries, continents, countries, and / or populations. More specifically, a reference panel can include data representing haplotypes that exhibit genetic diversity among variants within alleles of the haplotypes.

[0040] Further details about the persona group system will now be provided in connection with illustrative diagrams showing example embodiments and implementations. For example, Figure 1 shows a schematic diagram of a computing system 100 on which a customized genotype assignment system 104 and a sequencing system 106 operate according to one or more embodiments. As shown, the computing system 100 includes one or more server devices 102 connected to user client devices 108 and sequencing devices 114 via a network 112. While Figure 1 shows an embodiment of the customized genotype assignment system 104, this disclosure describes alternative embodiments and configurations below.

[0041] 1, server device 102, user client device 108, and sequencing device 114 are connected via network 112. Thus, each of the components of computing system 100 can communicate via network 112. Network 112 includes any suitable network with which computing devices can communicate. An exemplary network is described in more detail below with respect to FIG. 12.

[0042] As illustrated by FIG. 1 , the sequencing device 114 includes a device for sequencing a genomic sample or other nucleic acid polymer. In some embodiments, the sequencing device 114 utilizes computer-implemented methods and systems (described herein) either directly or indirectly on the sequencing device 114 to analyze oligonucleotides extracted from the genomic sample to generate data. More specifically, the sequencing device 114 receives and analyzes nucleic acid sequences extracted from the genomic sample within a nucleotide-sample slide (e.g., a flow cell). In one or more embodiments, the sequencing device 114 utilizes SBS to sequence the genomic sample or other nucleic acid polymer. In some embodiments, the sequencing device 114 communicates directly with the user client device 108, in addition to or as an alternative to communicating via the network 112. Additionally, as illustrated in FIG. 1 , in one or more embodiments, the sequencing device 114 includes a customized genotype assignment system 104.

[0043] As further illustrated by FIG. 1 , the server device 102 can generate, receive, analyze, store, and transmit digital data, such as nucleobase call or nucleotide read data. As illustrated in FIG. 1 , the sequencing device 114 can transmit (and the server device 102 can receive) various data from the sequencing device 114, including data representing nucleotide reads. The server device 102 can also communicate with the user client device 108. In particular, the server device 102 can transmit data about nucleotide reads, nucleobase calls, genomic samples, and / or reference panels to the user client device 108. As further illustrated in FIG. 1 , the server device 102 can include a customized genotype assignment system 104. In one or more embodiments, as described further below, the customized genotype assignment system 104 generates a target variant reference panel that includes one or more target variant positions. Accordingly, the server device 102 can further transmit data representing the target variant reference panel to the user client device 108.

[0044] In some embodiments, server device 102 comprises a distributed collection of servers, where server device 102 includes multiple server devices distributed across network 112 and located in the same or different physical locations. Additionally, server device 102 may comprise a content server, an application server, a communication server, a web hosting server, or another type of server.

[0045] In some cases, the server device 102 is located at or near the same physical location as the sequencing device 114, or remotely from the sequencing device 114. Indeed, in some embodiments, the server device 102 and the sequencing device 114 are integrated into the same computing device. The server device 102 can execute the sequencing system 106 or a customized gene assignment system 104 to generate, receive, analyze, store, and transmit digital data, such as by receiving base call data or determining variant calls based on analyzing such base call data.

[0046] 1, user client device 108 can generate, store, receive, and transmit digital data. In particular, user client device 108 can receive nucleotide reads, nucleic acid base calls, genotype calls, sequencing metrics, and / or targeted variant reference panel data from server device 102 and / or sequencing device 114. Accordingly, user client device 108 can present data regarding genotype calls to a user associated with user client device 108 within a graphical user interface.

[0047] 1 can include various types of client devices. For example, in some embodiments, user client device 108 includes a non-mobile device, such as a desktop computer or server, or other type of client device. In yet other embodiments, user client device 108 includes a mobile device, such as a laptop, tablet, mobile phone, or smartphone. Further details about user client device 108 are described below with respect to FIG. 12.

[0048] 1 , the user client device 108 includes a sequencing application 110. The sequencing application 110 may be a web application or a native application (e.g., a mobile application, a desktop application) stored and executed on the user client device 108. The sequencing application 110 may include instructions that (when executed) cause the user client device 108 to receive data from the customized genotype assignment system 104 and present data from the sequencing device 114 and / or the server device 102. Additionally, the sequencing application 110 may instruct the user client device 108 to display data for the genotype calls, such as genotype calls for target variants from a variant call file (VCF).

[0049] 1 , the customized gene assignment system 104 may be located on the user client device 108 or on the sequencing device 114 as part of the sequencing application 110. Thus, in some embodiments, the customized gene assignment system 104 is implemented (e.g., located completely or partially) on the user client device 108. As noted above, in still other embodiments, the customized gene assignment system 104 is implemented by one or more other components of the computing system 100, such as the sequencing device 114. Notably, the customized gene assignment system 104 can be implemented in a variety of different ways across the server device 102, the network 112, the user client device 108, and the sequencing device 114.

[0050] 1 depicts components of computing system 100 communicating over network 112, in certain implementations, components of computing system 100 may also communicate directly with one another, bypassing the network. For example, as previously described, in some embodiments, user client device 108 may communicate directly with sequencing device 114. Additionally, in some embodiments, user client device 108 communicates directly with customized genetic assignment system 104. Furthermore, customized genetic assignment system 104 may access one or more databases housed on or accessed by server device 102 or elsewhere within computing system 100.

[0051] As described above, in one or more embodiments, the customized genotype assignment system 104 generates and / or utilizes a target variant reference panel to assign genotype calls. According to one or more embodiments, Figure 2A shows an overview of a customized genotype assignment system 104 that generates a target variant reference panel for a target variant, and Figure 2B shows an overview of a customized genotype assignment system 104 that utilizes a target variant reference panel to assign genotype calls for a target variant.

[0052] As shown in Figure 2A, for example, customized genotype assignment system 104 generates reference panel 202. Reference panel 202 includes digital representations of haplotypes from genomic samples 200a, 200b, and 200c. While Figure 2A includes three genomic samples for illustrative purposes, it is understood that in one or more embodiments, reference panel 202 can include varying amounts of a variety of genomic samples.

[0053] 2A, the customized genotype assignment system 104 can generate a reference panel 202 to include phased alleles for the genomic samples 200a-200c. Illustratively, the customized genotype assignment system 104 can determine which alleles from the genomic samples 200a-200c correspond to maternal and paternal haplotypes. Thus, as shown in FIG. 2A, the reference panel 202 can include both maternal and paternal copies of each allele.

[0054] In addition to the different haplotypes from genomic samples 200a-200c, as further shown in FIG. 2A, customized genotype assignment system 104 generates reference panel 202, which includes marker variants such as SNPs and small indels (e.g., 10 base pairs or less, 50 base pairs or less). To mark individual marker variants in the corresponding genomic samples, reference panel 202 includes marker variant indicators 201a, 201b, 201c, and 201d at the genomic coordinates of the corresponding marker variants. In particular, FIG. 2A illustrates open or hollow circles within alleles of individual genomic samples 200a-200c to represent marker variant indicators 201a-201d. For illustrative purposes, open or hollow circles represent marker variant indicators in which a particular allele of a genomic sample includes the corresponding marker variant, and the absence of such open or hollow circles represents marker variant indicators in which a particular allele of a genomic sample does not include the corresponding marker variant. Indeed, in one or more embodiments, the reference panel 202 includes data representations of other marker variant indices of marker variants present in either or both alleles corresponding to the maternal haplotype or the paternal haplotype.

[0055] As also shown in FIG. 2A , the customized genotype assignment system 104 adds target variant positions 204 to the reference panel 202. More specifically, the customized genotype assignment system 104 adds the target variant positions 204 to the reference panel 202 as part of generating the target variant reference panel. In one or more embodiments, the target variant positions 204 are data fields for indicating the presence or absence of a target variant for maternal and paternal alleles of a genomic sample. In particular, FIG. 2A shows a dotted circle aligned with the alleles of genomic samples 200 a-200 c to represent the target variant positions 204. Indeed, as shown in FIG. 2A , the customized genotype assignment system 104 adds a target variant position 204 for each genomic sample or for each allele of each genomic sample.

[0056] Upon adding the target variant position 204, the customized genotype assignment system 104 phases the alleles 206 of the genomic samples 200a-200c, as further shown in FIG. 2A. More specifically, in one or more embodiments, the customized genotype assignment system 104 phases the alleles associated with the target variant to identify genomic sequences containing the target variant for either or both the maternal and paternal alleles. Thus, the customized genotype assignment system 104 can identify the presence or absence of the target variant for the maternal and paternal alleles of each genomic sample in the target variant reference panel. As shown in FIG. 2A, for example, the phased alleles 206 of the genomic samples 200a-200c include different patterns indicative of different alleles corresponding to different haplotypes.

[0057] In addition to phasing the alleles of different genomic samples, in one or more embodiments, the customized genotype assignment system 104 adds a target variant indicator to the target variant position 204. In particular, FIG. 2A shows a black circle alongside the alleles of genomic samples 200a-200c to represent the target variant indicator in the target variant position 204 to indicate that the target variant is present in a particular allele. In effect, the customized genotype assignment system 104 generates a target variant indicator that indicates whether the genomic sample contains the target variant. Furthermore, in one or more embodiments, the customized genotype assignment system 104 adds an indicator to the target variant position 204 for either the maternal allele or the paternal allele of each genomic sample in the target variant reference panel.

[0058] By adding target variant indices to target variant positions 204 and phasing the alleles of genomic samples 200a-200c, customized genotype assignment system 104 generates a target variant reference panel 208 that includes target variant indices at the target variant positions. Thus, target variant reference panel 208 includes data about the target variant at each allele associated with the target variant. As shown in FIG. 2A , for example, target variant reference panel 208 is represented as a file. Indeed, in one or more embodiments, customized genotype assignment system 104 generates a VCF with target variant positions 204 as rows in the VCF and target variant indices as "0" for unaffected alleles and "1" for affected alleles.

[0059] Referring now to FIG. 2B , the customized genotype assignment system 104 can utilize a target variant reference panel to assign genotype calls indicating the presence or absence of a target variant in a target genomic sample 216. Illustratively, as shown in FIG. 2B , the customized genotype assignment system 104 identifies nucleotide reads 210 corresponding to the target genomic sample 216. In one or more embodiments, the customized genotype assignment system 104 utilizes a sequencing system and / or one or more sequencing devices to identify nucleic acid segments or oligonucleotides extracted from the genomic sample and generate data. Illustratively, in some embodiments, the sequencing device or customized genotype assignment system 104 receives and analyzes oligonucleotides extracted from the target genomic sample 216 in a nucleotide-sample slide (e.g., a flow cell). Additionally or alternatively, the customized genotype assignment system 104 can receive nucleotide reads for the target genomic sample 216 from a third-party sequencing system or from a sequencing device controlled by a separate entity.

[0060] As also shown in FIG. 2B , the customized genotype assignment system 104 can align nucleotide reads 210 with a reference genome 212 to determine variant calls or sequences within specific genomic regions of the target genomic sample 216. Furthermore, the customized genotype assignment system 104 can identify one or more aligned nucleotide reads corresponding to the target genomic region of the target variant. Because alignment of nucleotide reads 210 with the reference genome 212 can result in inaccurate variant calls or no calls for some genomic regions, the customized genotype assignment system 104 can rely on a target variant reference panel 214 as a substitute for nucleotide reads covering the target genomic region. Thus, as shown in FIG. 2B , the customized genotype assignment system 104 can further utilize the target variant reference panel 214 to determine genotype calls for the target genomic sample 216, particularly for difficult-to-call genomic regions.

