WO2013166517A1 - Methods for determining absolute genome-wide copy number variations of complex tumors - Google Patents

Abstract

Methods for interpreting absolute copy number of complex tumors and for determining the copy number of a genomic region at a detection position of a target sequence in a sample are disclosed. In certain aspects, genomic regions of a target sequence in a sample are sequenced and measurement data for sequence coverage is obtained. Sequence coverage bias is corrected and may be normalized against a baseline sample. Hidden Markov Model (HMM) segmentation, scoring, and output are performed, and in some embodiments population-based no-calling and identification of low-confidence regions may also be performed. A total copy number value and region-specific copy number value for a plurality of regions are then estimated.

Description

METHODS FOR DETERMINING ABSOLUTE GENOME-WIDE COPY NUMBER

VARIATIONS OF COMPLEX TUMORS

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of, and claims the benefit of priority of U.S. Application Ser. No. 13/270, 989 filed on October 1 1 , 2011 , which claims the benefit of priority of U.S. Provisional Application Ser. No. 61/503,327, filed on June 30, 201 1 and U.S. Provisional Application Ser. No. 61/392,567, filed on October 13, 2010. The instant application also claims the benefit of priority of U.S. Provisional Application Serial No. 61/643,225, filed May 4^lh, 2012. The entire contents of each of which is hereby incorporated by reference in their entirety as if fully set forth herein.

BACKGROUND

[0002] Genomic abnormalities are often associated with various genetic disorders, degenerative diseases, and cancer. For example, the deletion or muitipli cation of copies of genes and the deletion or amplifications of genomic fragments or specific regions are common occurrences in cancer. For instance, alterations in proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumor igenesis. The identification and cloning of specific genomic regions associated with cancer and various genetic disorder is therefore of interest both to the study of tumorigenesis and in developing better means of diagnosis and prognosis.

[0003J Identification of polynucleotides that correspond to copy number alterations in cancerous, pre-cancerous, or low metastatic potential cells relative to normal cells of the same tissue type, provides the basis for diagnostic tools, facilitates drug discovery by providing for targets for candidate agents, and further serves to identify therapeutic targets for cancer therapies that are more tailored for the type of cancer to be treated.

[0004] In diagnostic genome sequencing, the computational complexity involved in sequence analysis of three billion base pairs in the human genome is further compounded by the accuracy requirements of clinical diagnostics such that 60 billion or more sequence data points must be analyzed to provide one accurate genome sequence. This complexity was dealt with in early sequencing methods by generating sequence data from thousands of isolated, very long fragments of DNA, thereby preserving the contextual integrity of the sequence information and reducing the redundant testing required for accurate data. However, this approach, used to generate the first complete human genome, cost hundreds of millions of dollars per genome due to the up-front complexity of preparing the genome fragments and the relative high cost of many individual biochemical tests.

[0005] In addition, contextual information in the genome is compounded by the presence of two distinct copies of the genome in each human cell such that accurate clinical analysis and diagnosis requires the ability to distinguish DNA sequence as a function of genome copy. Thus, a major challenge is to distinguish sequence differences between the two unique copies of the three billion DNA bases interspersed with millions of inherited single nucleotide polymorphisms (SNPs), hundreds of thousands of short insertions and deletions and hundreds of spontaneous mutations.

[0006] However, high degrees of aneuploidy, stromal (normal) contamination and genomic heterogeneity make estimating absolute total and lesser allele copy number from whole genome sequence read data challenging for tumor samples. Despite some progress in this area, robust methods remain elusive. For example, some approaches have been developed that aid in the identification of copy number variants ("CNV") within a complete DNA sequence, and to aid in the confidence of the identification based on comparison of the sequence with reference sequences or multiple different copies of the sequence. In these approaches identification of copy number and its validation is based on different sets of samples, and the data used in such approaches is relatively error-prone and known to harbor certain artifactual biases.

SUMMARY

[0007] In certain aspects, the present disclosure provides methods for determining the copy number of a genomic region at a detection position of a target polynucleotide sequence in a sample, said method comprising: obtaining measurement data for the sequence coverage for said sample; correcting the measurement data for sequence coverage bias; wherein the sequence coverage bias corcection comprises performing ploidy-aware baseline correction; and estimating a total copy number value and region-specific copy number value for a plurality of genomic regions. In one embodiment, the method comprises performing Hidden Markov Model (HMM) segmentation, scoring, and output. In another embodiment, the method comprises performing population-based no-calling and identification of low-confidence regions.

[0008] In certain aspects, the techniques and/or methods described herein provide a series of steps (e.g., a model) for interpretation of absolute copy number of complex tumors. In some embodiments, computer logic is configured to perform the model for processing total and allele- specific read depth data that result in an easily-interpretable graphical representation of a tumor sample, as well as an automated analysis process. The analysis is based on a model that assumes homogeneity of the tumor portion of a sample to the majority of the genome but allows for a portion of the sample to be affected by tumor heterogeneity. The result data obtained by performing the final model may be input to a separate model-based segmentation process (e.g. an HMM) - for example, the result data can be used as initial input states to the model-based segmentation process and the state interpretations can be used to annotate the final set of segments. Due to the separation of the modeling process and the final segmentation, the visualization of the tumor can be presented to a user; if problematic, an alternative model can be substituted for the automatically derived model.

[0009] In one aspect, the method further comprises normalization of sequence coverage by comparison to a baseline sample.

[00010] In one aspect, the method further comprises the determination of the sequence coverage by measuring sequence coverage depth at every position of the genome of the sample.

