DETERMINING PATHOGENIC RFC1 EXPANSIONS FROM SEQUENCING DATA

Disclosed herein include systems, devices, and methods for determining repeat expansion status (e.g., pathogenic, carrier, and benign) of a locus of a gene of interest (e.g., at, or at about, chr4:39348424 of hg38 for RFC1). After aligning sequence reads to a sequence graph, the number of occurrences of repeat sequences satisfying predetermined criteria and the frequency of a pathogenic repeat sequence can be determined, which are in turn used to determine a repeat expansion status.

BACKGROUND

Field

This disclosure relates generally to the field of processing sequence data, and more particularly to determining repeats.

Background

A biallelic intronic AAGGG repeat expansion in the replication factor C subunit (RFC1) gene can cause familial cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and late-onset ataxia. Current diagnosis methods of this pathogenic AAGGG expansion are time consuming and cannot be performed in large-scale. One method is clinical whole genome sequencing (cWGS) screening by manual examination of reads from alignment. However, in this high GC genomic region, the base quality of pair-end reads drops, which makes manual examination more difficult and error-prone, especially when there are pathogenic AAGGGs or other high-GC repeat patterns. There is a need for an automated and accurate method to identify this pathogenic expansion from sequencing data, including cWGS data.

SUMMARY

Disclosed herein include methods for determining a replication factor C subunit 1 (RFC1) repeat expansion status. In some embodiments, a method for determining RFC1 repeat expansion status is under control of a processor (such as a hardware processor or a virtual processor) and comprises: (a) receiving a plurality of sequence reads generated from a sample obtained from a subject. The method can comprise: (b) aligning the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of RFC1. The sequence graph can comprise a repeat sequence representation flanked by non-repeat sequences of the locus of RFC1. The plurality of aligned sequence reads can comprise the plurality of sequence reads and alignments of the plurality of sequence reads to the sequence graph. The method can comprise: (c) determining a number of occurrences of a plurality of repeat sequences in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold and a first quality threshold. The method can comprise: (d) determining a frequency indication of a number of occurrences of a pathogenic repeat sequence relative to a total number of occurrences of the plurality of repeat sequences. The method can comprise: (e) determining a status of a repeat expansion at the locus of RFC1 of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences.

In some embodiments, the method comprises: determining the subject has zero, one, or two alleles with a repeat expansion at the locus of RFC1 using the plurality of aligned sequence reads. In some embodiments, the repeat expansion at the locus of RFC1 is at about chr4:39348424 of hg38, or a corresponding position of another reference genome sequence.

In some embodiments, the status of the repeat expansion at the locus of RFC1 is a pathogenic status, a carrier status, or a benign status. In some embodiments, the repeat expansion is associated with or causes a disease. The disease can be cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). In some embodiments, the method comprises: confirming the status of the repeat expansion at the locus of RFC1 of the subject using one or more diagnosis methods. The one or more diagnosis methods can comprise polymerase chain reaction (PCR) and Sanger sequencing, southern blots, and linkage analysis.

In some embodiments, the subject has two alleles with repeat expansion at the locus of RFC1. In some embodiments, determining the status of the repeat expansion at the locus of RFC1 comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than a first status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the status of the repeat expansion at the locus of RFC1 as pathogenic status.

In some embodiments, determining the status of the repeat expansion at the locus of RFC1 comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold and greater than or equal to a second status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprises: determining the status of the repeat expansion at the locus of RFC1 as carrier status.

In some embodiments, determining the status of the repeat expansion at the locus of RFC1 comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold and greater than or equal to a second status threshold. Determining the status of the repeat expansion at the locus of RFC1 comprises can comprise: determining a frequency indication of a number of occurrences of the pathogenic repeat sequence and a sequence with a high sequence similarity to the pathogenic repeat sequence is greater than a third status threshold. For example, the pathogenic repeat sequence and the sequence similar to the pathogenic repeat sequence can differ by one base. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining a frequency indication of a number of occurrences of the pathogenic sequence is greater than a fourth status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the status of the repeat expansion at the locus of RFC1 as carrier status.

In some embodiments, determining the status of the repeat expansion at the locus of RFC1 comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than a second status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the status of the repeat expansion at the locus of RFC1 as benign status.

In some embodiments, the subject has one allele of RFC1 with repeat expansion at the locus of RFC1. In some embodiments, determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads comprises: determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than a first status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the status of the repeat expansion at the locus of RFC1 as pathogenic status.

In some embodiments, determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads comprises: determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold. Determining the status of the repeat expansion at the locus of RFC1 can comprise: determining the status of the repeat expansion at the locus of RFC1 as carrier status.

In some embodiments, the method comprises: selecting the aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 based on the alignments of the aligned sequence reads. In some embodiments, the aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 comprise the aligned sequence reads that are (i) in-repeat reads or (ii) flanking reads each with an overlap to the repeat expansion at the locus of RFC1 greater than a repeat expansion overlap threshold. The repeat expansion overlap threshold can be about 60 base pairs. The aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 can comprise (i) all the aligned sequence reads that are in-repeat reads and (ii) not all of the aligned sequence reads that are flanking reads.

In some embodiments, the subject has zero allele with repeat expansion at the locus of RFC1. The status of the repeat expansion at the locus of RFC1 can be benign status.

In some embodiments, the repeat expansion at the locus of RFC1 comprises greater than a threshold total copies of one or more repeat sequences. The threshold total copies of one or more repeat sequences can be 20 total copies of one or more repeat sequences. In some embodiments, each of the plurality of repeat sequences has a number of occurrences greater than or equal to the first occurrence threshold with each occurrence having a number of bases each having a quality score greater than or equal to the first quality threshold. The first occurrence threshold can be 2. The first quality threshold can be about 20. The number of bases of the repeat sequence each having a quality score greater than the first quality threshold can be 5. In some embodiments, each of the occurrences has a number of bases each having a quality score greater than or equal to a second quality threshold. The second quality threshold can be about 20. The number of bases of each of the occurrences having a quality score greater than the second quality threshold can be 3.

In some embodiments, the repeat sequence representation is degenerate. The repeat sequence representation can be AARRG. The repeat sequence representation can be at least 5 bases in length. The repeat sequence representation can be 5 bases in length. The repeat sequence representation can be 6 bases in length. Each of the plurality of repeat sequences can be at least 5 bases in length. Each of the plurality of repeat sequences can be 5 bases in length. Each of the plurality of repeat sequences can be 6 bases in length. The pathogenic repeat sequence can be AAGGG or ACAGG. The pathogenic repeat sequence can have a GC content of at least 60%.

In some embodiments, the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is a percentage of the number of occurrences of the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences can be a ratio of the number of occurrences of the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

In some embodiments, the plurality of sequence reads is aligned to the locus of RFC1. In some embodiments, receiving the plurality of sequence reads generated from the sample obtained comprises: aligning a second plurality of sequence reads comprising the plurality of sequence reads to a reference genome sequence. Receiving the plurality of sequence reads generated from the sample obtained can comprise: selecting the plurality of sequence reads from the second plurality of sequence reads, wherein the plurality of sequence reads is aligned to the locus of RFC1.