[0061] 2B , for example, customized genotype assignment system 104 accesses target variant reference panel 214. Illustratively, in one or more embodiments, customized genotype assignment system 104 compares (i) marker variants represented by a subset of aligned nucleotide reads that flank or surround a target genomic region of a target variant to (ii) corresponding marker variants within alleles of genomic samples 200a-200c represented by target variant reference panel 214. To represent such marker variants, target variant reference panel 214 includes marker variant indices 201a, 201b, 201c, and 201d within alleles of genomic samples 200a, 200b, and 200c. As noted above, a white or open circle represents a marker variant indices where a particular allele of a genomic sample includes the corresponding marker variant, and the absence of such a white or open circle represents a marker variant indices where a particular allele of a genomic sample does not include the corresponding marker variant.

[0062] 2B, both genomic sample 200c and target genomic sample 216 include open or hollow circles representing marker variants corresponding to marker variant index 201a on both maternal and paternal alleles. In contrast, both genomic sample 200c and target genomic sample 216 include a single open or hollow circle representing a marker variant corresponding to marker variant index 201a on a single allele. Alternatively, genomic sample 200c and target genomic sample 216 do not include such open or hollow circles, as marker variant indexes 201c and 201d represent that those alleles do not include the corresponding marker variant.

[0063] To facilitate comparison of marker variants between the target genomic sample 216 and the genomic samples 200a-200c represented by the target variant reference panel 214, the customized genotype assignment system 104 can limit the marker variants to be compared to a threshold distance from the target variant. Indeed, in one or more embodiments, the customized genotype assignment system 104 identifies marker variants within a threshold number of nucleobases from the target variant or target genomic region. For example, in some cases, the customized genotype assignment system 104 identifies marker variants (i) within a threshold number of nucleobases upstream of the target genomic region (e.g., 10, 50, 200 nucleobases) and / or (ii) within a threshold number of nucleobases downstream from the target genomic region (e.g., 10, 50, 200 nucleobases).

[0064] Based on such a comparison of marker variants, customized genotype assignment system 104 can phase the nucleotide reads of target genomic sample 216 to identify corresponding alleles in the maternal and paternal haplotypes. For example, the alleles of target genomic sample 216 contain the same marker variants as the alleles of genomic sample 200c, as indicated by different patterns indicating different alleles in target variant reference panel 214.

[0065] As further illustrated by FIG. 2B , the customized genotype assignment system 104 can assign a genotype call 218 for a target variant in a target genomic sample by comparing marker variants of the target variant reference panel with marker variants of the target genomic sample. More specifically, the customized genotype assignment system 104 determines the genotype call 218 by statistically inferring haplotypes (e.g., represented as values ​​between 0 and 1) that are likely to be present in a genomic region of the target genomic sample based on the target variant reference panel 214. Illustratively, the customized genotype assignment system 104 utilizes statistical inference and haplotypes that include marker variants from the target variant reference panel 214 to identify haplotypes from the target variant reference panel that are likely to be present in a genomic region. Furthermore, the customized genotype assignment system 104 can utilize the haplotypes identified from the target variant reference panel 214 to determine a genotype call for the target genomic sample.

[0066] As described above, many existing sequencing systems are unable to make genotype calls or make inaccurate genotype calls for difficult-to-call genomic regions that contain repeat expansions. Figure 3 illustrates such difficult-to-call genomic regions. More specifically, Figure 3 illustrates nucleotide reads of a genomic sample that are misaligned with a genomic region that contains a repeat expansion, according to one or more embodiments.

[0067] 3 , for example, a sequencing system aligns nucleotide reads corresponding to genomic sample 300a (e.g., HG04127) and genomic sample 300b (e.g., HG01506) to (i) a reference genome corresponding to target genomic region 302 and a repeat expansion in the RFC1 gene, and (ii) surrounding genomic regions 304a and 304b adjacent to target genomic region 302. Both genomic samples 300a and 300b are putative carriers of the repeat expansion variant corresponding to target genomic region 302. As shown, the sequencing system aligns nucleotide reads of genomic sample 300a with surrounding genomic regions 304a and 304b that have at least 10-fold coverage for genomic sample 300a, but inconsistently aligns nucleotide reads of genomic sample 300a with target genomic region 302. Similarly, the sequencing system aligns nucleotide reads of genomic sample 300b to surrounding genomic regions 304a and 304b, which have at least 4-fold coverage for genomic sample 300b, but inconsistently aligns nucleotide reads of genomic sample 300b to target genomic region 302. The alignment shows a read coverage hole within target genomic region 302, even though both genomic sample 300a and genomic sample 300b are putative carriers of the repeat expansion variant.

[0068] Thus, Figure 3 illustrates the poor nucleotide read data quality characteristic of some genomic regions exhibiting repeat expansions. In some cases, such extended repeats are impossible to accurately identify in genomic samples carrying target variants using existing sequencing systems. More specifically, alignment of nucleotide reads within target genomic region 302 with a reference genome is uncertain or impossible due to the nature of the repeats, which result in a variety of possible alignments. For example, as shown in Figure 3, genome samples 300a and 300b exhibit approximately 35 and 33 repeat units of AAGGG within target genomic region 302, respectively. For example, because a nucleotide fragment exhibiting "AGGGAAGGGAAG" may have a variety of alignments, existing sequencing systems find it difficult or even impossible to align the corresponding nucleotide reads with target genomic region 302 of the reference genome and determine the length of the repeat expansion.

[0069] As described above, the customized genotype assignment system 104 can utilize a targeted variant reference panel to assign more accurate genotype calls for targeted variants than existing sequencing systems, particularly for difficult-to-call genomic regions. According to one or more embodiments, Figure 4 shows a uniform manifold approximation and projection (UMAP) graph 400 of data points representing various genomic samples clustered according to SNPs or other marker variants. As shown by the targeted variant clusters 410 in the UMAP graph 400, genomic samples affected by the targeted variants tend to cluster together based on shared marker variants.

[0070] 4, in one or more embodiments, the customized genotype assignment system 104 performs principal component analysis (PCA) to cluster genomic samples based on the SNPs or other marker variants present in each genomic sample. The customized genotype assignment system 104 further utilizes UMAP to visualize the clusters of genomic samples. A UMAP graph 400 shows the results of such clustering.

[0071] As shown in FIG. 4 , for example, UMAP graph 400 depicts data points representing various genomic samples along UMAP-3D-1 axis 404 and UMAP-3D-2 axis 402 via dimensionality reduction. As indicated by the black circles representing particular data points, UMAP graph 400 includes data points representing genomic samples harboring variants 406 in the RFC1 gene that contain pathogenic repeat expansions. In particular, customized genotype assignment system 104 identifies target variant cluster 410, which includes data points representing genomic samples that contain at least one allele with the target variant in the RFC1 gene. In contrast, as indicated by the lighter-colored or gray circles representing particular data points, UMAP graph 400 also includes data points representing genomic samples that exhibit non-variants 408, or in other words, that are not affected by the target variant in the RFC1 gene.

[0072] Thus, UMAP graph 400 shows that SNPs or other marker variants constitute reliable evidence for the attribution of a genotype call to a target variant in RFC1. Illustratively, this is because genomic samples from target variant cluster 410 not only exhibit the same or similar nucleotides in the target genomic region of RFC1, but also exhibit similar or identical SNPs in other genomic regions adjacent to or surrounding the target genomic region (e.g., within 200 base pairs upstream or downstream from the target genomic region). Thus, UMAP graph 400 provides proof of concept that SNPs can be used to infer or identify genomic samples that exhibit an RFC1 pathogenic repeat.

[0073] To leverage such concepts using a unique reference panel specific to a target variant, the customized genotype assignment system 104 can generate a target variant reference panel that includes the target variant position. According to one or more embodiments, FIG. 5 shows the customized genotype assignment system 104 generating a reference panel 502 and adding the target variant position 518 to the reference panel 502 to generate a target variant reference panel 524. As described below, the customized genotype assignment system 104 can generate the target variant reference panel 524 that includes (i) phased alleles corresponding to the target variant indices in the target variant position 518, and (ii) marker variant indices for the marker variants phased according to the maternal and paternal haplotypes of the genomic sample.

[0074] 5, the customized genotype imputation system 104 generates a reference panel 502 that includes genomic samples 504, 506, and 508 of different haplotypes. In particular, the reference panel 502 includes alleles of genomic samples 504-508 that include marker variant indicators of SNPs 510, 512, and 516. However, genomic samples 504-508 and SNPs 510-516 are provided by way of example, and it is understood that the customized genotype imputation system 104 can generate reference panels and / or target variant reference panels that include various amounts of SNPs and genomic samples (e.g., 50,000; 100,000 SNPs), including genomic samples representing hundreds or thousands of haplotypes and thousands of SNPs.

[0075] As described above, in one or more embodiments, the customized genotype imputation system 104 generates a reference panel 502 that includes genomic samples with a variety of different haplotypes that represent genetic diversity. By way of example, the customized genotype imputation system 104 can generate a reference panel 502 that includes genomic samples 504-508 from various ancestries, continents, countries, and / or populations. Similarly, the customized genotype imputation system 104 can convert the reference panel 502 into a targeted variant reference panel that includes genomic samples 504-508 with marker variants from a variety of different ancestries, continents, countries, and / or populations.

[0076] As described above, in one or more embodiments, the customized genotype imputation system 104 can generate an output file (e.g., a VCF) containing data representing the reference panel 502 and / or the target variant reference panel 524. However, for purposes of illustration, FIG. 5 depicts the reference panel 502 and the target variant reference panel 524 as a collection of lines representing haplotypes of the genomic samples 504-508 and circles representing marker variant indices indicating the presence of SNPs 510-512. As indicated by the open or hollow circles representing the marker variant indices, the genomic sample 504 includes one copy of SNP 510 on both the maternal and paternal alleles, SNP 512 on both the maternal and paternal alleles, and SNP 516. In contrast, the genomic sample 506 includes one copy of SNP 512 and SNP 516 on both the maternal and paternal alleles. As also shown in FIG. 5, genomic sample 508 contains SNP 510 in both the maternal and paternal alleles, and one copy of SNP 512.

[0077] 5 depicts marker variant indices for SNPs as open or hollow circles, it is understood that in one or more embodiments, the reference panel 502 and / or target variant reference panel 524 may be represented in an output file (e.g., a VCF) that includes data fields with "0" reflecting the reference nucleobase and "1" reflecting the alternative nucleobase. Additionally, or alternatively, the customized genotype assignment system 104 may utilize an alternative binary scheme for marker variant indices. For example, the customized genotype assignment system 104 can generate a reference panel 502 and / or a target variant reference panel 524 that includes two cells or locations for a multi-allelic marker variant, where a "0" as the marker variant index at both locations reflects the reference nucleobase, "0" and "1" as the marker variant index at the first and second locations reflect a first alternative nucleobase, "1" and "1" as the marker variant index at the first and second locations reflect a second alternative nucleobase, and "1" and "0" as the marker variant index at the first and second locations reflect a third alternative nucleobase. Alternatively, by way of further example, in some embodiments, the customized genotype assignment system 104 can generate a reference panel 502 and / or a target variant reference panel 524 that includes a single cell or location for a multi-allelic marker variant, where the value "0" reflects the reference nucleobase, "1" reflects a first alternative nucleobase, "2" reflects a second alternative nucleobase, and / or "3" reflects a third alternative nucleobase.