[00011] In one aspect, the method further comprises correction of the sequence bias by calculating window-averaged coverage.

[00012] In one aspect, the method further comprises adjustments to account for GC bias in the library construction and sequencing process.

[00013] In a further embodiment, the method further comprises performing adjustments based on additional weighting factor associated with individual mappings to compensate for bias.

[00014] In one aspect, the method further comprises steps performed by a sequencing machine, said steps comprising: a) providing a plurality of amplicons, wherein: i) each amplicon comprises multiple copies of a fragment of the target nucleic acid, ii) each amplicon comprises a plurality of interspersed adaptors at predetermined sites

the fragment, each adaptor comprising at least one anchor probe hybridization site, and iii) said plurality of amplicons comprise fragments that substantially cover the target nucleic acid; b) providing a random array of said amplicons fixed to a surface at a density such that at least a majority of said amplicons are optically resolvable; c) hybridizing one or more anchor probes to said random array; d) hybridizing one or more sequencing probes to said random array to form perfectly matched duplexes between said one or more sequencing probes and fragments of target nucleic acid; e) Iigating the anchor probes to the sequencing probes; and f) identifying at least one nucleotide adjacent to at least one interspersed adaptor; and g) repeating steps (c) through (f) until a nucleotide sequence of said target nucleic acid is identified.

{000151 In one aspect, the method further comprises determining the measurement data by performing steps comprising: a) determining reads representing the sequences of a plurality of approximately random fragments of the genome in a sample, wherein said plurality provides a sampling of the genome of the sample whereby oa average a base position of the genome is sampled one or more times; b) obtaining mapping data for said reads by mapping said reads to the reference genome, or by mapping said reads to an assembled sequence (e.g., such as the assembled sequence of the sample itself or the assembled sequence of a related baseline sample); and c) obtaining coverage data by measuring the intensity of said reads along the reference genome or along the assembled sequence , wherein the measurement data comprises the mapping data and the coverage data.

[00016] In a further embodiment, the metiiod further comprises generation of an initial model that estimates the number of states and their means based on the overall coverage distribution.

[00017] In a further embodiment, the method further comprises optimization of an initial model by sequentially adding states to the model and then sequentially removing states from the model, or combination thereof.

[00018] In a further embodiment, the normalization further comprises the determination of normalized corrected coverage.

[00019] In a further embodiment, the method further comprises determining sequence coverage by segmental duplications and obtaining confidence measurements to f actionally attribute the mapping to each detection location.

[00020] In one aspect, the method comprises HMM calculations performed to determine a ploidy number at each detection position.

[00021] In a further embodiment, the method further comprises generating a plurality of states of a Hidden Markov Model (HMM) that correspond to respective copy numbers, wherein if the sample is a normal sample, then performing HMM segmentation, scoring, and output, including: initializing a mean of an emission distribution of the HMM for each state with copy number N greater than zero to N/2 multiplied by the median of the coverage in a portion of the sample expected to be diploid; and initializing the mean of the emission distribution for the state with copy number 0 to a positive value smaller than that used for the state with copy number 1.

[00022] In a further embodiment, the method further comprises generating plural states of an HMM that correspond to respective copy numbers, wherein if the sample is a tumor sample, then performing HMM segmentation, scoring, and output, including: estimating the number of states and a mean of each state based on a distribution of the coverage to generate an initial model for the HMM; optimizing the initial model by modifying the number of states in the model as well as optimizing the parameters of each state; and modifying the number of states in the model by sequentially adding states to the model and then sequentially removing states, or a combination thereof.

{00023] In a further embodiment, the method urther comprises adjusting the initial model that comprises: a) adding a new state between a pair of states if adding said new state improves a likelihood associated with the HMM beyond a first predetermined threshold; b) repeating step (a) recursively between each pair of states until no more additions are possible; c) removing a state from the HMM if removal of said state does not decrease the likelihood beyond a second predetermined threshold; and d) repeating step (c) iteratively for all the states.

[00024] A further embodiment comprises a computer-readable non-transitory storage medium having instructions stored thereon for determining the copy number of a genomic region at a detection position of a target polynucleotide sequence in a sample, the instructions when executed by a computer processor causing the processor to perform the operations of: obtaining measurement data for the sequence coverage for said sample using data generated from mate- pair mappings; correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction; and based at least on the corrected measurement data, estimating a total copy number value and region-specific copy number value for each of a plurality of genomic regions.

[00025] A further embodiment comprises a computer-readable non-transitory storage medium having instructions tangibly embodied thereon, the instructions when executed by a computer processor causing the processor to perform the operations of: obtaining measurement data for sequence coverage for a biological sample comprising a target sequence; correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction; based on the corrected measurement data, performing Hidden Markov Model (HMM) segmentation, scoring, and output; based on the HMM scoring and output, performing population- based no-calling and identification of low- confidence regions; and estimating a total copy number value and region-specific copy number value for a plurality of regions.

[00026] A further embodiment comprises a system for determining copy number variation of a genomic region at a detection position of a target sequence, comprising: a. a computer processor; and b. a computer-readable storage medium coupled to said processor, the storage medium having instructions tangibly embodied thereon, the instructions when executed by said processor causing said processor to perform the operations of: obtaining measurement data for the sequence coverage for said sample using data generated from mate-pair mappings; correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction; and based at least on the corrected measurement data, estimating a total copy number value and region-specific copy number value for each of a plurality of genomic regions.