In some embodiments, the plurality of sequence reads comprises sequence reads that are about 100 base pairs to about 1000 base pairs in length each. The plurality of sequence reads can comprise paired-end sequence reads. The plurality of sequence reads can comprise single-end sequence reads. The plurality of sequence reads can be generated by targeted sequencing and/or whole genome sequencing (WGS), optionally wherein the WGS is clinical WGS (cWGS). In some embodiments, the sample comprises cells, cell-free DNA, cell-free fetal DNA, amniotic fluid, a blood sample, a biopsy sample, or a combination thereof. In some embodiments, the reference genome sequence comprises a reference human genome sequence. The subject can be a human subject.

Disclosed herein include systems for determining a repeat expansion status of a gene of interest. In some embodiments, a system for determining repeat expansion status of a gene of interest comprising: non-transitory memory configured to store executable instructions; and a processor (e.g., a hardware processor or a virtual processor) in communication with the non-transitory memory. The processor can be programmed by the executable instructions to perform: (a) receiving a plurality of sequence reads generated from a sample obtained from a subject. The processor can be programmed by the executable instructions to perform: (b) aligning the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of a gene of interest. The sequence graph can comprise a repeat sequence representation flanked by non-repeat sequences of the locus of the gene. The plurality of aligned sequence reads can comprise the plurality of sequence reads. The plurality of aligned sequence reads can comprise alignments of the plurality of sequence reads to the sequence graph. The processor can be programmed by the executable instructions to perform: (c) determining a number of occurrences of a plurality of repeat sequences in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold and a first quality threshold. The processor can be programmed by the executable instructions to perform: (d) determining a frequency indication of a number of occurrences of a pathogenic repeat sequence relative to a total number of occurrences of the plurality of repeat sequence. The processor can be programmed by the executable instructions to perform: (e) determining a status of a repeat expansion at the locus of the gene of interest of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences.

In some embodiments, the gene of interest is replication factor C subunit 1 (RFC1). The locus of the gene of interest with the repeat expansion is at about chr4:39348424 of hg38, or a corresponding position of another reference genome sequence. The repeat expansion can be associated with or causes a disease. The disease can be cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). The repeat sequence representation can be AARRG. The pathogenic repeat sequence can be AAGGG or ACAGG.

In some embodiments, the processor is programmed by the executable instructions to perform: determining the subject has zero, one, or two alleles with a repeat expansion at the locus of the gene of interest using the plurality of aligned sequence reads. In some embodiments, the status of the repeat expansion at the locus of the gene of interest is a pathogenic status, a carrier status, or a benign status. The repeat expansion can be associated with or causes a disease. The disease can be a neurologic disease. In some embodiments, the processor is programmed by the executable instructions to perform: receiving conformation of the status of the repeat expansion at the locus of the gene of interest of the subject determined using one or more diagnosis systems. The one or more diagnosis systems can comprise polymerase chain reaction (PCR) and Sanger sequencing, southern blots, and linkage analysis.

In some embodiments, the subject has two alleles with repeat expansion at the locus of the gene of interest. In some embodiments, determining the status of the repeat expansion at the locus of the gene of interest comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than a first status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as pathogenic status.

In some embodiments, determining the status of the repeat expansion at the locus of the gene of interest comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold and greater than or equal to a second status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as carrier status.

In some embodiments, determining the status of the repeat expansion at the locus of the gene of interest comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold and greater than or equal to a second status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining a frequency indication of a number of occurrences of the pathogenic repeat sequence and a sequence with a high sequence similarity to the pathogenic repeat sequence is greater than a third status threshold. For example, the pathogenic repeat sequence and the sequence similar to the pathogenic repeat sequence differs by one base. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining a frequency indication of a number of occurrences of the pathogenic sequence is greater than a fourth status threshold Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as carrier status.

In some embodiments, determining the status of the repeat expansion at the locus of the gene of interest comprises: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than a second status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as benign status.

In some embodiments, the subject has one allele of the gene of interest with repeat expansion at the locus of the gene of interest. In some embodiments, determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads comprises: determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than a first status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as pathogenic status.

In some embodiments, wherein determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads comprise: determining the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to a first status threshold. Determining the status of the repeat expansion at the locus of the gene of interest can comprise: determining the status of the repeat expansion at the locus of the gene of interest as carrier status.

In some embodiments, the processor is programmed by the executable instructions to perform: selecting the aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest based on the alignments of the aligned sequence reads. In some embodiments, the aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest comprise the aligned sequence reads that are (i) in-repeat reads or (ii) flanking reads each with an overlap to the repeat expansion at the locus of the gene of interest greater than a repeat expansion overlap threshold, optionally wherein the repeat expansion overlap threshold is about 60 base pairs. The aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest can comprise (i) all the aligned sequence reads that are in-repeat reads and (ii) not all of the aligned sequence reads that are flanking reads.

In some embodiments, the subject has zero allele with repeat expansion at the locus of the gene of interest. The status of the repeat expansion at the locus of the gene of interest can be benign status.

In some embodiments, the repeat expansion at the locus of the gene of interest comprises greater than a threshold total copies of one or more repeat sequences. The threshold total copies of one or more repeat sequences can be 20 total copies of one or more repeat sequences. In some embodiments, each of the plurality of repeat sequences has a number of occurrences greater than or equal to the first occurrence threshold with each occurrence having a number of bases each having a quality score greater than or equal to the first quality threshold. The first occurrence threshold can be 2. The first quality threshold can be about 20. The number of bases of the repeat sequence each having a quality score greater than the first quality threshold can be 5. In some embodiments, each of the occurrences has a number of bases each having a quality score greater than or equal to a second quality threshold. The second quality threshold can be about 20. The number of bases of each of the occurrences having a quality score greater than the second quality threshold can be 3.

In some embodiments, the repeat sequence representation is degenerate. The repeat sequence representation and/or each of the plurality of repeat sequences can be at least 5 bases

in length. The repeat sequence representation and/or each of the plurality of repeat sequences can be 5 bases in length. The repeat sequence representation and/or each of the plurality of repeat sequences can be 6 bases in length. In some embodiments, the pathogenic repeat sequence has a GC content of at least 60%.

In some embodiments, wherein the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is a percentage of the number of occurrences of the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences or a ratio of the number of occurrences of the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

In some embodiments, the plurality of sequence reads is aligned to the locus of the gene of interest. In some embodiments, receiving the plurality of sequence reads generated from the sample obtained comprises: aligning a second plurality of sequence reads comprising the plurality of sequence reads to a reference genome sequence. Receiving the plurality of sequence reads generated from the sample obtained can comprise: selecting the plurality of sequence reads from the second plurality of sequence reads, wherein the plurality of sequence reads is aligned to the locus of the gene of interest.

In some embodiments, the plurality of sequence reads comprises sequence reads that are about 100 base pairs to about 1000 base pairs in length each. The plurality of sequence reads comprises paired-end sequence reads. The plurality of sequence reads comprises single-end sequence reads. In some embodiments, the plurality of sequence reads is generated by targeted sequencing and/or whole genome sequencing (WGS), optionally wherein the WGS is clinical WGS (cWGS). In some embodiments, the sample comprises cells, cell-free DNA, cell-free fetal DNA, amniotic fluid, a blood sample, a biopsy sample, or a combination thereof. In some embodiments, the reference genome sequence comprises a reference human genome sequence. The subject can be a human subject.

Details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims. Neither this summary nor the following detailed description purports to define or limit the scope of the inventive subject matter.