[0078] 5, the customized genotype assignment system 104 utilizes SNPs 510-516 as marker variants for the assignment of genotype calls for the presence or absence of a target variant in a target genomic sample. However, in one or more embodiments, the customized genotype assignment system 104 can utilize other marker variants, such as marker variants in the form of deletions, insertions, duplications, inversions, translocations, or CNVs. In some cases, the customized genotype assignment system 104 can generate a reference panel 502 that includes data fields having values ​​(e.g., a range of values) that identify various marker variant types.

[0079] As shown in FIG. 5 , the customized genotype assignment system 104 generates the target variant reference panel 524 in part by adding target variant positions 518. As briefly discussed above, the target variant positions 518 can correspond to a variety of target variants. For example, the target variants can include biallelic variants or multiallelic variants. Furthermore, in one or more embodiments, the target variants include repeat expansions (e.g., STR expansions or VNTR expansions). Regardless of whether the target variants constitute repeat expansions, in some cases the target variants constitute pathogenic variants.

[0080] More specifically, in one or more embodiments, the target variants can include variants of various genes. By way of example, in some embodiments, target variants may include, but are not limited to, variants of the replication factor C subunit 1 (RFC1) gene, the cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene, the cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene, the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, the survival motor neuron 1 (SMN1) gene, the survival motor neuron 2 (SMN2) gene, the glucosylceramide beta (GBA) gene, the blood group Rh(CE) (RHCE) gene, the lipoprotein (A) (LPA) gene, the fragile X mental retardation 1 (FMR1) gene, the hexosaminidase subunit alpha (HEXA) gene, the hemoglobin subunit alpha 1 (HBA1) gene, the hemoglobin subunit alpha 2 (HBA2) gene, or the hemoglobin subunit beta (HBB) gene.

[0081] Regardless of the gene or target genomic region, in some embodiments, the target variant can include a deletion, insertion, duplication, inversion, translocation, or CNV transmitted within a population. For example, in one or more embodiments, the customized genotype assignment system 104 uses target variants inherited from ancestral haplotypes to support sufficient data for a target variant reference panel with target variant positions specific to the target variant. Thus, in some embodiments, de novo variants may not support the target variant reference panel. Because the customized genotype assignment system 104 detects variants based on a target variant reference panel that includes various genomic samples, new mutations in the target genomic sample will not be present in a sufficient number of haplotypes to support a functional version of the target variant reference panel. Thus, the new variant will not be present in the target variant reference panel or will have only limited representation.

[0082] To ensure sufficient haplotype data, in one or more embodiments, the customized genotype assignment system 104 uses a target variant reference panel specific to the target variant that meets one or more thresholds. For example, in some cases, the target variant must meet one or more relative thresholds that depend on the number of genomic samples in the target variant reference panel, including a threshold carrier frequency, a threshold linkage disequilibrium (LD) for a particular marker variant, or a threshold mutation rate. In one or more embodiments using a target variant reference panel representing approximately 3,000 genomic samples to support the assignment of genotype calls, the target variant exhibits a threshold carrier frequency of approximately 2% of the genomic samples; 2 The threshold LD in is 0.75, thereby mimicking a strong founder effect; the threshold mutation rate is 1.29 x 10 -8 Mutations / base pairs / meiosis must be shown.

[0083] Indeed, in some embodiments, the customized genotype assignment system 104 determines a threshold carrier frequency, threshold linkage disequilibrium, or threshold mutation rate relative to the number of genomic samples represented by the target variant reference panel. For example, a target variant reference panel representing a relatively large number of genomic samples may facilitate a relatively low threshold carrier frequency, a relatively low threshold linkage disequilibrium, or a relatively low threshold mutation rate. Thus, other appropriate measures for the threshold carrier frequency, threshold LD, or threshold mutation rate may be used beyond the examples provided above. As described below, Figure 7 provides examples of different threshold carrier frequencies for target variants depending on the number of genomic samples represented by the target variant reference panel.

[0084] 5, in one or more embodiments, the customized genotype assignment system 104 adds target variant positions 518 to the reference panel 502 by adding one or more data fields associated with the target variant. As described above, the customized genotype assignment system 104 can generate the target variant reference panel 524 as a VCF file and can utilize various binary schemes to represent nucleotides in genomic coordinates. By way of example, in some embodiments, each target variant position 518 can be a field containing a target variant index of either "0" or "1," where "0" represents the reference nucleobase and "1" represents the alternative nucleobase.

[0085] By using a variety of different target variant indicators, the customized genotype assignment system 104 can generate a target variant reference panel for biallelic or multiallelic target variants. For example, by using two fields for two target variant positions, the customized genotype assignment system 104 can represent multiallelic target variants. Indeed, as shown in Figure 5, a pair of dotted circles in each allele of genomic samples 504, 506, and 508 represents target variant position 518 as two target variant positions (e.g., data fields) that together can contain or facilitate a binary code indicating the presence or absence of a multiallelic target variant for a given genomic sample.

[0086] To illustrate how such binary codes at two target variant positions indicate multi-allelic target variants, in some embodiments, a "0" as a target variant index at both target variant positions represents a reference nucleobase (e.g., A). In contrast, a "0" as a target variant index at a first target variant position and a "1" as a target variant index at a second target variant position represent a first alternative nucleobase (e.g., G). Furthermore, a "1" as a target variant index at a first target variant position and a "1" as a target variant index at a second target variant position represent a second alternative nucleobase (e.g., T). A "1" as a target variant index at a first target variant position and a "0" as a target variant index at a second target variant position represent a third alternative nucleobase (e.g., C).

[0087] As an alternative to multiple target variant positions, in some embodiments, the customized genotype assignment system 104 indicates the presence or absence of a multi-allelic target variant using a non-binary code at a single target variant position. Although not depicted in Figure 5, in some embodiments, a "0" as a target variant index at a target variant position represents a reference nucleobase (e.g., A), a "1" as a target variant index at a target variant position represents a first alternative nucleobase (e.g., G), a "2" as a target variant index at a target variant position represents a second alternative nucleobase (e.g., T), and a "3" as a target variant index at a target variant position represents a first alternative nucleobase (e.g., G).

[0088] 5, for example, targeted variant reference panel 524 includes target variant indicators 526a and 526b as filled circles on one allele and 528a and 528b as filled circles on another allele to indicate that genomic sample 504 includes a particular haplotype of a multi-allelic target variant on both the maternal and paternal alleles. Conversely, targeted variant reference panel 524 includes a pair of dashed circles on both alleles of genomic sample 506 to indicate that genomic sample 506 does not include a multi-allelic target variant on either the maternal or paternal allele. Additionally, the target variant reference panel 524 includes a target variant indicator 550 as a black circle on an allele of the genomic sample 508 to indicate that the genomic sample 508 contains one copy of the multi-allelic target variant on the maternal allele or the paternal allele, and a dotted circle on one allele of the genomic sample 508 to indicate that the genomic sample 508 does not contain the multi-allelic target variant on one allele.

[0089] 5, in addition to adding the target variant position 518, in some embodiments, the customized genotype imputation system 104 phases the alleles of the genomic samples 504-508 along with the target variant index at the target variant position for the target variant. By phasing the alleles of the genomic samples 504-508, the customized genotype imputation system 104 determines the presence or absence of the target variant in the corresponding alleles present on the maternal and paternal haplotypes of the genomic samples 504-508. To phase such alleles, in some cases, the customized genotype imputation system 104 executes a haplotype phasing model, such as the Segmented HAPlotype Estimation and Imputation Tool (SHAPEIT), to infer haplotypes from the genotype data corresponding to the genomic samples 504-508.

[0090] Because both alleles of a homozygous genomic sample contain copies of the target variant and corresponding target variant indices in the target variant reference panel, in some embodiments, the customized genotype assignment system 104 phases heterozygous alleles of a subset of genomic samples, such as genomic sample 508, in which the alleles are heterozygous for the target variant. Indeed, in some cases, the customized genotype assignment system 104 does not phase homozygous alleles of a subset of genomic samples, such as genomic samples 504 and 506. In contrast, in some embodiments, the customized genotype assignment system 104 executes a haplotype phasing model to phase the alleles of the genomic samples represented by the target variant reference panel 524, regardless of the zygosity of the genomic sample for the target variant, and the data representing the phased alleles in the target variant reference panel also includes target variant indices at the target variant positions relative to the target variant.

[0091] As further illustrated by Figure 5, the customized genotype assignment system 104 can compare the nucleotide reads of the target genomic sample 532 to a target variant reference panel 524. As described below with respect to Figure 8, the customized genotype assignment system 104 can utilize the target variant reference panel 524 to assign genotype calls for target variants in the target genomic sample. More specifically, the customized genotype assignment system 104 can utilize the target variant reference panel 524 to determine phased genotype calls for both the maternal and paternal copies from the target genomic sample 532.

[0092] 5, the customized genotype assignment system 104 generates a genotype call indicating that the target genomic sample 532 contains a multi-allelic target variant and has the same haplotype as the genomic sample 508. Indeed, similar to the genomic sample 508, the target genomic sample 532 includes a target variant indicator 552 as a black circle on the allele to indicate that the target genomic sample 532 contains one copy of the multi-allelic target variant on the maternal allele or the paternal allele, and a dotted circle on one allele to indicate that the target genomic sample 532 does not contain the multi-allelic target variant on one allele.

[0093] As described above, the customized genotype assignment system 104 can generate an output file that includes a target variant reference panel. According to one or more embodiments, FIG. 6 shows a client device 600 presenting a portion of an exemplary VCF that includes a target variant reference panel 601 within a graphical user interface. As described below, the target variant reference panel 601 includes indices of nucleobase calls at genomic coordinates for various genomic samples and target variant indices at target variant locations that indicate whether a particular allele of the genomic sample represents the target variant.

[0094] As shown in Figure 6, for example, target variant reference panel 601 includes a chromosome column 602, a coordinate column 604, a target variant column 605, a reference nucleobase column 606, an alternative nucleobase column 608, a format column 610, and a genomic sample column 612. While Figure 6 shows a client device 600 presenting a portion of target variant reference panel 601, it will be understood that target variant reference panel 601 can include information about alleles across the genome, and the genomic coordinates provided are merely exemplary.

[0095] As further shown in Figure 6, chromosome column 602 includes chromosome information for each row. For example, in Figure 6, client device 600 presents rows for nucleobase calls for genomic coordinates on chromosome 4. Additionally, coordinate column 604 includes partial genomic coordinates for each row indicating which genomic coordinates correspond to the nucleobase call information within that row. Specifically, as shown in the graphical user interface depicted in Figure 6, client device 600 presents genomic coordinates from chr4:39348321 to chr4:39348429.

[0096] Additionally, client device 600 presents information regarding reference nucleobases (e.g., non-variant nucleotide bases) in reference nucleobase sequence 606, such as a one-letter code (e.g., A, C, T, G) in each cell representing a reference base from a reference genome at a corresponding genomic coordinate. Furthermore, client device 600 presents information regarding alternative nucleobases (e.g., variant nucleotide bases) in alternative nucleobase sequence 608, such as a one-letter code (e.g., A, C, T, G) in each cell representing the most common alternative or called alternative nucleobase at a corresponding genomic coordinate.

[0097] As further shown in Figure 6, client device 600 further presents information about the format of the nucleobase call provided in format column 610, and values ​​for the phased nucleobase call in genome sample column 612 for a particular genomic sample. As shown in Figure 6, target variant reference panel 601 includes the text "GT" to indicate a genotype call format for an allele value of "0" or "1" in genome sample column 612. More specifically, a value of "0" indicates that the nucleobase call is a reference nucleobase from reference nucleobase sequence 606. In contrast, a value of "1" indicates that the nucleobase call is an alternative nucleobase from alternative nucleobase sequence 608. The symbol "|" between values ​​in genome sample column 612 indicates a phased genotype call.