[00027] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00G28] The following drawing(s) are representational of one format for presentation of the data provided from embodiments of the invention. These drawings are not intended to limit in any way the implementation of aspects of the invention as described herein, but rather to aid in clarification of the underlying concepts of the invention.

[00029] FIG. 1 depicts a generalized block diagram illustrating a system for calling variation in a sample containing target sequences according to an embodiment of the present disclosure. [00030] FIG. 2 depicts a generalized flow chart illustrating the CNV calling method according to an embodiment of the present disclosure.

{00031] FIG. 3 depicts a generalized computer system incorporating and operative according to certain aspects of the instant disclosure.

[00032] FIG. 4A and 4B illustrate exemplary sequencing systems.

[00033] FIG. 5 illustrates an exemplary computing device that can be used in, or in conjunction with, a sequencing machine and/or a computer system.

[00034] FIG. 6 is a diagram of the 1 component tumor model.

[00035] FIG. 7 is a diagram of the 2 component tumor model.

[00036] FIG. 8 is a diagram of an exemplary embodiment of determination of read coverage and segmentation.

[00037] FIG. 9 is a diagram of an exemplary initial state estimation logic.

[00038] FIG. 1 OA - IOC are illustrations of the exemplary results indicating the robustness of the process containing varying percentages of tumor and normal tissue.

[00039] FIG. 1 1 illustrates the strong statistical correlation between tumors with high average copy number and high variability.

DETAILED DESCRIPTIONS

[00040] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[00041] Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods.

[00042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. ΑΠ publications mentioned herein are incoiporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

[00043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[00044] Exemplary Sequencing Methods

[00045] An exemplary method for sequencing target nucleic acids includes sample

preparation involving extracting and fragmenting target nucleic acids from a DNA sample to produce fragmented target nucleic acid templates that will generally include one or more adaptors. The target nucleic acid templates are optionally subjected to amplification methods to form nucleic acid nanoballs, which are typically disposed on a surface or substrate for puipose of analysis. The substrate may yield patterned or random arrangements of nucleic acid nanoballs. Methods for forming nucleic acid nanoballs are described in Published Patent Application Nos. WO2007120208, WO2006073504, WO2007133831, and US2007099208, and U.S. patent application Ser. Nos. 11/679,124; 1 1/981 ,761 ; 11/981,661 ; 11 /981,605; 1 1/981 ,793; 1 1/981 ,804; 1 1/451,691 ; 1 1/981,607; 11/981 ,767; 1 1 /982,467; 1 1/451,692; 12/335,168; 1 1/541 ,225;

1 1/927,356; 1 1 /927,388; 11/938,096; 1 1/938,106; 10/547,214; 1 1/981 ,730; 11/981,685;

1 1 /981 ,797; 12/252,280; 1 1/934,695; 1 1/934,697; 11/934,703; 12/265,593; 12/266,385;

1 1 /938,213; 11/938,221; 12/325,922; 12/329,365; and 12/335,188. all of which are incorporated herein by reference in their entirety for all purposes and in particular for all teachings related to forming nucleic acid nanoballs. Methods for forming arrays of nucleic acid nanoballs are described in Published Patent Application Nos. WO2007120208, WO2006073504,

WO2007133831, and US2007099208, and U.S. patent application Ser. Nos. 1 1/679,124;

11/981 ,761 ; 1 1/981 ,661 ; 1 1/981,605; 11/981 ,793; 1 1/981,804; 1 1/451 ,691 ; 1 1/981,607;

1 1/981,767; 1 1/982,467; 1 1/451,692; 12/335, 168; 1 1/541 ,225; 1 1/927,356; 11/927,388;

1 1/938,096; 1 1/938,106; 10/547,214; 1 1 /981 ,730; 11/981 ,685; 1 1/981 ,797; 12/252,280; 1 1 /934,695: 1 1/934,697; 1 1/934,703; 12/265,593; 12/266,385; 1 1/938,213; 11/938,221 ;

12/325,922; 12/329,365; and 12/335,188, all of which are incorporated herein by reference in their entirety for all purposes and in particular for all teachings related to forming arrays of nucleic acid nanoballs. Methods of using nucleic acid nanoballs in sequencing reactions and in the detection of particular target sequences are also described in U.S. patent application Ser. Nos. 1 1/679,124; 1 1/981 ,761 ; 11/981 ,661 ; 11/981 ,605; 1 1/981,793; 1 1/981,804; 1 1 /451,691 ;

1 1/981,607; 1 1/981 ,767; 1 1/982,467; 1 1/451,692; 12/335,168; 11/541 ,225; 1 1/927,356;

1 1/927,388; 11/938,096; 11/938,106; 10/547,214; 1 1/981,730; 1 1/981,685; 1 1/981 ,797;

12/252,280; 11/934,695; 11/934,697; 1 1/934,703; 12/265,593; 12/266,385; 1 1/938,213;

1 1/938,221 ; 12/325,922; 12/329,365; and 12/335,188, each of which is herein incorporated by reference in its entirety for all purposes and in particular for all teachings related conducting sequencing reactions on nucleic acid nanoballs. As will be appreciated, any of the sequencing methods described herein and known in the ait can be applied to nucleic acid templates and/or nucleic acid nanoballs in solution or to nucleic acid templates and/or nucleic acid nanoballs disposed on a surface and/or in an array.