DETAILED DESCRIPTION

All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

Disclosed herein include methods for determining a replication factor C subunit 1 (RFC1) repeat expansion status. In some embodiments, a method for determining RFC1 repeat expansion status is under control of a processor (such as a hardware processor or a virtual processor) and comprises: (a) receiving a plurality of sequence reads generated from a sample obtained from a subject. The method can comprise: (b) aligning the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of RFC1. The sequence graph can comprise a repeat sequence representation flanked by non-repeat sequences of the locus of RFC1. The plurality of aligned sequence reads can comprise the plurality of sequence reads and alignments of the plurality of sequence reads to the sequence graph. The method can comprise: (c) determining a number of occurrences of a plurality of repeat sequences in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold and a first quality threshold. The method can comprise: (d) determining a frequency indication of a number of occurrences of a pathogenic repeat sequence relative to a total number of occurrences of the plurality of repeat sequences. The method can comprise: (e) determining a status of a repeat expansion at the locus of RFC1 of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences.

Disclosed herein include systems for determining a repeat expansion status of a gene of interest. In some embodiments, a system for determining repeat expansion status of a gene of interest comprising: non-transitory memory configured to store executable instructions; and a processor (such as a hardware processor or a virtual processor) in communication with the non-transitory memory. The processor can be programmed by the executable instructions to perform: (a) receiving a plurality of sequence reads generated from a sample obtained from a subject. The processor can be programmed by the executable instructions to perform: (b) aligning the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of a gene of interest. The sequence graph can comprise a repeat sequence representation flanked by non-repeat sequences of the locus of the gene. The plurality of aligned sequence reads can comprise the plurality of sequence reads. The plurality of aligned sequence reads can comprise alignments of the plurality of sequence reads to the sequence graph. The processor can be programmed by the executable instructions to perform: (c) determining a number of occurrences of a plurality of repeat sequences in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold and a first quality threshold. The processor can be programmed by the executable instructions to perform: (d) determining a frequency indication of a number of occurrences of a pathogenic repeat sequence relative to a total number of occurrences of the plurality of repeat sequence. The processor can be programmed by the executable instructions to perform: (e) determining a status of a repeat expansion at the locus of the gene of interest of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences.

Determining Pathogenic RFC1 Expansion

A biallelic intronic AAGGG repeat expansion in the replication factor C subunit (RFC1) gene is recently discovered as the cause of familial cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and a frequent cause of late-onset ataxia. In the general population, this repeat expansion is very divergent with many different benign repeat patterns. Current diagnosis methods for this pathogenic AAGGG expansion include polymerase chain reaction (PCR) and Sanger sequencing, Southern blots and linkage analysis, but all of these methods are time consuming and cannot be performed in large-scale. Compared to these methods, clinical whole genome sequencing (cWGS) screening makes it possible for this expansion to be identified in a massive parallel way. However, there is no automated and accurate way to identify this pathogenic expansion from cWGS data. Currently, this pathogenic expansion is identified by manual examination of reads from alignment, such as alignment by ExpansionHunter (Dolzhenko, E. et al. ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data. Genome Biol 21, 102 (2020). https://doi.org/10.1186/s13059-020-02017-z, the content of which is incorporated herein by reference in its entirety). When the sample size is large, this manual screening becomes extremely time-consuming.

Furthermore, in this high GC genomic region, the base quality of pair-end reads drops. This quality drop is correlated with what the repeat pattern is. This makes manual screening even more difficult and error-prone, especially when there are pathogenic AAGGGs or other high-GC repeat patterns. Because of that, there was an argument that it is not possible to accurately diagnose this pathogenic repeat from pair-end cWGS data.

Disclosed herein are methods to automatically and accurately screen this pathogenic expansion from sequencing data, such as cWGS data. There is no other method to screen this pathogenic repeat from sequencing data. The method implements a logic described herein to minimize the artifact that comes from decreased data quality in this region. For example, for one cWGS sample, a prior method determined more than 80 different repeat patterns and the percentage of AAGGG was only about 64%. In comparison, for the same sample, the method of the present disclosure determined only a few different repeat patterns and the percentage of AAGGG was more than 90%. The method implements rules to separate real pathogenic repeats from benign ones. The method can quickly screen a large population and output patients with double expanded AAGGG repeats, carriers with expanded AAGGG repeats on one haplotype and benign repeats on the other haplotype, and normal people with no expanded AAGGG on either haplotype. On one 30x cWGS sample, the method took less than one second to output the screening result (after ExpansionHunter had been run).

Repeat expansions in RFC1 has been identified as a cause for CANVAS. In GRCh38, RFC1 has (AAAAG)11repeats in chr4:39348424-39348479. There can be many other benign expansion patterns, such as (AAAAG)expand possibly (AAAGG)exp. Homozygous (AAGGG)expand (ACAGG)exphave been identified as pathogenic. Diagnosis methods for pathogenic RFC1 expansions include PCR & Sanger sequencing, Southern blots, linkage analysis, and clinical whole-genome sequencing (cWGS) screening. PCR & Sanger sequencing utilizes different primers for mutants and wildtypes. However, long-range PCR is more error-prone in repetitive regions. Southern blots are more difficult than PCR. Southern blots can confirm the presence of biallelic large expansions. Southern blots do not tell what expansions they are. Regarding linkage analysis, family-based studies have identified a few linkage disequilibrium (LD) single nucleotide polymorphisms (SNPs) for mutants. However, a few pedigrees do not guarantee the SNPs are in LD with the pathogenic repeats.

This pathogenic expansion can be identified with cWGS screening by manual examination of reads from, for example, ExpansionHunter (EH) alignment. The screening can be performed through ExpansionHunter, on a constructed repeat graph of (AARRG)n, where R is A or G (FIG.1), such that the reference AAAAG and pathogenic AAGGG can be genotyped simultaneously. In graph alignments, reads that overlap with repeats can be categorized as spanning reads, in-repeat reads, and flanking reads as illustrated inFIG.1. A spanning read can occur when a repeat is shorter than the read length such that the read includes the repeat and the flanking regions on both end of the repeat. An in-repeat read can occur when the repeat is longer than the read length such that the entire read (whether a single-end sequencing read or a pair-end sequencing read) or one read of a pair-end sequencing read includes only a portion of the repeat and no flanking region of the repeat. A flanking read includes a portion of the repeat and the flanking region on one end of the repeat.

To identify pathogenic expansion with cWGS screening by manual examination, all reads that overlap with the repeats can be visualized through an EH visualization tool. The reads can be examined to see if the reads include pathogenic (AAGGG)n.FIG.2shows the visualization of reads of a human patient sample that overlap with the repeat. The human patient FAM-092-D12 was confirmed by PCR to have two expanded AAGGG haplotypes. As shown inFIG.2, the reads were either in-repeat reads and flanking reads mostly containing AAGGG. Because the repeat is longer than the read length, no spanning read was observed. However, manual examination can be difficult and inaccurate. For example, two different haplotypes can be difficult to differentiate. Patterns similar to pathogenic repeats (e.g., AAAGG) may result in false positives calls.FIG.3shows the visualization of reads of a patient sample that overlap with the repeat. The human patient FAM-062-F08 was a presumed patient. However, PCR did not validate the presumption. As illustrated inFIG.3, it is difficult to tell what repeats they are from bare eyes. PCR suggested AAGAG repeats instead of AAGGG repeats.