[0098] In addition to the genotype calls for the marker variants and other genomic coordinates, the client device 600 also presents a target variant column 605 containing identifiers for the target variants. As shown in FIG. 6 , the target variant reference panel 601 includes an identifier for RCF1 at genomic coordinate chr4:39348425. In some embodiments, chr4:39348425 represents a placeholder genomic coordinate for the target variant location, rather than an actual genomic coordinate within the reference genome. Indeed, the row corresponding to chr4:3934825 includes cells or fields representing exemplary target variant locations for each of genomic samples HG00096, HG00097, HG00099, HG00100, and HG00101.

[0099] In particular, as shown in the target variant reference panel 601, the row corresponding to chr4:3934825 includes "0" and "1" values ​​as target variant indices for the presence or absence of the target variant in genomic samples HG00096, HG00097, HG00099, HG00100, and HG00101. By separating the "0" and "1" values ​​with a "|" as a straight bar symbol, the target variant reference panel 601 includes phased target variant indices for the maternal allele and the paternal allele of each genomic sample. Thus, the client device 600 provides information about the target variants in the target variant reference panel 601 via a graphical user interface.

[0100] In some embodiments, as part of improving the accuracy of genotype calls for target variants, the customized genotype assignment system 104 can use target variant reference panels representing different numbers of genomic samples. According to one or more embodiments, FIG. 7 shows a graph 700 plotting the non-reference match rate at which a sequencing system accurately assigns target variants of various allele frequencies based on target variant reference panels representing different numbers of genomic samples. As shown by FIG. 7 , the non-reference match rate curve indicates that the customized genotype assignment system 104 more accurately assigns genotype calls for target variants as the genomic sample size of the target variant reference panel increases. Graph 700 further illustrates how the customized genotype assignment system 104 can use the non-reference match rate and allele frequency to determine a threshold carrier frequency depending on the genomic sample size of the target variant reference panel.

[0101] To test the accuracy of imputation for different reference panels, for example, researchers removed specific target variants from data representing target genotype samples sequenced by a sequencing device. The customized genotype imputation system 104 then imputed the genotype calls of the target variants from the target genome samples based on corresponding target variant reference panels of various genome sample sizes. As shown in Figure 7, the first target variant reference panel corresponding to the non-reference concordance curve 706d contains approximately 100 genome samples, the second target variant reference panel corresponding to the non-reference concordance curve 706c contains approximately 500 genome samples, the third target variant reference panel corresponding to the non-reference concordance curve 706b contains approximately 1,000 genome samples, and the fourth target variant reference panel corresponding to the non-reference concordance curve 706a contains approximately 2,500 genome samples.

[0102] As shown in graph 700, graph 700 includes non-reference match rate values ​​along a non-reference match rate axis 702 and allele frequency values ​​along an allele frequency axis 704. In particular, non-reference match rate axis 702 represents the accuracy of genotype call assignments for non-reference match rates ranging from 0 to 1.0 (e.g., 0 represents no match, and 1.0 represents full match). In graph 700, such non-reference match rate values ​​represent the quotient of (i) the true positive rate at which the sequencing system attributes the target variant, divided by (ii) the sum of the false positive rate, true positive rate, and false negative rate at which the sequencing system attributes the target variant, which can be expressed as TPR / FPR+TPR+FNR. Furthermore, allele frequency axis 704 represents the allele frequency (also referred to as carrier frequency) for the target variant ranging from 0.00 to 0.05.

[0103] According to the non-reference concordance rate axis 702 and the allele frequency axis 704 of the graph 700, the customized genotype assignment system 104 improves the accuracy of genotype call assignments for target variants as the number of genomic samples represented by the target variant reference panel increases. In particular, the non-reference concordance rate curve 706d for the customized genotype assignment system 104 using the first target variant reference panel representing 100 genomic samples shows the lowest non-reference concordance rate for assigning target variants removed across allele frequencies for the target variants. In contrast, the non-reference concordance rate curve 706a for the customized genotype assignment system 104 using the fourth target variant reference panel representing 2,500 genomic samples shows the highest non-reference concordance rate for assigning target variants removed across allele frequencies for the target variants. Indeed, for each of the non-reference match rate curves 706a, 706b, and 706c, the non-reference match rate increases with allele frequency before reaching a plateau with maximum match at an allele frequency of approximately 0.02.

[0104] Thus, in some embodiments, the customized genotype assignment system 104 can accurately assign genotype calls for target variants exhibiting a threshold carrier frequency of at least 2% by using a target variant reference panel representing 500 or more genomic samples. Indeed, as shown by the non-reference concordance curve 706a, the customized genotype assignment system 104 can accurately assign genotype calls for relatively less common target variants (e.g., having a carrier frequency of 2% or less) by using a target variant reference panel including 2,500 genomic samples. Furthermore, in some embodiments, the customized genotype assignment system 104 can accurately assign genotype calls for target variants exhibiting a threshold carrier frequency of at least 5% by using a target variant reference panel representing approximately 100 or more genomic samples. Indeed, as shown by the non-reference concordance curve 706d, the customized genotype assignment system 104 can accurately assign genotype calls for relatively more common target variants (e.g., having a carrier frequency of 5% or less) by using a target variant reference panel representing 100 genomic samples.

[0105] As described above, the customized genotype assignment system 104 can further utilize a target variant reference panel. According to one or more embodiments, FIG. 8 illustrates a customized genotype assignment system 104 that utilizes a target variant reference panel to assign a genotype call indicating the presence or absence of a target variant in a target genomic sample. In overview, the customized genotype assignment system 104 (i) identifies nucleotide reads for the target genomic sample, (ii) accesses a target variant reference panel that includes target variant indices within target variant positions for phased alleles of the genomic sample of different haplotypes, and (iii) assigns a genotype call for the target variant in the target genomic sample based on comparing the alleles of the haplotypes represented by the target variant reference panel to the nucleotide reads for the target genomic sample.

[0106] As shown in FIG. 8 , for example, the customized genotype assignment system 104 performs operation 802 of identifying nucleotide reads for a target genome sample. In some cases, for example, the customized genotype assignment system 104 receives data representing nucleotide reads for a genome sample sequenced by a sequencing device. Such data for the nucleotide reads includes sequences of nucleic acid base calls determined by the sequencing device. After receiving the read data, the customized genotype assignment system 104 can align the nucleotide reads with a reference genome. Based on the aligned nucleotide reads, the customized genotype assignment system 104 can determine one or more nucleic acid base calls for genomic coordinates and genomic regions of the target genome sample relative to the reference genome.

[0107] 8, the customized genotype assignment system 104 performs operation 806 of assigning a genotype call to the target variant based on a comparison of the nucleotide reads to the target variant reference panel. Illustratively, in one or more embodiments, the customized genotype assignment system 104 accesses the target variant reference panel 808, such as by accessing a VCF stored locally or on one or more client devices within the computing system 100. In one or more embodiments, the customized genotype assignment system 104 provides and / or receives the target variant reference panel 808 over a network.

[0108] As shown in Figure 8, target variant reference panel 808 includes filled circles representing target variant indices within target variant positions for phased alleles of genomic samples 810a, 810b, and 810c of different haplotypes. Target variant reference panel 808 further includes open or hollow circles representing marker variant indices for marker variants within the phased alleles of genomic samples 810a-810c. As shown, the phased alleles of genomic samples 810a-810c include different patterns indicative of different alleles corresponding to different haplotypes. Similarly, Figure 8 shows alleles of target genomic sample 812 including various patterns representing different alleles.

[0109] Based on a comparison of (i) a subset of nucleotide reads of the target genome sample 812 corresponding to the target genomic region for the target variant and (ii) alleles of genome samples 810a-810c in the target variant reference panel 808, the customized genotype assignment system 104 assigns a genotype call for the target genome sample 812. More specifically, in some embodiments, the customized genotype assignment system 104 assigns a genotype call corresponding to the genomic coordinates of the target genomic region based on marker variants surrounding or adjacent to the target genomic region for the target variant.

[0110] 8 , in one or more embodiments, operation 806 further includes operation 814, identifying SNPs within the nucleotide reads of the target genomic sample. More specifically, in one or more embodiments, the customized genotype assignment system 104 compares marker variants surrounding the target genomic region on the target genomic sample 812 to marker variants on genomic samples 810a-810c included in the target variant reference panel 808. Indeed, in one or more embodiments, the customized genotype assignment system 104 identifies marker variants within a threshold number of nucleic acid bases from the target variant. For example, in some cases, the customized genotype assignment system 104 identifies marker variants within a threshold number of nucleic acid bases upstream of the target genomic region (e.g., 50 base pairs, 200 base pairs, 500 base pairs) and / or within a threshold number of nucleic acid bases downstream from the target genomic region (e.g., 50 base pairs, 200 base pairs, 500 base pairs). As noted above, FIG. 8 shows marker variant indices for marker variants (eg, SNPs) as open or closed circles within phased alleles of genomic samples 810a-810c and target genomic sample 812.

[0111] To illustrate the comparison of marker variants, the customized genotype assignment system 104 can determine SNPs within genomic coordinates surrounding or adjacent to a target genomic region on the target genomic sample 812 and SNPs within genomic coordinates surrounding or adjacent to a target genomic region on genomic samples 810a-810c in the target variant reference panel 808. Based on the SNPs (or other marker variants) common between the haplotypes of the target genomic sample 812 and the haplotypes of genomic samples 810a-810c in the target variant reference panel 808, the customized genotype assignment system 104 statistically infers which nucleic acid bases or alleles are likely to be present within the target genomic region on the target genomic sample 812.

[0112] 8 , in some embodiments, operation 806 of assigning genotype calls includes operation 816 of determining phased alleles for target genomic sample 812. Illustratively, in one or more embodiments, customized genotype assignment system 104 phases nucleotide reads of target genomic sample 812 based on marker variants (e.g., SNPs) in the nucleotide reads of target genomic sample 812 and marker variants in genomic samples 810a-810c. By comparing marker variants and phasing nucleotide reads with respect to haplotypes in target variant reference panel 808, customized genotype assignment system 104 identifies alleles of target genomic sample 812 in the target genomic region that are also present in the maternal and paternal haplotypes of genomic samples 810a-810c.

[0113] For example, an allele in target genomic sample 812 contains the same marker variant as an allele in genomic sample 810c, as indicated by different patterns indicating different alleles in target variant reference panel 808. Because customized genotype assignment system 104 can identify alleles shared between target genomic sample 812 and one or more haplotypes of genomic samples 810a-810c and identify target variant indicators within the target variant positions for genomic samples 810a-810c in target variant reference panel 808, customized genotype assignment system 104 can generate a phased genotype call indicating the presence or absence of a target variant at a particular allele in target genomic sample 812. As indicated by the black circle representing the target variant indicator in target variant reference panel 808, customized genotype assignment system 104 can statistically infer that a particular allele in target genomic sample 812 contains a target variant because the corresponding allele in genomic sample 810c contains a target variant indicator at the target variant position. Indeed, by applying the haplotype phasing model and the genotype imputation model to the target variant reference panel 808, the customized genotype imputation system 104 can determine a phased genotype call indicating the presence or absence of a target variant in the allele of the target genome sample 812 corresponding to the maternal or paternal haplotype represented in the target variant reference panel 808.