[00046] Nucleotide sequencing processes are performed on the nucleic acid nanoballs, typically through sequencing-by-ligation techniques, including combinatorial probe anchor ligation ("cPAL") methods, which are described, for example, in Drmanac et ah, "Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanaoarrays,^'* Science 327:78-81, 2009 (Jan. 1 , 2010), as well as in published PCT patent applications

WO07/133831 , WO06/138257, WO06/138284, WO07/044245, WO08/070352, WO08/058282, WO08/070375; and published U.S. patent applications 2007-0037152 and 2008-0221832. In such methods, known labels, such as specific fragments containing a single molecule of a distinguishable iluorophore, are attached as labels according to well-understood rules to the target nucleic acid templates, then resequence indexed on the same types of DNA strand to provide the basis of overlapping data. The sequencing processes referred to herein are merely representative. In another embodiment, tagging is employed. Other processing techniques known or developed in the art may be employed. Then the collection of nucleic acid nanoballs on the substrate is irradiated with radiation to excite the iluorophores sufficient to cause the

fhiorophores associated with each specific label C, G, A or T to fluoresce at their unique wavelengths, from which a spatial image can be made by a camera, on a (standard or time-delay integration TDl) CCD array or a scanner in lieu of a CCD array, or other electronic current/voltage sensing techniques that may be employed in a sequencing machine. Other sensing mechanisms, such as impedance change sensors, may also be employed. The irradiation may be spectrum specific to excite only a selected fluorophore at a time, which can then be recorded by the camera, or the input to the camera may be filtered to sense and record only spectrum-specific received fluorescent radiation, or all fluorescent radiation can be sensed and recorded simultaneously on a color LCD array and then later analyzed for spectral content at each interrogation site in which there is a nucleic acid construct. The image acquisition yields a series of images of a plurality of interrogation sites that can be analyzed based on spectrum- specific fluorescence intensity through computer processing of the levels of intensity in a process herein denoted as base calling and explained in greater detail herein below. The cPAL and other sequencing methods can also be used to detect specific sequences, such as including Single Nucleotide Polymorphisms ("SNPs^'') in nucleic acid constructs, (which include nucleic acid nanoballs as well as linear and circular nucleic acid templates). The calls, or identification of the sequences of base calls, e.g., base calls may contain errors for reasons evident by the nature of the sequencing procedure. Using a computer process-based Reed-Solomon error correction, whether in the form of a computer processor performing a Reed-Solomon algorithm or in the form of a comparison mechanism using precompiled expected base call sequences, such as in a look-up table, errors can be identified, "nocall" sequences can be flagged and corrections can be made to y ield corrected base call sequences. It should be understood that the magnitude of the sites and stmctures herein depicted are merely a minute fraction of the magnitude of the sites and structures analyzed on a substrate, as they do not easily admit to illustration. For example the substrate may be a photolithographically etched, surface modified (SOM) 25 mm by 75 mm silicon substrate with grid-patterned arrays of about 300-nm spots for nucleic acid nanoballs binding to increase DNA content per array and improve image information density as compared to random genomic DNA arrays.

[00047J Sequencing probes may be detectably labeled with a wide variety of labels. Although the foregoing is primarily directed to embodiments in which the sequencing probes are labeled with fiuorophores. it will be appreciated that similar embodiments utilizing sequencing probes comprising other kinds of labels are encompassed by the present invention. Moreover, the processes according to the invention can be employed with unlabeled structures. [00048] In some embodiments, multiple cycles of cPAL (whether single, double, triple, etc.) will identify multiple bases in the regions of the target nucleic acid adjacent to the adaptors. (It is possible to employ a single cycle of cPAL to render multiple bases in an alternate design.) In brief, cPAL methods are repeatedly executed for interrogation of multiple bases within a target nucleic acid by cycling anchor probe hybridization and enzymatic ligation reactions with sequencing probe pools designed to detect nucleotides at varying positions removed from the interface between the adaptor and target nucleic acid. In any given cycle, the sequencing probes used are designed such that the identity of one or more of the bases at one or more positions is correlated with the identity of the label attached to that sequencing probe. Once the ligated sequencing probe, and hence the base or bases at the interrogation position or positions are detected, the ligated complex is stripped off of the nucleic acid nanoballs and a new cycle of adaptor and sequencing probe hybridization and ligation is conducted. By this mechanism, oversampled data are obtainable.

[00049] Selected Definitions

[00050] "Adaptor" refers to an engineered construct comprising "adaptor elements" where one or more adaptors may be interspersed within target nucleic acid in a library construct. The adaptor elements or features included in any adaptor vary widely depending on the use of the adaptors, but typically include sites for restriction endonuclease recognition and/or cutting, sites for primer binding (for amplifying the library constructs) or anchor primer binding (for sequencing the target nucleic acids in the library constructs), nickase sites, and the like. In some aspects, adaptors are engineered so as to comprise one or more of the following: 1) a length of about 20 to about 250 nucleotides, or about 40 to about 100 oligonucleotides, or less than about 60 nucleotides, or less than about 50 nucleotides; 2) features so as to be ligated to the target nucleic acid as at least one and typically two "arms"; 3) different and distinct anchor binding sites at the 5' and/or the 3 ' ends of the adaptor for use in sequencing of adjacent target nucleic acid; and 4) optionally one or more restriction sites. In one aspect, adaptors can be interspersed adaptors. By "interspersed adaptors" is meant herein oligonucleotides that are inserted at spaced locations within the interior region of a target nucleic acid. In one aspect, "interior" in reference to a target nucleic acid means a site internal to a target nucleic acid prior to processing, such as circularization and cleavage, that may introduce sequence inversions, or like transformations, which disrupt the ordering of nucleotides within a target nucleic acid. Use of interspersed adaptors facilitates sequence reconstruction and alignment, as sequence runs of 10 bases each from a single adaptor can allow 20, 30. 40, etc. bases to be read without alignment, per se.