Disclosed herein are methods to automatically and accurately screen the pathogenic expansion from sequencing data, such as cWGS data. On graph-aligned reads, how many times each 5-mer was observed in the repeat-overlapping part can be counted. In some embodiments, to minimize the noise from decreased data quality, the following criterion can be used: Each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold (e.g., 20). Referring toFIG.4A, a base with quality greater than or equal to 20 is shown capitalized. For example, of the four AAAAG/AAaAg/AaaaG 5-mers shown inFIG.4A, two had all five bases with quality greater than or equal to 20 (the 5-mers shown as AAAAG), one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAaAG), and one had two bases with quality greater than or equal to 20 (the 5-mer shown as AaaaG). The four AAAAG/AAaAg/AaaaG 5-mers satisfy this criterion and are counted. As another example, of the three AAGGG/AAGgG 5-mers shown inFIG.4A, two had all five bases with quality greater than or equal to 20 (the 5-mers shown as AAGGG), and one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAGgG). All three AAGGG/AAGgG 5-mers satisfy this criterion and are counted. As a further example, of the two remaining 5-mers shown inFIG.4A, one had all five bases with quality greater than or equal to 20 (the 5-mer shown as AGGGG), and one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAGaG). These two 5-mers do not satisfy the criterion that each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold of 20 because each type of these 5-mers has an occurrence of one. These two 5-mers are filtered and not counted.

In some embodiments, to minimize the noise from decreased data quality, the following criteria can be used: Each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold (e.g., 20), and each counted 5-mer has at least 3 bases having quality greater than or equal to a quality threshold (e.g., 20). Referring toFIG.4B, a base with quality greater than or equal to 20 is shown capitalized. For example, of the four AAAAG/AAaAg/AaaaG 5-mers shown inFIG.4B, two had all five bases with quality greater than or equal to 20 (the 5-mers shown as AAAAG), one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAaAG), and one had two bases with quality greater than or equal to 20 (the 5-mer shown as AaaaG). The AAAAG/AAaAg 5-mers satisfy the criteria and are counted. The AaaaG 5-mers does not satisfy the criteria (because this 5-mer only has two bases with quality greater than or equal to 20) and are filtered and not counted. As another example, of the three AAGGG/AAGgG 5-mers shown inFIG.4B, two had all five bases with quality greater than or equal to 20 (the 5-mers shown as AAGGG), and one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAGgG). All three AAGGG/AAGgG 5-mers satisfy the criteria and are counted. As a further example, of the two remaining 5-mers shown inFIG.4B, one had all five bases with quality greater than or equal to 20 (the 5-mer shown as AGGGG), and one had four bases with quality greater than or equal to 20 (the 5-mer shown as AAGaG). These two 5-mers do not satisfy the criterion that each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold of 20 because each type of these 5-mers had an occurrence of one) and are filtered and not counted.

Sequence reads of patient samples were processed and 5-mer counting was performed using the criteria. Real patients had almost 100% AAGGG (a pathogenic repeat). With the 5-mer filtering and counting rules, it was observed that 93% AAGGG in the PCR-validated patient FAM-062-F08 (SeeFIG.2and the accompanying description). The other 5-mers were AAAGG (2%) and GGGGG (5%). These other 5-mers are very likely to come from sequencing errors/artifacts.

As illustrated inFIGS.5A-5B, carriers need a better separation for the two haplotypes. In reads from the expanded allele, carriers had almost 100% AAGGG. For a patient who is a carrier with one expanded allele, sequence reads of the patient’s sample were processed and 5-mer filtering and counting using the criteria. With the 5-mer filtering the counting criteria, the patient was observed to have 75% AAGGG (FIG.5A). By performing 5-mer filtering and counting on the reads from the expanded allele using the criteria, the patient was observed to have 93% AAGGG. Referring toFIGS.6A-6B, spanning reads are from the short allele. For flanking reads with short overlaps with the repeats, it is uncertain which allele the reads come from. The remaining flanking reads shown inFIG.6Band the in-repeat reads come from the expanded allele.

The samples of 77 patients with exhibiting signs of repeat expansions such as anticipation (earlier onset or more severe phenotype in subsequent generations) and/or neuropathy symptoms underwent cWGS. These patients were suspected to have some pathogenic RFC1 repeat expansions. None of them had any clinical documentation for known expansions. Failure to identify repeat expansions in these samples may be because of insufficient testing or associated with previously unknown pathogenic repeat expansions. With a general ExpansionHunter screening, RFC1 was identified as “expanded” in a few patients. Previously, real RFC1 patients were identified by manually looking at the sequence reads aligned to the repeat one by one. The methods of the present disclosure can be used for automatic screening of RFC1 status (pathogenic, carrier, or benign and/or the number of expansions such as zero, one, or two). Patterns of RFC1 expansions in the 77-patient cohort were examined. When screening the repeats in RFC1, greater than or equal to 85% for AAAAG was used for reference alleles, and greater than or equal to 85% for AAGGG was used for pathogenic alleles. Repeat patterns were identified by looking at the expanded allele.FIG.7shows the percentage of 5-mer repeats in the expanded allele for samples with one short allele and one expanded allele in the 75-patient cohort. Table 1 shows screening results on the patient cohort using the following criteria:Pathogenic if two expanded alleles and greater than 85% AAGGG repeat.Carrier if one expanded allele and greater than 85% AAGGG repeat, or if two expanded alleles and 30%-85% AAGGG repeat.Benign if no expanded alleles, if one expanded allele and less than 85% AAGGG repeat, or if two expanded alleles and less than 30% AAGGG repeats.

TABLE 1Screening results on 77-patient cohortTotal# Pathogenic# Carriers# BenignTwo Expansions257*216One Expansion38-335Zero Expansions14--14* Six samples have been validated by PCR. PCR validation has not been performed for one sample.

Patterns of RFC1 repeats in the Polaris population of 150 unrelated healthy individuals were examined. The Polaris population is a diverse population of Europeans, East Asians, and Africans.FIG.8shows the percentage of 5-mer repeats in the expanded allele for samples with one short allele and one expanded allele for this Polaris population. The analysis shows a high frequency for AAGGG and many other patterns. Table 1 shows screening results on the Polaris population using the following criteria:Pathogenic if two expanded alleles and greater than 85% AAGGG repeat.Carrier if one expanded allele and greater than 85% AAGGG repeat, or if two expanded alleles and 30%-85% AAGGG repeat.Benign if no expanded alleles, if one expanded allele and less than 85% AAGGG repeat, or if two expanded alleles and less than 30% AAGGG repeats.

Determining RFC1 Repeat Expansion Status

FIG.9is a flow diagram showing an exemplary method900of determining a replication factor C subunit 1 (RFC1) repeat expansion status. The method900may be embodied in a set of executable program instructions stored on a computer-readable medium, such as one or more disk drives, of a computing system. For example, the computing system1100shown inFIG.11and described in greater detail below can execute a set of executable program instructions to implement the method900. When the method900is initiated, the executable program instructions can be loaded into memory, such as RAM, and executed by one or more processors of the computing system1100. Although the method900is described with respect to the computing system1100shown inFIG.11, the description is illustrative only and is not intended to be limiting. In some embodiments, the method900or portions thereof may be performed serially or in parallel by multiple computing systems.