[0114] As indicated immediately above, in one or more embodiments, the customized genotype assignment system 104 utilizes a haplotype phasing model to phase nucleotide reads from the target genomic sample 812. In one or more embodiments, the customized genotype assignment system 104 utilizes the Segmented Haplotype Estimation and Imputation Tool (SHAPEIT) to infer haplotypes from genotype data including the nucleotide reads of the target genomic sample 812 and the genomic sequences of genomic samples 810a-810c in the target variant reference panel 808. Illustratively, in one or more embodiments, the customized genotype assignment system 104 utilizes the SHAPEIT algorithm to perform a position-dependent Burrow Wheeler transform (PBWT) to efficiently select a set of associated haplotypes to be used to phase the nucleotide reads of the target genomic sample 812. Thus, the customized genotype assignment system 104 can preprocess and extract phase information from the set of associated haplotypes. In one or more embodiments, the customized genotype assignment system 104 can further utilize haplotype scaffold or parent haplotype data to phase nucleotide reads of the target genomic sample 812. Thus, the customized genotype assignment system 104 can utilize phase information from the set of related haplotypes, and optionally the haplotype scaffold or parent haplotype data, to write a VCF or BCF file that phases the target genomic sample 812. In one or more embodiments, the customized genotype assignment system 104 utilizes HTSlib to write the VCF or BCF file.

[0115] In some embodiments, for example, the customized genotype imputation system 104 uses SHAPEIT to phase haplotypes, as described in Olivier Delaneau, Jean-Francois Zagury et al., Scalable and Integrative Haplotype Estimation, Nat. Comm. (2019), which is incorporated herein by reference in its entirety.

[0116] Also as described above, in one or more embodiments, the customized genotype assignment system 104 applies a genotype assignment model, such as a hidden Markov model (HMM)-based genotype assignment model, to assign genotype calls for target regions corresponding to target variants. By way of example, in some embodiments, the customized genotype assignment system 104 can use the HMM-based genotype assignment model to identify associated haplotypes from genomic samples 810a-810c in the target variant reference panel 808. More specifically, the customized genotype assignment system 104 can utilize the HMM-based genotype assignment model to (i) compare marker variants corresponding to target genomic regions in the target genomic sample 812 with marker variants in haplotypes of the target genomic regions in genomic samples 810a-810c, and (ii) identify haplotypes that likely correspond to the target genomic regions present in the target genomic sample 812.

[0117] In one or more embodiments, the customized genotype imputation system 104 utilizes the Genotype Likelihood Imputation and Phasing mEthod (GLIMSSE) as a genotype imputation model, as described by Simone Rubinacci et al., "Efficient Phasing and Imputation of Low-coverage Sequencing Data Using Large Reference Panels," 53 Nature Genetics 120-126 (2021), which is incorporated herein by reference in its entirety. More specifically, in some embodiments, the customized genotype imputation system 104 uses GLIMPSE to determine posterior genotype likelihoods for target genomic regions corresponding to target variants in the target genomic sample 812. Indeed, in some embodiments, the customized genotype imputation system 104 performs SHAPEIT to phase nucleotide reads from the target genomic sample before performing GLIMPSE to impute genotype calls for the target variants based on the target variant reference panel.

[0118] As described above, in one or more embodiments, the customized genotype assignment system 104 generates a target variant reference panel that includes one or more target genomic regions (or genomic regions of interest) corresponding to the target variants and excludes other genomic coordinates or genomic regions. For example, in some embodiments, the customized genotype assignment system 104 restricts the target variant reference panel to include data representing haplotypes of a genomic sample corresponding to one or more target genomic regions corresponding to the target variants, but not data representing haplotypes outside the one or more target genomic regions. Indeed, in one or more embodiments, the customized genotype assignment system 104 includes data representing haplotypes from a genomic sample for multiple target genomic regions comprising different chromosomes in a target variant reference panel corresponding to multiple target variants. For example, the customized genotype assignment system 104 can generate a target variant reference panel that includes data representing different haplotypes corresponding to target variants of the CYP2D6 gene in a target genomic region (e.g., chr4:35149660-47004037). In some cases, the same target variant reference panel includes data representing different haplotypes corresponding to additional target variants for the RFC1 gene in additional target genomic regions (e.g., chr22:37149660 to 54004037).

[0119] Indeed, in one or more embodiments, the customized genotype imputation system 104 inputs data for such a target variant reference panel for only the target genomic region into a genotype imputation model (e.g., GLIMPSE). By reducing or eliminating unnecessary genomic regions and using a target variant reference panel that includes data limited to one or more target genomic regions, the customized genotype imputation system 104 uses less memory to store the target variant reference panel and reduces the computational time for running the genotype imputation model to impute genotype calls for the target variants.

[0120] As an alternative to GLIMPSE, in some embodiments, for example, the customized genotype imputation system 104 imputes haplotypes using a different HMM-based genotype imputation model (e.g., a model described in Genetic Variants Predictive of Cancer Risk, International Publication No. 2013 / 035 / 114(A1) (published March 14, 2013), or A. Kong et al., Detection of Sharing by Descent, Long-Range Phasing and Haplotype Imputation, Nat. Genet. 40, 1068-75 (2008)), both of which are incorporated herein by reference in their entireties. Additionally or alternatively, the customized genotype imputation system 104 imputes genotype calls using other available software, such as BEAGLE, MACH, or IMPUTE.

[0121] As further shown in Figure 8, the customized genotype assignment system 104 can optionally perform operation 818, which generates a prediction of whether the target genomic sample contains the target variant. Illustratively, in one or more embodiments, the customized genotype assignment system 104 can utilize the determined genotype call to generate a prediction of whether the target genomic sample contains a pathogenic variant at an allele present on the maternal haplotype or the paternal haplotype. As described below with respect to Figure 9, the customized genotype assignment system 104 can provide such a prediction to a client device via a graphical user interface.

[0122] In some embodiments, for example, the customized genotype assignment system 104 can utilize inheritance patterns associated with a condition or disease corresponding to a target variant to generate a prediction. Illustratively, the customized genotype assignment system 104 can determine whether a condition associated with a target variant is autosomal recessive, autosomal dominant, X-linked, Y-linked, codominant, or a variety of inheritance patterns. More specifically, the customized genotype assignment system 104 compares the inheritance patterns with the genotype call to generate a prediction. In some embodiments, the prediction indicates whether the target genomic sample is a carrier of the target variant at a particular allele, a case of the target variant at both alleles, or unaffected by the target variant at either allele.

[0123] After determining the imputed genotype calls, in one or more embodiments, the customized genotype assignment system 104 provides information about such imputed genotype calls for one or more target variants via a graphical user interface. According to one or more embodiments, FIG. 9 illustrates a client device 900 presenting a graphical user interface 901 containing information about the imputed genotype calls for the target variants. While FIG. 9 illustrates the graphical user interface 901 that is displayed when the client device 900 implements the computer-executable instructions of the customized genotype assignment system 104, rather than repeatedly referencing the computer-executable instructions that cause the client device 900 to perform specific operations for the customized genotype assignment system 104, the present disclosure describes the client device 900 or customized genotype assignment system 104 performing those operations in the following paragraphs.

[0124] 9 , for example, client device 900 provides data to target variant column 902, gene column 904, and carrier frequency column 906. Illustratively, target variant column 902 includes data identifying the target variant and a corresponding prediction. More specifically, client device 900 presents the genomic coordinates of the target variant and a prediction as to whether the target genomic sample contains the target variant (e.g., a pathogenic variant). Illustratively, in one or more embodiments, customized genotype assignment system 104 provides client device 900 with a prediction of whether a pathogenic variant is present in the target genomic sample at one or both alleles of the maternal and paternal haplotypes.

[0125] Therefore, based on the imputed genotype calls, the client device 900 can present a prediction regarding whether the target genomic sample is affected by one or more target variants. As shown in FIG. 9 , for example, the client device 900 presents a target variant column 902 including "Predicted: If" for a first target variant in the target genomic sample at genomic coordinate "chr4:39,287,456-39...." As shown in "Predicted: If," the customized genotype imputation system 104 predicts that the target genomic sample contains the first target variant in the RFC1 gene on both alleles. Thus, in some cases, the prediction indicates the target genomic sample's potential phenotype for the cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) spectrum. As further shown in FIG. 9 , the client device 900 presents a target variant column 902 including "Predicted: Carrier" for a second target variant in the target genomic sample at genomic coordinate "chr22:42,126,499-42...." As shown in "Predicted: Carrier," in some cases, the customized genotype assignment system 104 predicts that the target genomic sample contains a second target variant of the CYP2D6 gene on one allele. Thus, the prediction indicates that the target genomic sample carries a variant that is genetically indicative of neuroleptic malignant syndrome.

[0126] As further shown in FIG. 9 , the client device 900 presents annotations for the genes and carrier frequencies corresponding to the target variants and their corresponding predictions. For example, the client device 900 presents a gene column 904 including "RFC1" and "CYP2D6," which correspond to the predictions in the target variant column 902 for the first and second target variants, respectively. In addition to the specific gene identifications, the client device 900 presents carrier frequencies in a carrier frequency column 906. More specifically, the client device 900 presents a carrier frequency of 0.7% to 4% for the first target variant on the RFC1 gene and a carrier frequency of approximately 5% for the second target variant on the CYP2D6 gene. In some embodiments, the carrier frequencies represent the frequencies of the target variants from a genomic sample database or from metadata corresponding to a target variant reference panel. By providing predictions, genomic coordinates, genes, and carrier frequencies, the customized genotype assignment system 104 provides clinicians, test subjects, or others with important information indicating variant calls for specific genes.

[0127] 1-9, corresponding text, and examples provide several different methods, systems, devices, and non-transitory computer-readable media for a customized genotype assignment system 104. In addition to the above, one or more embodiments may also be described in terms of flowcharts including operations for achieving particular results, as shown in FIGS. 10-11. The operations in FIGS. 10-11 may be performed with more or fewer operations. Furthermore, operations may be performed in a different order. Furthermore, operations described herein may be repeated or performed in parallel with each other or with different instances of the same or similar operations.

[0128] As discussed above, Figure 10 shows a flowchart of a series of operations 1000 for generating a target variant reference panel in accordance with one or more embodiments. While Figure 10 shows operations according to one embodiment, alternative embodiments may omit, add, reorder, and / or modify any of the operations shown in Figure 10. The operations of Figure 10 may be performed as part of a method. Alternatively, a non-transitory computer-readable medium may contain instructions that, when executed by one or more processors, cause a computing device or system to perform the operations of Figure 10. In some embodiments, a system may perform the operations of Figure 10.

[0129] As shown in FIG. 10 , the series of operations 1000 includes an operation 1002 for generating a reference panel including marker variant indices corresponding to genomic samples of different haplotypes. In particular, operation 1002 can include generating a reference panel including marker variant indices for marker variants at genomic coordinates corresponding to genomic samples of different haplotypes. Specifically, in some cases, the at least one target variant position includes a target variant position for a target variant indices of a biallelic target variant. Further, in one or more embodiments, in some cases, the at least one target variant position includes multiple target variant positions for target variant indices of a multiallelic target variant.

[0130] Furthermore, in one or more embodiments, in some cases, the marker variant comprises a single nucleotide polymorphism (SNP).

[0131] As shown in FIG. 10 , the series of operations 1000 includes an operation 1004 for adding a target variant location to the reference panel, the target variant location indicating the presence or absence of the target variant in the genomic sample. In particular, operation 1004 may include adding at least one target variant location to the reference panel, the target variant location indicating the presence or absence of the target variant in the genomic sample. Specifically, in some cases, the target variant includes a repeat expansion. Additionally, in one or more embodiments, operation 1004 includes the target variant including a deletion, insertion, duplication, inversion, translocation, or copy number variation (CNV) transmitted within a population. Operation 1004 may further include the target variant satisfying one or more of a threshold carrier frequency, a threshold linkage disequilibrium (LD) with respect to a particular marker variant, or a threshold mutation rate.