[00051] "Amplicon" means the product of a polynucleotide amplification reaction. That is, it is a population of polynucleotides that are replicated from one or more starting sequences.

Amplicons may be produced by a variety of amplification reactions, including but not limited to polymerase chain reactions (PCRs), linear polymerase reactions, nucleic acid sequence-based amplification, circle dependant amplification and like reactions (see, e.g., U.S. Pat. Nos.

4,683,195; 4,965,188; 4,683,202; 4,800159; 5,210,015; 6,174,670; 5,399,491; 6,287,824 and 5,854,033; and US Pub. No. 2006/002471 1).

[00052] The term "base" when used in the context of identification refers to the purine or pyrimidine group (or an analog or variant thereof) that is associated with a nucleotide at a given position within a target nucleic acid. Thus, to call a base or to identify a nucleotide both refer to determining a data value identifying the purine or pyrimidine group (or an analog or variant thereof) at a specific position within a target nucleic acid. The purine and pyrimidine groups include the ibur main nucleotide bases of C, G, A, and T.

[00053] "Polynucleotide", "nucleic acid", "Oligonucleotide", "oligo" or grammatical equivalents used herein refers generally to at least two nucleotides covalently linked together in a linear fashion. A nucleic acid generally will contain phosphodiester bonds, although in some cases nucleic acid analogs may be included that have alternative backbones such as

phosphoramidite, phosphorodithioate, or methylphophoroamidite linkages; or peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with bicyciic structures including locked nucleic acids, positive backbones, non-ionic backbones and non-ribose backbones.

[00054] The term "reference polynucleotide sequence^", or simply "reference", refers to a known sequence of nucleotides of a reference organism. The reference may be an entire genome sequence (e.g., a reference genome) of a reference organism, a portion of a reference genome, a consensus sequence of many reference organisms, a compilation sequence based on different components of different organisms, a collection of genome sequences drawn from a population of organisms, or any other appropriate sequence. The reference may also include information regarding variations of the reference known to be found in a population of organisms. The reference organism may also be specific to the sample being sequenced, possibly a related individual or the same individual, separately drawn (possibly normal to complement cancer sequence).

[00055] "Sample polynucleotide sequence" refers to a nucleic acid sequence of a sample or target organism derived from a gene, a regulatory element, genomic DNA, cDNA, NAs including inRNAs, rRNAs, siRNAs, miRNAs, and the like, and/or from fragments thereof. A sample polynucleotide sequence may be a nucleic acid from a sample, or a secondary nucleic acid such as a product of an amplification reaction. For a sample polynucleotide sequence or a polynucleotide fragment to be "derived" from a sample polynucleotide (or any polynucleotide) can mean that the sample sequence/polynucleotide fragment is formed by physically, chemically, and/or enzymatically fragmenting a sample polynucleotide (or any other polynucleotide). To be "derived" from a polynucleotide may also mean that the fragment is the result of a replication or amplification of a particular subset of the nucleotide sequence of the source polynucleotide.

[00056] A "read" refers to a set of one or more data values that represent one or more nucleotide bases. A "mated read" (also referred to as "mate-pair") refers generally to a set of individual nucleotide reads originating from two distinct regions of genomic sequence (arms) located at opposite ends of a DNA fragment across a distance of a few hundred or thousand bases. The mated read may be generated during sequencing from a fragment of a larger contiguous polynucleotide (e.g., DNA) obtained from the sample organism to be variation called and/or reassembled.

[00057] "Mapping" refers to one or more data values that relate a read (e.g., such as a mated read) to zero, one or more locations in the reference to which the read is similar, e.g., by matching the instantiated read to one or more keys within an index corresponding to a location within a reference.

[00058] "Hybridization" refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The resulting (usually) double-stranded polynucleotide is a "hybrid" or "duplex." "Hybridization conditions" will typically include salt concentrations of less than about 1M, more usually less than about 500 mM and may be less than about 200 mM. Hybridization temperatures can be as low as 5^C C, but are typically greater than 22° C, and more typically greater than about 30° C, and typically in excess of 37° C. [00059] "Ligation" means to form a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically or chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon terminal nucleotide of one oligonucleotide with a 3' carbon of another nucleotide. Template driven ligation reactions are described in the following references: U.S. Pat. Nos. 4,883,750; 5,476,930; 5,593,826; and 5,871 ,921.

[00060] "Logic" refers to a set of instructions which, when executed by one or more processors (e.g., CPUs) of one or more computing devices and/or computer systems, are operable to perform one or more functionalities and/or to return data in the form of one or more results and/or data that is used by other logic elements. In various embodiments and

implementations, any given logic may be implemented as one or more software components that are executable by one or more processors (e.g., CPUs), as one or more hardware components such as Application-Specific Integrated Circuits (ASICs) and/or Field- Programmable Gate Arrays (FPGAs), or as any combination of one or more software components and one or more hardware components. The software component(s) of any paiticular logic may be implemented, without limitation, as a standalone or client-server software application, as a client in a client- server system, as a server in a client-server system, as one or more software modules, as one or more libraries of functions, and as one or more static and/or dynamically-linked libraries.