After the method900begins at block904, the method900proceeds to block908, where a computing system (such as the computing system1100described with reference toFIG.11) receives a plurality of sequence reads generated from a sample obtained from a subject. Sequence reads can be, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more base pairs (bps) in length each. For example, sequence reads are about 100 base pairs to about 1000 base pairs in length each. The sequence reads can comprise paired-end sequence reads. The sequence reads can comprise single-end sequence reads. The sequence reads can be generated by targeted sequencing. The sequence reads can be generated by whole genome sequencing (WGS). The sequence reads can be generated by whole genome sequencing (WGS). The WGS can be clinical WGS (cWGS). The sample can comprise cells, cell-free DNA, cell-free fetal DNA, amniotic fluid, a blood sample, a biopsy sample, or a combination thereof. The subject can be a human subject.

The sequence reads received can include only reads from the locus of RFC1. For example, the plurality of sequence reads can be aligned to the locus of RFC1. Alternatively, the sequence reads received can include reads from the locus of RFC1 and elsewhere. The computing system can align a second plurality of sequence reads comprising the plurality of sequence reads aligned to the locus of RFC1 to a reference genome sequence. The reference genome sequence can comprise a reference human genome sequence, such as hg19 or hg38. The computing system can select the plurality of sequence reads that are aligned to the locus of RFC1 from the second plurality of sequence reads.

The computing system can store the sequence reads in memory. The computing system can load sequence reads into memory. Sequence reads can be generated by techniques such as sequencing by synthesis, sequencing by binding, or sequencing by ligation. Sequence reads can be generated using instruments such as MINISEQ, MISEQ, NEXTSEQ, HISEQ, and NOVASEQ sequencing instruments from Illumina, Inc. (San Diego, CA).

The method900proceeds from block908to block912, where the computing system aligns the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of RFC1. The sequence graph can comprise a repeat sequence representation (e.g., AARRG where R is A or G) flanked by non-repeat sequences of the locus of RFC1. The plurality of aligned sequence reads can comprise or be associated with the plurality of sequence reads and alignments of the plurality of sequence reads to the sequence graph.

The method900proceeds from block912to block916, where the computing system determines a number of occurrences of a plurality of repeat sequences (e.g., AAGGG, AAAAG, AAAGG, AAGAG, AACGG, and ACGGG) in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold (e.g., 2) and a first quality threshold (e.g., 20). Determining or counting the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads using the first occurrence threshold and the first quality threshold is referred to herein as 5-mer filtering and counting. The 5-mer filtering and counting can be for both alleles or for the expanded allele.

A repeat expansion at the locus of RFC1 can comprise greater than a threshold total copies (e.g., 20 total copies) of one or more repeat sequences. The threshold total copies of one or more repeat sequences can be, for example, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more or less, total copies of one or more repeat sequences.

In some embodiments, each of the plurality of repeat sequences has a number of occurrences greater than or equal to (or greater than) a first occurrence threshold with each occurrence having a number of bases each having a quality score greater than or equal to a first quality threshold. The first occurrence threshold can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The first quality threshold can be, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, or more or less. The number of bases of the repeat sequence each having a quality score greater than (or greater than or equal to) the first quality threshold can be, for example, 4 or 5. For example, each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold (e.g., a first quality threshold) of 20. In some embodiments, each of the occurrences has a number of bases each having a quality score greater than or equal to (or a greater than) than a second quality threshold. The number of bases each having a quality score greater than or equal to (or greater than) the second quality threshold can be, for example, 2, 3, 4, or 5. The second quality threshold can be for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, or more or less. The number of bases of each of the occurrences having a quality score greater than the second quality threshold can be, for example, 2, 3, 4, or 5. For example, each counted 5-mer has at least 3 bases having quality greater than or equal to a quality threshold (e.g., a second quality threshold) of 20. In some embodiments, the first quality threshold and the second quality threshold are identical.

The repeat sequence representation can be degenerate. The repeat sequence representation can be AARRG, where R is A or G. The repeat sequence representation can be at least 5 bases in length. The repeat sequence representation can be 5 bases in length. The repeat sequence representation can be 6 bases in length. Each of the plurality of repeat sequences can be at least 5 bases in length. Each of the plurality of repeat sequences can be 5 bases in length. Each of the plurality of repeat sequences can be 6 bases in length. The repeat sequence representation and a repeat sequence can have an identical length.

The method900proceeds from block916to block920, where the computing system determines a frequency indication of a number of occurrences of a pathogenic repeat sequence (e.g., AAGGG or ACAGG), or one or more pathogenic repeat sequences, relative to a total number of occurrences of the plurality of repeat sequences. The pathogenic repeat sequence can be AAGGG or ACAGG. The pathogenic repeat sequence can have a GC content of at least (or greater than) 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more or less. The pathogenic repeat sequence can be at least 5 bases in length. The pathogenic repeat sequence can be 5 bases in length. The pathogenic repeat sequence can be 6 bases in length.

The frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences can be a percentage of the number of occurrences of the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences can be a ratio of the number of occurrences of the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

The method900proceeds from block920to block924, where the computing system determines a status of a repeat expansion (or a repeat expansion status) at the locus of RFC1 of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences. The repeat expansion status at the locus of RFC1 can be a pathogenic status, a carrier status, or a benign status. The computing system can determine the subject has zero, one, or two alleles with a repeat expansion at the locus of RFC1 using the plurality of aligned sequence reads. The repeat expansion at the locus of RFC1 can be at or at about chr4:39348424 of hg38, or a corresponding position of another reference genome sequence. The repeat expansion can be associated with or cause a disease. The disease can be cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) or ataxia.

Two expanded alleles. If both alleles are expanded, 5-mer counting of both expanded alleles can be performed. In some embodiments, the subject can have two alleles each with a repeat expansion at the locus of RFC1.

Two expanded alleles - Pathogenic status. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the pathogenic status.

Two expanded alleles - Carrier status. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold and is greater than or equal to (or greater than) a second status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the carrier status.

Two expanded alleles - Carrier status. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold and is greater than or equal to (or is greater than or equal to) a second status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less.

The computing system can determine a frequency indication of a number of occurrences of (1) the pathogenic repeat sequence and (2) a sequence with a high sequence similarity to the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences. The sequence similarity of the pathogenic repeat sequence and the sequence with a high sequence similarity to the pathogenic repeat sequence can be, or be about, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The pathogenic repeat sequence and the sequence similar to the pathogenic repeat sequence differs by one or more bases, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, bases. The sequence with a high sequence similarity to the pathogenic repeat sequence may be non-pathogenic and/or associated with (e.g., linked with) benign expansion.

The frequency indication of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a percentage of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a ratio of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

The computing system can determine frequency indication of a number of occurrences of the pathogenic repeat sequence and the sequence with a high sequence similarity to the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a third status threshold. The third status threshold can be, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less.

The computing system can determine the frequency indication of the number of occurrences of the pathogenic sequence is greater than (or greater than or equal to) a fourth status threshold. The fourth status threshold can be, for example, 5%, 10%, 15%, 20%, 25%, 30%, or more or less. Alternatively or additionally, the computing system can determine a frequency indication of a number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence. The frequency indication of number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a percentage of the number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a ratio of the number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences. The computing system can determine the frequency indication of the number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence is less than or equal to (or less than) a fifth status threshold. The fifth status threshold can be, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the carrier status.