[0132] Further, in one or more embodiments, the target variant comprises a variant of the replication factor C subunit 1 (RFC1) gene, the cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene, the cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene, the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, the survival motor neuron 1 (SMN1) gene, the survival motor neuron 2 (SMN2) gene, the glucosylceramide beta (GBA) gene, the blood group Rh(CE) (RHCE) gene, the lipoprotein (A) (LPA) gene, the fragile X mental retardation 1 (FMR1) gene, the hexosaminidase subunit alpha (HEXA) gene, the hemoglobin subunit alpha 1 (HBA1) gene, the hemoglobin subunit alpha 2 (HBA2) gene, or the hemoglobin subunit beta (HBB) gene.

[0133] 10 , the series of operations 1000 includes an operation 1006 for phasing alleles of a genomic sample based on the marker variants to determine the presence or absence of a target variant in the corresponding allele. In particular, operation 1006 may include phasing alleles of a genomic sample based on the marker variants to determine the presence or absence of a target variant in the corresponding allele present on the maternal haplotype and the paternal haplotype. Specifically, in some cases, phasing the alleles of a genomic sample includes phasing heterozygous alleles of a subset of the genomic sample.

[0134] As shown in FIG. 10 , the series of operations 1000 includes an operation 1008 for generating a target variant reference panel including a target variant index. In particular, operation 1008 can include generating a target variant reference panel including a target variant index within at least one target variant position for a phased allele of the genomic sample. Specifically, in some cases, generating the reference panel includes generating a phased reference panel including marker variant indexes for marker variants phased according to maternal and paternal haplotypes of the genomic sample. Further, in one or more embodiments, in some cases, the genomic samples of different haplotypes include genomic samples of different haplotypes that exhibit genetic diversity. In some cases, the target variant reference panel includes marker variant indexes for marker variants within the target genomic region for the target variant, and does not include additional marker variant indexes for additional marker variants outside the target genomic region.

[0135] Additionally, Figure 11 depicts a flowchart of a series of operations 1100 for utilizing a targeted variant reference panel to impute genotype calls, according to one or more embodiments. While Figure 11 depicts operations according to one embodiment, alternative embodiments may omit, add, reorder, and / or modify any of the operations depicted in Figure 11. The operations of Figure 11 may be performed as part of a method. Alternatively, a non-transitory computer-readable medium may include instructions that, when executed by one or more processors, cause a computing device or system to perform the operations of Figure 11. In some embodiments, a system may perform the operations of Figure 11.

[0136] 11, the series of operations 1100 includes an operation 1102 of identifying nucleotide reads for a target genetic sample. In particular, operation 1102 can include identifying nucleotide reads that correspond to the target genomic sample.

[0137] As shown in FIG. 11 , the series of operations 1100 includes operation 1104 for accessing a target variant reference panel including a target variant index. In particular, operation 1104 may include accessing a target variant reference panel including a target variant index within at least one target variant position for a phased allele of a genomic sample of a different haplotype. Specifically, in some cases, the target variant index indicates the presence or absence of a target variant at at least one target variant position for a phased allele of a genomic sample. In some cases, the target variant reference panel includes a marker variant index for a marker variant within a target genomic region relative to the target variant, and does not include additional marker variant indexes for additional marker variants outside the target genomic region.

[0138] 11 , the series of operations 1100 includes an operation 1106 for assigning a genotype call for a target variant in a target genomic sample based on a comparison of nucleotide reads to a target variant reference panel. In particular, operation 1106 may include assigning a genotype call for a target variant in a target genomic sample based on a comparison of nucleotide reads corresponding to the target genomic sample to the target variant reference panel. Specifically, operation 1106 may include determining phased alleles of the target genomic sample based on a comparison of nucleotide reads corresponding to the target genomic sample to the target variant reference panel, and assigning the genotype call by assigning a phased genotype call for the target variant in the target genomic sample based on the phased alleles of the target genomic sample.

[0139] Additionally, in one or more embodiments, operation 1106 includes attributing a genotype call to the target variant by generating a prediction of whether the target genomic sample contains the target variant. Further, in some embodiments, generating the prediction includes predicting whether the target genomic sample contains a pathogenic variant at an allele present on the maternal haplotype or the paternal haplotype.

[0140] Operation 1106 may further include assigning a genotype call by identifying one or more single nucleotide polymorphisms (SNPs) within the nucleotide reads corresponding to the target genomic sample as one or more marker variants in a target variant reference panel for the target variant, and determining a genotype call further based on the one or more SNPs within the nucleotide reads. Further, operation 1106 may include assigning a genotype call for the target variant by assigning a genotype call for a repeat extension. Further, operation 1106 may include assigning the genotype call utilizing a genotype assignment model.

[0141] The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. Particularly applicable techniques involve attaching nucleic acids to fixed positions within an array so that their relative positions do not change, and repeatedly imaging the array. For example, embodiments in which images are obtained in different color channels corresponding to different labels used to distinguish one nucleotide base type from another are particularly applicable. In some embodiments, the process of determining the nucleotide sequence of a target nucleic acid can be an automated process. A preferred embodiment involves sequencing-by-synthesis (SBS) technology.

[0142] SBS technology generally involves the enzymatic extension of nascent nucleic acid chain by repeatedly adding nucleotide to template chain.In the traditional method of SBS, a single nucleotide monomer can be provided to target nucleic acid in the presence of polymerase in each delivery.However, in the method described herein, in the presence of polymerase during delivery, two or more kinds of nucleotide monomers can be provided to target nucleic acid.

[0143] SBS can utilize nucleotide monomers with terminator moieties or nucleotide monomers lacking any terminator moiety. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides, as described in more detail below. In methods using nucleotide monomers without terminators, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. In SBS techniques utilizing nucleotide monomers with terminator moieties, the terminators can be effectively irreversible under the sequencing conditions used, as in conventional Sanger sequencing using dideoxynucleotides, or the terminators can be reversible, as in the sequencing method developed by Solexa (now Illumina, Inc.).

[0144] SBS techniques can use nucleotide monomers that have a label moiety or lack a label moiety. Therefore, incorporation events can be detected based on the properties of the label, such as the fluorescence of the label, the properties of the nucleotide monomer, such as molecular weight or charge, or by-products of nucleotide incorporation, such as the release of pyrophosphate. In embodiments in which two or more different nucleotides are present in the sequencing reagent, the different nucleotides can be distinguishable from one another, or alternatively, the two or more different labels can be distinguishable under the detection technique used. For example, the different nucleotides present in the sequencing reagent can have different labels, which can be distinguished using appropriate optical systems, as exemplified by the sequencing method developed by Solexa (now Illumina, Inc.).

[0145] A preferred embodiment is pyrosequencing, which detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M., and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375),363, U.S. Patent No. 6,210,991, U.S. Patent No. 6,258,568, and U.S. Patent No. 6,274,320, the disclosures of which are incorporated herein by reference in their entireties. In pyrosequencing, released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurase, and the level of generated ATP is detected via luciferase-generated photons. Nucleic acids to be sequenced can be attached to features in an array, and the array can be imaged to capture chemiluminescent signals generated by nucleotide incorporation into the array features. Images can be obtained after treating the array with a specific nucleotide type (e.g., A, T, C, or G). Images obtained after the addition of each nucleotide type differ in terms of which features in the array are detected. These differences in the images reflect the different sequence content of the features on the array. However, the relative positions of each feature remain unchanged in the image. Images can be stored, processed, and analyzed using the methods described herein.For example, images obtained after treating the array with each different nucleotide type can be processed in the same manner as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.

[0146] In another exemplary type of SBS, cyclic sequencing is achieved by stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels, as described, for example, in International Publication No. 04 / 018497 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated by reference. This approach has been commercialized by Solexa (now Illumina Inc.) and is also described in International Publication Nos. 91 / 06678 and 07 / 123,844, each of which is incorporated by reference herein. The availability of fluorescently labeled terminators, both of whose ends can be reversed and from which the fluorescent labels are cleaved, facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

[0147] Preferably, in reversible terminator-based sequencing embodiments, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example, by cleavage or degradation. Images can be taken after incorporation of the label into arrayed nucleic acid features. In certain embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array, with each nucleotide type bearing a spectrally distinct label. Four images can then be obtained, each using a detection channel selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, with images of the array being obtained between each addition step. In such embodiments, each image shows nucleic acid features incorporating a particular type of nucleotide. Because the sequence content of each feature is different, different features may or may not be present in different images. However, the relative positions of the features remain unchanged within the image. Images obtained from such reversible terminator-SBS methods can be stored, processed, and analyzed as described herein. Following the imaging step, the label can be removed, and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removing the label after detection in a particular cycle and before subsequent cycles has the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described below.

[0148] In certain embodiments, some or all of the nucleotide monomers can contain reversible terminators. In such embodiments, the reversible terminator / cleavable fluorophore can comprise a fluorophore attached to the ribose moiety via a 3' ester bond (Metzker, Genome Res. 15:1767-1776 (2005), which is incorporated herein by reference). Another approach separates the terminator chemistry from the cleavage of the 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 a reversible terminator that uses a small 3' allyl group to block extension but can be easily deblocked by brief treatment with a palladium catalyst. The fluorophore was attached to the group via a photocleavable linker that can be easily cleaved by 30 seconds of exposure to long-wavelength UV light. Therefore, either disulfide reduction or photocleavage can be used as a cleavable linker. Another approach to reversible termination is the use of a natural terminator followed by the placement of a bulky dye on the dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and / or electrostatic hindrance. The presence of one incorporation event prevents further binding unless the dye is removed. Cleavage of the dye removes the fluorophore, effectively reversing the terminus. Examples of modified nucleotides are also described in U.S. Pat. Nos. 7,427,673 and 7,057,026, the disclosures of which are incorporated herein by reference in their entireties.

[0149] Additional exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Application Publication No. 2007 / 0166705, U.S. Patent Application Publication No. 2006 / 0188901, U.S. Patent No. 7,057,026, U.S. Patent Application Publication No. 2006 / 0240439, U.S. Patent Application Publication No. 2006 / 0281109, WO 05 / 065814, U.S. Patent Application Publication No. 2005 / 0100900, WO 06 / 064199, WO 07 / 010,251, U.S. Patent Application Publication No. 2012 / 0270305, and U.S. Patent Application Publication No. 2013 / 0260372, the disclosures of which are incorporated herein by reference in their entireties.

[0150] Some embodiments may utilize detection of four different nucleotides using fewer than four different labels. For example, SBS may be performed using the methods and systems described in the incorporated document, U.S. Patent Application Publication No. 2013 / 0079232. As a first example, pairs of nucleotide types may be detected at the same wavelength but may be distinguished based on differences in intensity for one member of the pair or based on a change to one member of the pair (e.g., via chemical, photochemical, or physical modification) that results in the appearance or disappearance of a distinct signal compared to the signal detected for the other member of the pair. As a second example, three of the four different nucleotide types may be detected under certain conditions, while the fourth nucleotide type may be devoid of a detectable label under those conditions or may be minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid may be determined based on the presence of their corresponding signals, and incorporation of the fourth nucleotide type into a nucleic acid may be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type can include a label that is detected in two different channels, while the other nucleotide type is detected in no more than one of the channels. The foregoing three exemplary configurations 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 that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and second channels (e.g., dTTP having at least one label that is detected in both channels when excited by the first and / or second excitation wavelength), and a fourth nucleotide type that is not detected or minimally detected in either channel (e.g., unlabeled dGTP).