During execution, the instructions of any particular logic may be embodied as one or more computer processes, threads, fibers, and any other suitable run-time entities that can be instantiated on the hardware of one or more computing devices and can be allocated computing resources that may include, without limitation, such as memory, CPU time, storage space, and network bandwidth.

[00061] "Primer" means an oligonucleotide, either natural or synthetic, which is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a DNA polymerase. [00062] "Probe" means generally an oligonucleotide that is complementary to an oligonucleotide or target nucleic acid under investigation. Probes used in certain aspects of the claimed invention are labeled in a way that permits detection, e.g., with a fluorescent or other optically-discernable tag.

[00063] "Sequence determination" (also referred to as "sequencing") in reference to a target nucleic acid means detennination of information relating to the sequence of nucleotide bases in the target nucleic acid. Such information may include the identification or determination of partial as well as full sequence information of the target nucleic acid. The sequence inforaiation may be determined with varying degrees of statistical reliability or confidence. In one aspect, sequencing includes the determination of the identity and ordering of a plurality of contiguous nucleotides in a target nucleic acid starting from different nucleotides in the target nucleic acid. Sequencing and the various steps thereof may be performed by a sequencing machine that comprises a reaction subsystem and an imaging subsystem. The reaction subsystem includes flow devices (on which biochemical reactions take place between various reagents, buffers, etc. and a biochemical sample or fragments derived therefrom) and various other components (e.g., such tubing, valves, injectors, actuators, motors, and the like) that are configured to dispose the reagents, buffers, sample fragments, etc. on, or in, the flow device. The imaging subsystem comprises a camera, a microscope (and/or appropriate lenses and tubing), a stage that holds the flow device during sequencing, and various other components (e.g., such as motors, actuators, robotic arms, etc.) for placing and adjusting the flow device on the stage as well as adjusting the relative positions of the camera and the microscope.

[00064] "Target nucleic acid" means a nucleic acid of (typically) unknown sequence from a gene, a regulatory element, genomic DNA, cDNA, RNAs including mRNAs. rRNAs. siRNAs, miRNAs and the like and fragments thereof. A target nucleic acid may be a nucleic acid from a sample, or a secondary nucleic acid such as a product of an amplification reaction. Target nucleic acids can be obtained from virtually any source and can be prepared using methods known in the art. For example, target nucleic acids can be directly isolated without amplification, isolated by amplification using methods known in the art, including without limitation polymerase chain reaction (PCR), strand displacement amplification (SDA), multiple

displacement amplification (MDA), rolling circle amplification (RCA), rolling circle

amplification (RCR) and other amplification (including whole genome amplification) methodologies. Target nucleic acids may also be obtained through cloning, including but not limited to cloning into vehicles such as plasmids, yeast, and bacterial artificial chromosomes. In some aspects, the target nucleic acids comprise mRNAs or cDNAs. In certain embodiments, the target DNA is created using isolated transcripts from a biological sample. Target nucleic acids can be obtained from a sample using methods known in the art. As will be appreciated, the sample may comprise any number of substances, including, but not limited to, bodily fluids such as, for example, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred. Methods of obtaining target nucleic acids from various organisms are well known in the art. Samples comprising genomic DNA of humans find use in many embodiments. In some aspects such as whole genome sequencing, about 20 to about 1 ,000,0000 or more genome-equivalents of DNA are preferably obtained to ensure that the population of target DNA fragments sufficiently covers the entire genome.

[00065] Exemplary Methods of Genome Sequencing and CNV Estimation

[00066] The present invention is directed to methods for estimating copy number variants of genomic regions of interest at a detection position in a target sequence in a sample, which find use in a wide variety of applications as described herein.

[00067] The methods of the present disclosure may also include extracting and fragmenting target nucleic acids from a sample and/or sequencing the target nucleic acids for which CNV estimation is perfonned. These fragmented nucleic acids are used to produce target nucleic acid templates that generally include one or more adaptors. The target nucleic acid templates are subjected to amplification methods to form nucleic acid concatemers such as, for example, nucleic acid nanoballs.

[00068] In one aspect, nucleic acid templates can comprise target nucleic acids and multiple interspersed adaptors, also referred to herein as "library constructs," "circular templates", "circular constructs", "target nucleic acid templates", and other grammatical equivalents. The nucleic acid template constructs are assembled by inserting adaptors molecules at a multiplicity of sites throughout each target nucleic acid. The interspersed adaptors permit acquisition of sequence information from multiple sites in the target nucleic acid consecutively or

simultaneously. [00069] In further embodiments, nucleic acid templates formed from a plurality of genomic fragments can be used to create a library of nucleic acid templates. Such libraries of nucleic acid templates will in some embodiments encompass target nucleic acids that together encompass all or part of an entire genome. That is, by using a sufficient number of starting genomes (e.g. genomes of cells), combined with random fragmentation, the resulting target nucleic acids of a particular size that are used to create the circular templates sufficiently "cover" the genome, although as will be appreciated, on occasion, bias may be introduced inadvertently to prevent the entire genome from being represented.

[00070] Further embodiments and examples of methods of constructing nucleic acid templates are described in U.S. application Ser. Nos. 11/679, 124; 1 1 /981 ,761; 1 1/981 ,661 ; 1 1/981 ,605; 1 1/981,793; 1 1/981 ,804; 1 1/451,691 ; 1 1/981,607; 1 1/981 ,767; 1 1/982,467; 1 1/451 ,692;

12/335,168; 1 1 /541,225; 1 1/927,356; 11 /927,388; 1 1/938,096; 1 1 /938,106; 10/547,214;

11/981 ,730; 1 1/981 ,685; 11/981 ,797; 12/252,280; 11/934,695; 1 1/934,697; 1 1/934,703;

12/265,593; 12/266,385; 1 1/938,213; 11/938,221 ; 12/325,922; 12/329,365; and 12/335,188, each of which is herein incorporated by reference in its entirety for all purposes and in particular for all teachings related to constructing nucleic acid templates of the techniques described herein.