In some embodiments, the computing system determines the frequency indication of the number of occurrences of the pathogenic sequence is less than or equal to (or less than) the fourth status threshold. Alternatively or additionally, the computing system can determine the frequency indication of the number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence is greater than (or greater than or equal to) the fifth status threshold. The computing system can determine the repeat expansion status at the locus of RFC1 as the benign status.

For example, the pathogenic repeat sequence is AAGGG, and the sequence with a high sequence similarity to the pathogenic repeat sequence is AAAGG. The two sequences have a sequence identity of 80%. The two sequences differ by one base. The percentage of the AAGGG repeat sequence and the AAAGG repeat sequence out of all the repeat sequences can be greater than 80%. The percentage of the AAGGG repeat sequence out of all repeat sequences can be greater than 10%. Alternatively or additionally, the percentage of the AAAGG repeat sequence out of all the repeat sequences can be less than or equal to 90%. The computing system can determine the repeat expansion status at the locus of RFC1 as the carrier status. If the percentage of the AAGGG repeat sequence out of all repeat sequences is less than or equal to 10% and/or the percentage of the AAAGG repeat sequence out of all the repeat sequences is greater than 90%, the computing system can determine the repeat expansion status at the locus of RFC1 as the benign status.

Two expanded alleles - Benign status. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than (or less than or equal to) a second status threshold. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the benign status.

One expanded allele. If one allele is expanded and one allele is not expanded, 5-mer counting of the expanded allele can be performed. In some embodiments, the subject has one allele of RFC1 with repeat expansion at the locus of RFC1.

One expanded allele - Pathogenic status. To determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads, the computing system can determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the pathogenic status.

One expanded allele - Carrier status. To determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads, the computing system can determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1. To determine the repeat expansion status at the locus of RFC1, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of RFC1 as the carrier status.

To determine the repeat expansion status at the locus of RFC1 as the pathogenic status or the carrier status when there is one expanded allele, the computing system can select the aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 based on the alignments of the aligned sequence reads. The aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 can comprise the aligned sequence reads that are (i) in-repeat reads or (ii) flanking reads each with an overlap to the repeat expansion at the locus of RFC1 greater than a repeat expansion overlap threshold. The repeat expansion overlap threshold can be, for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or more or less, base pairs. The aligned sequence reads that are from the one allele of RFC1 with repeat expansion at the locus of RFC1 can comprise (i) all the aligned sequence reads that are in-repeat reads and (ii) not all of the aligned sequence reads that are flanking reads. An in-repeat read can occur when the repeat is longer than the read length such that the entire read (whether a single-end sequencing read or a pair-end sequencing read) or one read of a pair-end sequencing read includes only a portion of the repeat and no flanking region of the repeat. A flanking read includes a portion of the repeat and the flanking region on one end of the repeat.

No expanded allele. If both alleles are not expanded, 5-mer counting of both alleles is can be performed. In some embodiments, the subject has zero allele with repeat expansion at the locus of RFC1. The repeat expansion status at the locus of RFC1 can be benign status.

In some embodiments, the computing system can generate a user interface (UI), such as a graphical user interface, comprising or representing any results (including intermediate results) of the method900. For example, the UI can comprise or represent the repeat expansion status. The UI can include, for example, a dashboard. The UI can include one or more UI elements. A UI element can comprise or represent the repeat expansion status. A UI element can be a window (e.g., a container window, browser window, text terminal, child window, or message window), a menu (e.g., a menu bar, context menu, or menu extra), an icon, or a tab. A UI element can be for input control (e.g., a checkbox, radio button, dropdown list, list box, button, toggle, text field, or date field). A UI element can be navigational (e.g., a breadcrumb, slider, search field, pagination, slider, tag, icon). A UI element can informational (e.g., a tooltip, icon, progress bar, notification, message box, or modal window). A UI element can be a container (e.g., an accordion). In some embodiments, the computing system can generate a report comprising or representing any results (including intermediate results) of the method900, such as the repeat expansion status.

The computing system can cause one or more diagnosis methods to be performed to confirm of the repeat expansion status at the locus of RFC1 of the subject. The UI or the report can comprise or indicate that one or more diagnosis methods should be performed to confirm the repeat expansion status at the locus of RFC1 of the subject. The computing system can receive confirmation of the repeat expansion status at the locus of RFC1 of the subject determined using one or more diagnosis methods. The one or more diagnosis methods can comprise polymerase chain reaction (PCR) and Sanger sequencing, southern blots, and linkage analysis.

In some embodiments, any threshold of the method900(such as an occurrence threshold (e.g., a first occurrence threshold), a quality threshold (e.g., a first quality threshold, or a second quality threshold), a threshold total copies, a status threshold (e.g., a first status threshold, a second status threshold, a third status threshold, a fourth status threshold, or a fifth status threshold), or a repeat expansion overlap threshold) can be determined using a number of samples, such as 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, or more or less, samples.

The method900ends at block928.

Determining Repeat Expansion Status of a Gene of Interest

FIG.10is a flow diagram showing an exemplary method1000of determining a status of a repeat expansion (also referred to herein as repeat expansion status) of a gene of interest. The method1000may be embodied in a set of executable program instructions stored on a computer-readable medium, such as one or more disk drives, of a computing system. For example, the computing system1100shown inFIG.11and described in greater detail below can execute a set of executable program instructions to implement the method1000. When the method1000is initiated, the executable program instructions can be loaded into memory, such as RAM, and executed by one or more processors of the computing system1100. Although the method1000is described with respect to the computing system1100shown inFIG.11, the description is illustrative only and is not intended to be limiting. In some embodiments, the method1000or portions thereof may be performed serially or in parallel by multiple computing systems.

After the method1000begins at block1004, the method1000proceeds to block1008, where a computing system (such as the computing system1100described with reference toFIG.11) receives a plurality of sequence reads generated from a sample obtained from a subject. Sequence reads can be, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more base pairs (bps) in length each. For example, sequence reads are about 100 base pairs to about 1000 base pairs in length each. The sequence reads can comprise paired-end sequence reads. The sequence reads can comprise single-end sequence reads. The sequence reads can be generated by targeted sequencing. The sequence reads can be generated by whole genome sequencing (WGS). The sequence reads can be generated by whole genome sequencing (WGS). The WGS can be clinical WGS (cWGS). The sample can comprise cells, cell-free DNA, cell-free fetal DNA, amniotic fluid, a blood sample, a biopsy sample, or a combination thereof. The subject can be a human subject.

The sequence reads received can include only reads from the locus of the gene of interest. For example, the plurality of sequence reads can be aligned to the locus of the gene of interest. Alternatively, the sequence reads received can include reads from the locus of the gene of interest and elsewhere. The computing system can align a second plurality of sequence reads comprising the plurality of sequence reads aligned to the locus of the gene of interest to a reference genome sequence. The reference genome sequence can comprise a reference human genome sequence, such as hg19 or hg38. The computing system can select the plurality of sequence reads that are aligned to the locus of the gene of interest from the second plurality of sequence reads.

The computing system can store the sequence reads in memory. The computing system can load sequence reads into memory. Sequence reads can be generated by techniques such as sequencing by synthesis, sequencing by binding, or sequencing by ligation. Sequence reads can be generated using instruments such as MINISEQ, MISEQ, NEXTSEQ, HISEQ, and NOVASEQ sequencing instruments from Illumina, Inc. (San Diego, CA).