[0151] Furthermore, as described in incorporated document U.S. Patent Application Publication No. 2013 / 0079232, sequencing data can be obtained using a single channel. In such so-called single-dye sequencing methods, a first nucleotide type is labeled but the label is removed after the first image is generated, and a second nucleotide type is labeled only after the first image is generated. A third nucleotide type retains its label in both the first and second images, and a fourth nucleotide type remains unlabeled in both images.

[0152] Some embodiments may utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate and identify the incorporation of oligonucleotides. The oligonucleotides typically have different labels that correlate with the identity of specific nucleotides in the sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained after treating an array of nucleic acid features with labeled sequencing reagents. Each image shows nucleic acid features that incorporate a specific type of label. Because the sequence content of each feature varies, different features may or may not be present in different images, but the relative positions of the features remain constant within the image. Images obtained from ligation-based sequencing methods can be stored, processed, and analyzed as described herein. Exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Nos. 6,1069,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties.

[0153] Some embodiments can utilize nanopore sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35:917-925 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope." Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,892; Soni, GV & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K., "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR, "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties.)The data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein. Specifically, the data can be processed as images according to the exemplary processing of optical and other images described herein.

[0154] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing polymerase and a γ-phosphate-labeled nucleotide, as described, for example, in U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (each of which is incorporated herein by reference), or nucleotide incorporation can be detected using zero-mode waveguides, as described, for example, in U.S. Patent No. 7,315,019 (each of which is incorporated herein by reference), and fluorescent nucleotide analogs and engineered polymerases, as described, for example, in U.S. Patent No. 7,405,281 and U.S. Patent Application Publication No. 2008 / 0108082 (each of which is incorporated herein by reference). Illumination can be restricted to a zeptoliter-scale volume around the surface-tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, MJ et al., "Zero-mode waveguides for single-molecule analysis at high concentrations." Science, 299, 682-686 (2003); Lundquist, PM et al., "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al., "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties).Images resulting from such methods can be stored, processed, and analyzed as described herein.

[0155] Some SBS embodiments involve the detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on the detection of released protons can use commercially available electrical detectors and related technology from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082 (A1), 2009 / 0127589 (A1), 2010 / 0137143 (A1), or 2010 / 0282617 (A1), each of which is incorporated herein by reference. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be easily adapted to substrates used for detecting protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons used for detecting protons.

[0156] The SBS method described above can be advantageously performed in a multiplex format, allowing multiple different target nucleic acids to be manipulated simultaneously. In certain embodiments, the different target nucleic acids can be processed in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids are typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent binding, binding to beads or other particles, or binding to a surface-bound polymerase or other molecule. The array can contain a single copy of the target nucleic acid at each site (also referred to as a feature), or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be generated by amplification methods such as bridge amplification or emulsion PCR, which are described in more detail below.

[0157] The methods described herein can be used to detect, for example, at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 , 5,000,000 features / cm 2 Arrays having features of any of a variety of densities can be used, including 1000, ...2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000

[0158] An advantage of the methods described herein is that they provide rapid and efficient detection of multiple target nucleic acids in parallel. Accordingly, the present disclosure provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art, such as those exemplified above. Accordingly, the integrated system of the present disclosure can include fluidic components capable of delivering amplification and / or sequencing reagents to one or more immobilized DNA fragments, including components such as pumps, valves, reservoirs, and fluid lines. A flow cell can be configured and / or used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768(A1) and U.S. Patent Application No. 13 / 273,666, each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used in the amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system can be used to deliver sequencing reagents in the amplification methods described herein and in the sequencing methods exemplified above. Alternatively, an integrated system may include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and sequencing the nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina Inc., San Diego, CA) and the apparatus described in U.S. Patent Application No. 13 / 273,666, which is incorporated herein by reference.

[0159] The sequencing system described above sequences nucleic acid polymers present in a sample received by the sequencing device. As defined herein, the term "sample" and its derivatives are used in the broadest sense and include any sample, culture, etc. suspected of containing a target. In some embodiments, a sample includes DNA, RNA, PNA, LNA, chimeric, or hybrid forms of nucleic acid. A sample can include any biological, clinical, surgical, agricultural, air, or water sample containing one or more nucleic acids. The term also includes any isolated nucleic acid sample, such as genomic DNA, fresh-frozen, or formalin-fixed, paraffin-embedded nucleic acid sample. It is also contemplated that a sample can be derived from a single individual, a collection of nucleic acid samples from genetically related members, nucleic acid samples from genetically unrelated members, nucleic acid samples from a single individual (matched), such as a tumor sample and a normal tissue sample, or a sample from a single source containing two different forms of genetic material, such as maternal and fetal DNA obtained from a maternal subject, or the presence of contaminating bacterial DNA in a sample containing plant or animal DNA. In some embodiments, the source of nucleic acid material can include nucleic acid obtained from a newborn, for example, as typically used in newborn screening.

[0160] A nucleic acid sample can include high molecular weight material, such as genomic DNA (gDNA). A sample can include low molecular weight material, such as nucleic acid molecules obtained from FFPE or archived DNA samples. In another embodiment, the low molecular weight material includes enzymatically or mechanically fragmented DNA. A sample can include cell-free circulating DNA. In some embodiments, a sample can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissection, surgical resection, and other clinical or laboratory samples. In some embodiments, a sample can be an epidemiological, agricultural, forensic, or pathogenic sample. In some embodiments, a sample can include nucleic acid molecules obtained from animals, such as humans or mammalian sources. In another embodiment, a sample can include nucleic acid molecules obtained from non-mammalian sources, such as plants, bacteria, viruses, or fungi. In some embodiments, the source of the nucleic acid molecules can be an archived or extinct sample or species.

[0161] Additionally, the methods and compositions disclosed herein may be useful for amplifying nucleic acid samples with low-quality nucleic acid molecules, such as degraded and / or fragmented genomic DNA from forensic samples. In one embodiment, a forensic sample may include nucleic acids obtained from a crime scene, from a missing persons DNA database, from a laboratory associated with a forensic investigation, or may include forensic samples obtained by law enforcement agencies, one or more military services, or any such personnel. A nucleic acid sample may be crude DNA, including purified samples or lysates, derived from, for example, oral swabs, paper, cloth, or other substrates that may be impregnated with saliva, blood, or other bodily fluids. Thus, in some embodiments, a nucleic acid sample may contain small amounts of DNA or fragmented portions of DNA, such as genomic DNA. In some embodiments, target sequences may be present in one or more bodily fluids, including, but not limited to, blood, sputum, plasma, semen, urine, and serum. In some embodiments, target sequences may be obtained from hair, skin, tissue samples, autopsies, or remains of victims. In some embodiments, nucleic acids containing one or more target sequences may be obtained from deceased animals or humans. In some embodiments, the target sequence can comprise nucleic acid obtained from non-human DNA, such as microbial, plant, or entomological DNA. In some embodiments, the target sequence or amplified target sequence is for human identification purposes. In some embodiments, the present disclosure generally relates to methods for identifying features of forensic samples. In some embodiments, the present disclosure generally relates to human identification methods using one or more target-specific primers disclosed herein or one or more target-specific primers designed using the primer design criteria outlined herein. In one embodiment, a forensic sample or human identification sample comprising at least one target sequence can be amplified using any one or more of the target-specific primers disclosed herein or using the primer criteria outlined herein.

[0162] Components of the customized genotype assignment system 104 may include software, hardware, or both. For example, components of the customized genotype assignment system 104 may include one or more instructions stored on a computer-readable storage medium and executable by a processor of one or more computing devices (e.g., user client device 108, client device 600). When executed by one or more processors, the computer-executable instructions of the customized genotype assignment system 104 can cause the computing device to perform the bubble detection methods described herein. Alternatively, components of the customized genotype assignment system 104 may include hardware, such as a dedicated processing device, for performing a particular function or group of functions. Additionally or alternatively, components of the customized genotype assignment system 104 may include a combination of computer-executable instructions and hardware.

[0163] Additionally, components of the customized genotype assignment system 104 that perform the functions described herein with respect to the customized genotype assignment system 104 may be implemented, for example, as part of a standalone application, as a module of an application, as a plug-in to an application, as a library function that can be called by other applications, and / or as a cloud computing model. Thus, components of the customized genotype assignment system 104 may be implemented as part of a standalone application on a personal computing device or a mobile device. Additionally, or alternatively, components of the customized genotype assignment system 104 may be implemented in any application that provides sequencing services, including, but not limited to, Illumina BaseSpace, Illumina DRAGEN, Illumina TruSight software, ExpansionHunter, or Graph ExpansionHunter. "Illumina," "BaseSpace," "DRAGEN," "TruSight," "ExpansionHunter," and "Graph ExpansionHunter" are registered trademarks or trademarks of Illumina, Inc. in the United States and / or other countries.

[0164] Embodiments of the present disclosure may include or utilize special purpose or general purpose computers, including computer hardware such as, for example, one or more processors and system memory, as discussed in more detail below. Embodiments within the scope of the present disclosure also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. In particular, one or more of the processes described herein may be embodied in a non-transitory computer-readable medium and implemented at least in part as instructions executable by one or more computing devices (e.g., any of the media content access devices described herein). Generally, a processor (e.g., a microprocessor) receives instructions from a non-transitory computer-readable medium (e.g., memory, etc.) and executes those instructions, thereby performing one or more processes, including one or more of the processes described herein.

[0165] Computer-readable media may be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer-readable media that store computer-executable instructions are non-transitory computer-readable storage media (devices). Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example, and not limitation, embodiments of the present disclosure may include at least two distinctly different kinds of computer-readable media: non-transitory computer-readable storage media (devices) and transmission media.

[0166] Non-transitory computer-readable storage media (devices) include RAM, ROM, EEPROM, CD-ROM, solid state drives (SSD) (e.g., based on RAM), flash memory, phase-change memory (PCM), other types of memory, other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code means in the form of computer-executable instructions or data structures and that can be accessed by a general-purpose or special-purpose computer.

[0167] A "network" is defined as one or more data links that enable the transport of electronic data between computer systems and / or modules and / or other electronic devices. When information is transferred or provided to a computer over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless), the computer properly views the connection as a transmission medium. Transmission media can be used to transport desired program code means in the form of computer-executable instructions or data structures and can include networks and / or data links that can be accessed by a general-purpose or special-purpose computer. Combinations of the above should also be included within the scope of computer-readable media.

[0168] Furthermore, upon reaching various computer system components, program code means in the form of computer-executable instructions or data structures may be automatically transferred from transmission media to non-transitory computer-readable storage media (devices) (or vice versa). For example, computer-executable instructions or data structures received over a network or data link may be buffered in RAM within a network interface module (e.g., a NIC) and then eventually transferred to computer system RAM and / or less volatile computer storage media (devices) within the computer system. Thus, it should be understood that non-transitory computer-readable storage media (devices) may be included in computer system components that also (or even primarily) utilize transmission media.

[0169] Computer-executable instructions include, for example, instructions and data that, when executed by a processor, cause a general-purpose computer, a special-purpose computer, or a special-purpose processing device to perform a certain function or group of functions. In some embodiments, computer-executable instructions are executed on a general-purpose computer to transform the general-purpose computer into a special-purpose computer that implements elements of the present disclosure. Computer-executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code. While the subject matter has been described in language specific to structural features and / or methodological acts, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the described features or acts described above. Rather, the described features and acts are disclosed as example forms of implementing the claims.