[00071] Nucleic acid templates of the techniques described herein may be double stranded or single stranded, and they may be linear or circular. In some embodiments, libraries of nucleic acid templates are generated, and in further embodiments, the target sequences contained among the different templates in such libraries together cover all or part of an entire genome. As will be appreciated, these libraries of nucleic acid templates may comprise diploid genomes or they may be processed using methods known in the art to isolate sequences from one set of parental chromosomes over the other. As will also be appreciated by those of skill in the art, single stranded circular templates in libraries may together comprise both strands of a -chromosome or chromosomal region (i.e., both "Watson" and "Crick" strands), or circles comprising sequences from one strand or the other may be isolated into their own libraries using methods known in the art.

[00072] For any of sequencing methods known in the art and described herein using nucleic acid templates, the techniques described herein provide methods for determining at least about 10 to about 200 bases in target nucleic acids. In further embodiments, the techniques described herein provide methods for determining at least about 20 to about 180, about 30 to about 160, about 40 to about 140, about 50 to about 120, about 60 to about 100, and about 70 to about 80 bases in target nucleic acids. In still further embodiments, sequencing metliods are used to identify 5, 10, 15, 20, 25, 30 or more bases adjacent to one or both ends of each adaptor in a nucleic acid template.

[00073] Overview OF TECHNIQUES FOR CNV CALLING

[00074] CNV calling for normal and tumor samples share some features but also have differences, in some embodiments, both types of samples are subject to the following three steps.

[00075] 1 ) Computation of sequence coverage.

[00076] 2) Estimation and correction of bias in coverage:

a. Modeling of coverage bias;

b. Correction of modeled bias;

c. Coverage smoothing.

[00077] 3) Normalization of coverage by comparison to a baseline sample of set of samples. Following this, both normal and tumor samples are segmented using Midden Markov Models

(HMMs), but with different models for the two sample types in the following steps:

[00078] 4A) HMM segmentation, scoring and output for normal samples;

4B) Modifications to HMM segmentation, scoring and output for tumor samples;

Finally, normal samples are subjected to a 'no -calling' process which identifies CNV calls that are suspect in the following step:

[00079] 5) Population-based no-calling/identification of low-confidence regions.

[00080] In various embodiments, the above steps of CNV calling may be performed by different types of logic that is executed on one or more computing devices.

[00081] EXAMPLE EMBODIMENTS OF TECHNIQUES FOR CNV CALLING

J00082] 1. Computation of sequence coverage

[00083] As used hereinafter, "DNB^" refers to the sequence of a nucleic acid nanoball irom which one or more reads (e.g., such as a mated read) have been sequenced. It is noted that in the reads sequenced from a biological sample or fragments thereof, a DNB is represented as one or more reads that may or may not cover the entire sequence that constitutes the DNB. For example, in one embodiment the DNB is represented by a mated read that comprises two or more ami reads, from opposite ends of the NB, that are separated by an unknown sequence of a few hundred bases.

[00084] in one aspect, all mate-pair constraint-satisfying paired-end (e.g., foil DNB) mappings are used to compute sequence coverage. In a certain embodiment, a unique paired end mapping contributes a single count to each base of the reference that is aligned to the DNB. A reference base aligned to a nonunique paired-end mapping is weighted (e.g., is given a fractional count) based on the estimated probability that the mapping is the correct location of the DNB in the reference. Fractional attribution of DNBs in proportion io the confidence in each mapping thus provides the ability to give reasonable coverage estimates in regions where mappings are non- unique.

[OODSSj in one aspect, each position / of the reference genome R receives the following coverage value c,-:

where M, is the set of mappings over all DNBs such, that a called base i each mapping is aligned to position i_f DNB_m is the DNB described by mapping , N(m) is the set of all mappings involving DN.B_!}!, and a is the probability that a DNB is generated in a fashion that does not allow it to map to the reference.

[00086] According to the techniques described herein, computer logic (e.g., such as CNV caller 18 in FIG. 1 and/or a component thereof such as coverage calculation logic 22) computes the coverage values for the positions (or loci) in the reference genome based on the DNB mappings. The computer logic then includes the computed coverage values in measurement data that is used in subsequent processing.

[00087] 2. Estimation and correction of bias coverage (Sample-internal manipulation of^* coverage)

[00088] Curreatty, genome sequencing can result in coverage bias that may affect estimation of copy number. One element of the bias involves the GC content over intervals approximately the length of the ini tial DM A fragment that becomes a DNB (e.g., approximateiy 400bp)_s tho ugh other factors are known as well, in one embodiment, it is generally preferred to model and correct for such biases prior to or as pari of copy number estimation. |OO089J In another embodiment, it is desirable to apply some smoothing to short scale fluctuations in coverage, which may be at least partly specific to an individual library of circles or DNBs.

(00090) There are several approaches to bias correction and smoothing that may be used. Ail of the operations and steps in these approaches may be performed b computer logic (e.g., such as C V cal ler 18 in FIG, 1 and/or a component thereof such as GC correction logic 34) based on measurement data that includes, but may not be limited to. coverage values for each position in the reference genome.

f 00091] Approach^: Post- hoc coverage correction:

[00092) In one embodiment, the sequence coverage as described above is smoothed by window- averaging and then adjusted to account for GC bias in the library construction and sequencing process.