The method1000proceeds from block1008to block1012, where the computing system aligns the plurality of sequence reads to a sequence graph to generate a plurality of aligned sequence reads. The sequence graph can represent a locus of a gene of interest (e.g., RFC1). The sequence graph can comprise a repeat sequence representation (e.g., AARRG where R is A or G if the gene of interest is RFC1) flanked by non-repeat sequences of the locus of the gene. The plurality of aligned sequence reads can comprise the plurality of sequence reads. The plurality of aligned sequence reads can comprise or be associated with alignments of the plurality of sequence reads to the sequence graph.

The method1000proceeds from block1012to block1016, where the computing system determines a number of occurrences of a plurality of repeat sequences (e.g., AAGGG, AAAAG, AAAGG, AAGAG, AACGG, and ACGGG if the gene of interest is RFC1) in aligned sequence reads of the plurality of aligned sequence reads using a first occurrence threshold (e.g., 2) and a first quality threshold (e.g., 20). Determining or counting the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads using the first occurrence threshold and the first quality threshold is referred to herein as n-mer filtering and counting, where n is the length of the repeat sequence. The n-mer filtering and counting can be for both alleles or for the expanded allele.

A repeat expansion at the locus of the gene of interest can comprise greater than a threshold total copies of one or more repeat sequences. The threshold total copies of one or more repeat sequences can be, for example, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more or less, total copies of one or more repeat sequences.

In some embodiments, each of the plurality of repeat sequences has a number of occurrences greater than or equal to (or greater than) a first occurrence threshold with each occurrence having a number of bases each having a quality score greater than or equal to (or greater than) a first quality threshold. The first occurrence threshold can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The first quality threshold can be, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, or more or less. The number of bases of the repeat sequence each having a quality score greater than (or greater than or equal to) the first quality threshold can be, for example, 4, 5, 6, 7, 9, 9, 10, or more. For example, each type of 5-mer has two occurrences with all 5 bases having quality (Q) greater than or equal to a quality threshold (e.g., a first quality threshold) of 20. In some embodiments, each of the occurrences has a number of bases each having a quality score greater than or equal to (or greater than) than a second quality threshold. The number of bases each having a quality score greater than or equal to (or greater than) than the second quality threshold can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The second quality threshold can be for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, or more or less. The number of bases of each of the occurrences having a quality score greater than the second quality threshold can be, for example, 2, 3, 4, or 5. For example, each counted 5-mer has at least 3 bases having quality greater than or equal to a quality threshold (e.g., a second quality threshold) of 20. In some embodiments, the first quality threshold and the second quality threshold are identical.

The repeat sequence representation can be degenerate. The repeat sequence representation can be AARRG, where R is A or G, if the gene of insert is RFC1. The repeat sequence representation can be 5, 6, 7, 8, 9, 10, or more, bases in length. Each of the plurality of repeat sequences can be 5, 6, 7, 8, 9, 10, or more, bases in length. The repeat sequence representation and a repeat sequence can have an identical length.

The method1000proceeds from block1016to block1020, where the computing system determines a frequency indication of a number of occurrences of a pathogenic repeat sequence (e.g., AAGGG or ACAGG if the gene of interest is RFC1), or one or more pathogenic repeat sequences, relative to a total number of occurrences of the plurality of repeat sequence. The pathogenic repeat sequence can have a GC content of at least (or greater than) 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more or less. The pathogenic repeat sequence can be 5, 6, 7, 8, 9, 10, or more, bases in length.

The frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences can be a percentage of the number of occurrences of the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences can be a ratio of the number of occurrences of the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

The method1000proceeds from block1020to block1024, where the computing system determines a repeat expansion status at the locus of the gene of interest of the subject using the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences. The computing system can determine the subject has zero, one, or two alleles with a repeat expansion at the locus of the gene of interest using the plurality of aligned sequence reads. The repeat expansion status at the locus of the gene of interest is a pathogenic status, a carrier status, or a benign status. The repeat expansion can be associated with or causes a disease. The disease can be, for example, late-onset ataxia, cerebellar ataxia , sensory neuronopathy, bilateral vestibulopathy, or cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). The disease can be a cancer, a non-cancer disease, a neurological disease, a neurodegenerative disease, an autoimmune disease, Alzheimer’s disease, Parkinson’s Disease, dementia, rheumatoid arthritis, or inflammation. The disease can be Huntington disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy spinocerebellar ataxias, fragile X, fragile X tremor ataxia syndrome, other fragile sites, myotonic dystrophy type 1, Huntington disease-like 2, spinocerebellar ataxia type 8, Fuchs corneal dystrophy, Friedreich ataxia, FRAXE mental retardation, oculopharyngeal muscular dystrophy, myotonic dystrophy type 1, spinocerebellar ataxia type 10, spinocerebellar ataxia type 31, spinocerebellar ataxia type 36, frontotemporal dementia/amyotrophic lateral sclerosis, or EPM1 (myoclonic epilepsy). The gene of interest can be Huntingtin, androgen receptor (AR) gene, ATN1, ATXN1, ATXN2, ATXN3, ATXN10, CACNA1A, ATXN7, TBP gene, PPP2R2B, TK2, BEAN, NOP56, JPH3, FRDA, CSTB, PABP2, TCF4, or C9ORF72.

In some embodiments, the gene of interest is replication factor C subunit 1 (RFC1). The locus of the gene of interest with a repeat expansion can be at, or at about, chr4:39348424 of hg38, or a corresponding position of another reference genome sequence. The repeat expansion can be associated with or causes a disease. The disease can be cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) or ataxia. The repeat sequence representation can be AARRG. The pathogenic repeat sequence can be AAGGG or ACAGG.

Two expanded alleles. If both alleles are expanded, 5-mer counting of both expanded alleles can be performed. In some embodiments, the subject can have two alleles each with a repeat expansion at the locus of the gene of interest.

Two expanded alleles - Pathogenic status. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the pathogenic status.

Two expanded alleles - Carrier status. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold and greater than or equal to (or greater than) a second status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the carrier status.

Two expanded alleles - Carrier status. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold and is greater than or equal to (or is greater than or equal to) a second status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less.

The computing system can determine a frequency indication of a number of occurrences of (1) the pathogenic repeat sequence and (2) a sequence with a high sequence similarity to the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences. The sequence similarity of the pathogenic repeat sequence and the sequence with a high sequence similarity to the pathogenic repeat sequence can be, or be about, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The pathogenic repeat sequence and the sequence similar to the pathogenic repeat sequence differs by one or more bases, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, bases. The sequence with a high sequence similarity to the pathogenic repeat sequence may be non-pathogenic and/or associated with (e.g., linked with) benign expansion.

The frequency indication of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a percentage of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a ratio of the number of occurrences of (1) the pathogenic repeat sequence and (2) the sequence with a high sequence similarity to the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences.

The computing system can determine frequency indication of a number of occurrences of the pathogenic repeat sequence and the sequence with a high sequence similarity to the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a third status threshold. The third status threshold can be, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less.