[0170] Those skilled in the art will appreciate that the present disclosure may be implemented in networked computing environments having many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, cellular phones, PDAs, tablets, pagers, routers, switches, etc. The present disclosure may also be implemented in distributed system environments where tasks are performed by both local and remote computer systems that are linked through a network (either by hardwired data links, wireless data links, or a combination of hardwired and wireless data links). In a distributed system environment, program modules may be located in both local and remote memory storage devices.

[0171] Embodiments of the present disclosure may also be implemented in a cloud computing environment. As used herein, "cloud computing" is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources. For example, cloud computing may be used in markets to provide ubiquitous, convenient, on-demand access to a shared pool of configurable computing resources. The shared pool of configurable computing resources can be quickly configured through virtualization, exposed with low management effort or service provider interaction, and then scaled accordingly.

[0172] Cloud computing models can consist of various characteristics, such as, for example, on-demand self-service, wide area network access, resource pooling, rapid elasticity, and measured service. Cloud computing models can also expose various service models, such as, for example, Software as a Service (SaaS), Platform as a Service (PaaS), and Infrastructure as a Service (IaaS). Cloud computing models can also be deployed using different deployment models, such as private cloud, community cloud, public cloud, and hybrid cloud. As used herein and in the claims, a "cloud computing environment" is an environment in which cloud computing is employed.

[0173] FIG. 12 shows a block diagram of a computing device 1200 that may be configured to perform one or more of the processes described above. It will be understood that one or more computing devices, such as computing device 1200, may implement the genotype assignment system 104 and the sequencing system 106. As shown by FIG. 12, computing device 1200 may include a processor 1202, a memory 1204, a storage device 1206, an I / O interface 1208, and a communication interface 1210, and may be communicatively coupled via a communication infrastructure 1212. In particular embodiments, computing device 1200 may include fewer or more components than those shown in FIG. 12. The following paragraphs describe in more detail the components of computing device 1200 shown in FIG. 12.

[0174] In one or more embodiments, processor 1202 includes hardware for executing instructions, such as those comprising a computer program. By way of example and not limitation, to execute instructions for dynamically modifying a workflow, processor 1202 may retrieve (or fetch), decode, and execute instructions from an internal register, an internal cache, memory 1204, or storage device 1206. Memory 1204 may be volatile or non-volatile memory used to store data, metadata, and programs for execution by the processor. Storage device 1206 includes storage, such as a hard disk, flash disk drive, or other digital storage device, for storing data or instructions for implementing the methods described herein.

[0175] I / O interface 1208 enables a user to provide input to, receive output from, and otherwise transfer data to and receive data from computing device 1200. I / O interface 1208 may include a mouse, a keypad or keyboard, a touchscreen, a camera, an optical scanner, a network interface, a modem, other known I / O devices, or a combination of such I / O interfaces. I / O interface 1208 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 particular embodiments, I / O interface 1208 is configured to provide graphical data to a display for presentation to a user. The graphical data may represent one or more graphical user interfaces and / or any other graphical content that may be useful in a particular implementation.

[0176] Communications interface 1210 may include hardware, software, or both. In any case, communications interface 1210 may provide one or more interfaces for communications (e.g., packet-based communications, etc.) between computing device 1200 and one or more other computing devices or networks. By way of example and not limitation, communications interface 1210 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.

[0177] Additionally, communication interface 1210 can facilitate communication with various types of wired or wireless networks. Communication interface 1210 can also facilitate communication using various communication protocols. Communication infrastructure 1212 may also include hardware, software, or both that couple components of computing device 1200 to one another. For example, communication interface 1210 can use one or more networks and / or protocols to enable multiple computing devices connected by a particular infrastructure to communicate with each other to perform one or more aspects of the processes described herein. By way of example, a sequencing process can enable multiple devices (e.g., client device, sequencing device, and server device) to exchange information such as sequencing data and error notifications.

[0178] In the foregoing specification, the present disclosure has been described with reference to specific exemplary embodiments thereof. Various embodiments and aspects of the present disclosure will be described with reference to the details discussed herein, and the accompanying drawings illustrate various embodiments. The above description and drawings are illustrative of the present disclosure and should not be construed as limiting the disclosure. Numerous specific details are set forth to provide a thorough understanding of various embodiments of the present disclosure.

[0179] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects as illustrative only and not restrictive. For example, methods described herein may be implemented using fewer or more steps / actions, or the steps / actions may be performed in a different order. Additionally, steps / actions described herein may be repeated or performed in parallel with each other, or with different occurrences of the same or similar steps / actions. The scope of the present application is therefore indicated by the appended claims, rather than the foregoing description. All changes that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

Claims

1. 1. A computer-implemented method comprising: generating a reference panel comprising marker variant indices for marker variants at genomic coordinates corresponding to genomic samples of different haplotypes; adding at least one target variant position to the reference panel, the target variant position being indicative of the presence or absence of the target variant within the genomic sample; phasing the alleles of the genomic sample based on the marker variants to determine the presence or absence of the target variants in corresponding alleles present on maternal and paternal haplotypes; generating a target variant reference panel comprising a target variant index within the at least one target variant position for the phased allele of the genomic sample; 20. A computer-implemented method comprising:

2. 2. The computer-implemented method of claim 1, wherein the at least one target variant position comprises a target variant position for a target variant index of a biallelic target variant.

3. 2. The computer-implemented method of claim 1, wherein the at least one target variant position comprises a plurality of target variant positions for a target variant index of a multi-allelic target variant.

4. 2. The computer-implemented method of claim 1, wherein phasing the alleles of the genomic sample comprises phasing heterozygous alleles of a subset of the genomic sample.

5. The computer-implemented method of claim 1 , wherein the marker variant comprises a single nucleotide polymorphism (SNP).

6. 2. The computer-implemented method of claim 1, wherein generating the reference panel comprises generating a phased reference panel comprising the marker variant indices for marker variants phased according to the maternal haplotypes and the paternal haplotypes of the genomic sample.

7. The computer-implemented method of claim 1 , wherein the genomic samples of different haplotypes comprise genomic samples of different haplotypes that represent genetic diversity.

8. The computer-implemented method of claim 1 , wherein the target variant is a repeat expansion.

9. 2. The computer-implemented method of claim 1, wherein the target variant comprises a deletion, insertion, duplication, inversion, translocation, or copy number variation (CNV) that is transmitted within a population.

10. The target variant is a gene encoding a replication factor C subunit 1 (RFC1) gene, a cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene, a cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene, a cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, a survival motor neuron 1 (SMN1) gene, a survival motor neuron 2 (SMN2) gene, a glucosylceramide 2. The computer-implemented method of claim 1, comprising a variant of the beta (GBA) gene, blood group Rh (CE) (RHCE) gene, lipoprotein (A) (LPA) gene, fragile X mental retardation 1 (FMR1) gene, hexosaminidase subunit alpha (HEXA) gene, hemoglobin subunit alpha 1 (HBA1) gene, hemoglobin subunit alpha 2 (HBA2) gene, or hemoglobin subunit beta (HBB) gene.

11. 10. The computer-implemented method of claim 1, wherein the target variant satisfies one or more of a threshold carrier frequency, a threshold linkage disequilibrium (LD) with respect to a particular marker variant, or a threshold mutation rate.

12. 1. A system comprising: at least one processor; a non-transitory computer-readable medium, the non-transitory computer-readable medium, when executed by the at least one processor, providing the system with: identifying nucleotide reads corresponding to the target genomic sample; accessing a target variant reference panel comprising target variant indices within at least one target variant position for phased alleles of the genomic samples of different haplotypes; assigning a genotype call for a target variant in the target genomic sample based on a comparison of the nucleotide reads corresponding to the target genomic sample to the target variant reference panel; A system that executes the following.

13. 13. The system of claim 12, wherein the target variant indicator indicates the presence or absence of the target variant at the at least one target variant position for the phased allele of the genomic sample.

14. When executed by the at least one processor, the system: determining phased alleles of the target genomic sample based on a comparison of the nucleotide reads corresponding to the target genomic sample to the target variant reference panel; 13. The system of claim 12, further comprising instructions to: impute a phased genotype call for the target variant in the target genomic sample based on the phased alleles of the target genomic sample.

15. 13. The system of claim 12, further comprising instructions that, when executed by the at least one processor, cause the system to impute the genotype call of the target variant by generating a prediction of whether the target genomic sample contains the target variant.

16. 16. The system of claim 15, further comprising instructions that, when executed by the at least one processor, cause the system to generate the prediction by predicting whether the target genomic sample contains a pathogenic variant at an allele present on a maternal haplotype or a paternal haplotype.

17. When executed by the at least one processor, the system: identifying one or more single nucleotide polymorphisms (SNPs) within the nucleotide reads corresponding to the target genomic sample as one or more marker variants within the target variant reference panel for the target variant; determining the genotype call further based on the one or more SNPs within the nucleotide reads; 13. The system of claim 12, further comprising instructions to impute a genotype call by executing:

18. 13. The system of claim 12, further comprising instructions that, when executed by the at least one processor, cause the system to impute the genotype call for the target variant by imputing a genotype call for a repeat extension.

19. 13. The system of claim 12, further comprising instructions that, when executed by the at least one processor, cause the system to utilize a genotype imputation model to impute the genotype call.

20. A non-transitory computer-readable medium that, when executed by at least one processor, causes a computing device to: identifying nucleotide reads corresponding to the target genomic sample; accessing a target variant reference panel comprising a target variant index within at least one target variant position for a phased allele of the genomic sample; assigning a genotype call for a target variant in the target genomic sample based on a comparison of the nucleotide reads corresponding to the target genomic sample to the target variant reference panel; A non-transitory computer-readable medium storing instructions for causing a

21. 21. The non-transitory computer-readable medium of Claim 20, wherein the target variant indicator indicates the presence or absence of the target variant at the at least one target variant position for the phased allele of a genomic sample.

22. When executed by the at least one processor, the computing device: determining phased alleles of the target genomic sample based on the comparison of the nucleotide reads corresponding to the target genomic sample to the target variant reference panel; assigning a phased genotype call for the target variant in the target genomic sample based on the phased alleles of the target genomic sample; 21. The non-transitory computer-readable medium of claim 20, further comprising instructions to cause execution of:

23. 21. The non-transitory computer-readable medium of Claim 20, further comprising instructions that, when executed by the at least one processor, cause the computing device to impute the genotype call for the target variant by generating a prediction of whether the target genomic sample contains the target variant.

24. When executed by the at least one processor, the computing device is provided with a set of genes encoding a replication factor C subunit 1 (RFC1) gene, a cytochrome P450 family 2 subfamily D member 6 (CYP2D6) gene, a cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene, a cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, a survival motor neuron 1 (SMN1) gene, a survival motor neuron 2 (SMN2) gene, a glucosylceramide beta (GBA) gene, a blood 21. The non-transitory computer-readable medium of claim 20, further comprising instructions for imputing the genotype call to the target variant by imputing the genotype call to the Rh(CE) (RHCE) gene, the lipoprotein(A) (LPA) gene, the fragile X mental retardation 1 (FMR1) gene, the hexosaminidase subunit alpha (HEXA) gene, the hemoglobin subunit alpha 1 (HBA1) gene, the hemoglobin subunit alpha 2 (HBA2) gene, or the hemoglobin subunit beta (HBB) gene.

25. 21. The non-transitory computer-readable medium of Claim 20, wherein the target variant reference panel comprises marker variant indices for marker variants within a target genomic region for the target variant, and does not comprise additional marker variant indices for additional marker variants outside the target genomic region.

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