The computing system can determine the frequency indication of the number of occurrences of the pathogenic sequence is greater than (or greater than or equal to) a fourth status threshold. The fourth status threshold can be, for example, 5%, 10%, 15%, 20%, 25%, 30%, or more or less. Alternatively or additionally, the computing system can determine a frequency indication of a number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence. The frequency indication of number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a percentage of the number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence out of the total number of occurrences of the plurality of repeat sequences. The frequency indication of the sequence with a high sequence similarity to the pathogenic repeat sequence, relative to the total number of occurrences of the plurality of repeat sequences, can be a ratio of the number of occurrences of the sequence with a high sequence similarity to the pathogenic repeat sequence over the total number of occurrences of the plurality of repeat sequences. The computing system can determine the frequency indication of the number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence is less than or equal to (or less than) a fifth status threshold. The fifth status threshold can be, for example, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the carrier status.

In some embodiments, the computing system determines the frequency indication of the number of occurrences of the pathogenic sequence is less than or equal to (or less than) the fourth status threshold. Alternatively or additionally, the computing system can determine the frequency indication of the number of occurrences of the sequence with a high sequence similarity to the pathogenic sequence is greater than (or greater than or equal to) the fifth status threshold. The computing system can determine the repeat expansion status at the locus of RFC1 as the benign status.

Two expanded alleles - Benign status. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than (or less than or equal to) a second status threshold. The second status threshold can be, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the benign status.

2. One expanded allele. If one allele is expanded and one allele is not expanded, 5-mer counting of the expanded allele can be performed. In some embodiments, the subject has one allele of the gene of interest with repeat expansion at the locus of the gene of interest.

One expanded allele - Pathogenic status. To determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads, the computing system can determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of RFC1. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is greater than (or greater than or equal to) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the pathogenic status.

One expanded allele - Carrier status. To determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of sequence reads, the computing system can determine the number of occurrences of the plurality of repeat sequences in the aligned sequence reads of the plurality of aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of RFC1. To determine the repeat expansion status at the locus of the gene of interest, the computing system can determine the frequency indication of the number of occurrences of the pathogenic repeat sequence relative to the total number of occurrences of the plurality of repeat sequences is less than or equal to (or less than) a first status threshold. The first status threshold can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more or less. The computing system can determine the repeat expansion status at the locus of the gene of interest as the carrier status.

To determine the repeat expansion status at the locus of the gene of interest as the pathogenic status or the carrier status when there is one expanded allele, the computing system can select the aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest based on the alignments of the aligned sequence reads. The aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest can comprise the aligned sequence reads that are (i) in-repeat reads or (ii) flanking reads each with an overlap to the repeat expansion at the locus of the gene of interest greater than a repeat expansion overlap threshold. The repeat expansion overlap threshold can be, for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or more or less, base pairs. The aligned sequence reads that are from the one allele of the gene of interest with repeat expansion at the locus of the gene of interest can comprise (i) all the aligned sequence reads that are in-repeat reads and (ii) not all of the aligned sequence reads that are flanking reads. An in-repeat read can occur when the repeat is longer than the read length such that the entire read (whether a single-end sequencing read or a pair-end sequencing read) or one read of a pair-end sequencing read includes only a portion of the repeat and no flanking region of the repeat. A flanking read includes a portion of the repeat and the flanking region on one end of the repeat.

No expanded allele. If both alleles are not expanded, 5-mer counting of both alleles is can be performed. In some embodiments, the subject has zero allele with repeat expansion at the locus of the gene of interest. The repeat expansion status at the locus of the gene of interest can be benign status.

In some embodiments, the computing system can generate a user interface (UI), such as a graphical user interface, comprising or representing any results (including intermediate results) of the method1000. For example, the UI can comprise or represent the repeat expansion status. The UI can include, for example, a dashboard. The UI can include one or more UI elements. A UI element can comprise or represent the repeat expansion status. A UI element can be a window (e.g., a container window, browser window, text terminal, child window, or message window), a menu (e.g., a menu bar, context menu, or menu extra), an icon, or a tab. A UI element can be for input control (e.g., a checkbox, radio button, dropdown list, list box, button, toggle, text field, or date field). A UI element can be navigational (e.g., a breadcrumb, slider, search field, pagination, slider, tag, icon). A UI element can informational (e.g., a tooltip, icon, progress bar, notification, message box, or modal window). A UI element can be a container (e.g., an accordion). In some embodiments, the computing system can generate a report comprising or representing any results (including intermediate results) of the method900, such as the repeat expansion status.

The computing system can cause one or more diagnosis methods to be performed to confirm of the repeat expansion status at the locus of the gene of interest of the subject. The UI or the report can comprise or indicate that one or more diagnosis methods should be performed to confirm the repeat expansion status at the locus of the gene of interest of the subject. The computing system can receive conformation of the repeat expansion status at the locus of the gene of interest of the subject determined using one or more diagnosis systems. The one or more diagnosis systems can comprise polymerase chain reaction (PCR) and Sanger sequencing, southern blots, and linkage analysis.

In some embodiments, any threshold of the method1000(such as an occurrence threshold (e.g., a first occurrence threshold), a quality threshold (e.g., a first quality threshold, or a second quality threshold), a threshold total copies, a status threshold (e.g., a first status threshold, a second status threshold, a third status threshold, a fourth status threshold, or a fifth status threshold), or a repeat expansion overlap threshold) can be determined using a number of samples, such as 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, or more or less, samples.

The method1000ends at block1028.

Execution Environment

FIG.11depicts a general architecture of an example computing device1100configured for determining repeat expansion status of a gene of interest (e.g., RFC1). The general architecture of the computing device1100depicted inFIG.11includes an arrangement of computer hardware and software components. The computing device1100may include many more (or fewer) elements than those shown inFIG.11. It is not necessary, however, that all of these generally conventional elements be shown in order to provide an enabling disclosure. As illustrated, the computing device1100includes a processing unit1110, a network interface1120, a computer readable medium drive1130, an input/output device interface1140, a display1150, and an input device1160, all of which may communicate with one another by way of a communication bus. The network interface1120may provide connectivity to one or more networks or computing systems. The processing unit1110may thus receive information and instructions from other computing systems or services via a network. The processing unit1110may also communicate to and from memory1170and further provide output information for an optional display1150via the input/output device interface1140. The input/output device interface1140may also accept input from the optional input device1160, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

The memory1170may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit1110executes in order to implement one or more embodiments. The memory1170generally includes RAM, ROM and/or other persistent, auxiliary or non-transitory computer-readable media. The memory1170may store an operating system1172that provides computer program instructions for use by the processing unit1110in the general administration and operation of the computing device1100. The memory1170may further include computer program instructions and other information for implementing aspects of the present disclosure.

For example, in one embodiment, the memory1170includes a repeat expansion status determining module1174for determining repeat expansion status (e.g., pathogenic, carrier, or benign), such as the method900described with reference toFIG.9and the method1000described with reference toFIG.10. In addition, memory1170may include or communicate with the data store1190and/or one or more other data stores that store sequence reads being processed and repeat expansion status determined (and any intermediate results thereof).

Additional Considerations

One skilled in the art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods can be implemented in differing order. Furthermore, the outlined steps and operations are only provided as examples, and some of the steps and operations can be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Accordingly, phrases such as “a device configured to” are intended to include one or more recited devices. Such one or more recited devices can also be collectively configured to carry out the stated recitations. For example, “a processor configured to carry out recitations A, B and C can include a first processor configured to carry out recitation A and working in conjunction with a second processor configured to carry out recitations B and C. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.