[00093] Window-averaging is performed by computing the mean of the unsmoothed coverage values for ever position within a window. For windo length N, the averaged coverage reported at position i is: " c. ?N

{00094] From such smoothed coverage, in one embodiment a set ofadjustmeni factors is computed. GC content is computed over 1000 base pair windows (i.e. N-1000) every 1000 bases along each reference contig. Each window is assigned to one of 1000 bins based on the number of Gs and Cs present in the portion of the reference covered by the window. Let ff ' be the set of tabulated windows (equiva)ently, iheir center positions) and be the set of wi ndows with [G+ ~ b. The average uncorrected coverage for each bin b, is s¾., determined as:

Letting be the mean coverage over the full genome (C—

for each GC bin b a correction factor ^ is given as:

100095) In another embodiment, the eoiTection factors may be estimated using further smoothing operations. This may provide, e.g., greater robustness to small-sample variation or over tting, For instance, the temis _δ may be subjected to smoothing using splines, piecewise regression, sliding windo averaging, LOESS, etc. 0Θ896] The corrected, smoothed coverage for a 1000-base window centered at position i assigned to bin bi, is then computed as follows:

[00097] Corrected, smoothed coverage for larger windows, of length = ^* 1000 (n being a positive integer) can be computed as the mean, over the values for the contained 1000-base windows.

[00898] in addition to the above, it should be clear that many embodiment variations can exist. Window sizes and shifts may be changed. Certain positions may be i nored (and the

.corresponding windows either enlarged to achieve a fixed number of accepted positions or means taken only on the accepted positions), based on various characteristics such as sinictural annotation (e.g. repeats), excess or insufficient variability among multiple samples,

accessibility/uniqueness under the criteria used for mapping, depth of coverage in simulated data (measuring mapabiiity) etc. The mathematical mean may be replaced by median, mode or other summary statistics in appropriate locations. Correction factors may be computed based on the coverage of a single position rather than the average coverage for a window, with

smoothing/averaging being applied after correction rather than before.

|!§0§99| This class of exemplary approaches may be extended to consider multiple predictors of coverage by computing correction factors for m uliidimensional binning of positions on the genom e. For example, not only GC content on the scale of full DNB can be considered, but also on the scale of the individual DNB arms. Alternatively, separate correction factors can be computed for each predictor, corresponding to an assumption of independence of the effects.

[0Ο0ίΟ§] A proach 2: Mapping-level coverage correction:

{000101] In the second approach to bias correction and smoothing, individual mappings are given an additional weighting factor to compensate for bias prior to smoothing, DNBs

( mappings) that are more likely due to the bias than expected of a uniform random sampling are downweighted, while DNBs that are less likely due to the bias are upweighted (and may contribute more than a full count t the coverage computation). Letting q_m be the correction factor for mapping m (defined below), corrected coverage at position can be computed as:

O 0102J The correction factor q_m, is determined based on the odds ratio derived from a logistic regression model fit to discriminate mappings in the real dataset from, mappings in a dataset simulated with uniform random sampling of the reference genome. The model predicts whether a given mapping is real or simulated based on a b~spline of order 1 (piece wise linear) with knots at ever fifth percentile of the distribution of GC content in the combined (real+simulation) dataset. For example, the corresponding R code is:

model <-gmi(isReal~

bs(dnbG€pcnt,d:i¾0 egree^si1 ^

where the input dataset, d, is composed of equal numbers of records of unique paired-end simulated mappings and records of unique paired-end real mappings. For simulation records, tsReal-0; for real records, isReal^:::l , dnbGCpcnt is the percent GC in the portion of the reference spanned by the mapping.

[000103] Given the resulting model, the correction factor q_m is taken as the model-predicted sim:real odds ratio given the GC percentage tor mapping m . Thus, if a given GC percentage is three times more likely hi real data than in simulated data, real mappings with that GC conient are weighted by a factor of 1/3.

[0001 4] A similar odds-ratio based factor could be determined using a logistic model accounting for many properties of a mapping, including factors such as:

Composition of entire fragment (~50Obp);

Composition of genomic segments in final DNB ( -~89bp);

Choice of base at every position in final DNB ;

Oligomers at specific locations in original fragment;

Sequences adjacent to adaptors (ligation efficiency impact, e.g.);

Sequences at typical locations of restricti n enzyme cut sites;

Predicted physical characteristics;

Melting temperatures;

Flexibility/curvature; * Measure mieasurable/predieted characteristics of regions of the genome such as Histone binding and Methylatioa.

f 00105] The model could include not only linear effects of single measurements but also various transformations of single measurements (e.g. piecewise linear or polynomial fitting or binning) and interaction terms.

1000106] In a certain embodiment, model-corrected coverage is then smoothed via sliding window averaging, and rounded to an integer. The width of the window is configurable; the default value is 2kb. Average coverage is by default reported for abutting windows (e.g. for window shifts equal to the window width), but other shift amounts can be employed. Average corrected coverage is reported for the posi tion at the midpoint of each window.

[000107] Each contig (or a region of contiguous loci) of the reference genome is processed separately, so thai with default width - 2k each contig > 2kb in length results in coverage values at ikb, 3kb, 5kb, ... rela tive to the s tart of the contig. Thus, for such a. posi t ion

smoothed coverage is given as:

[000108] The first window of each contig staris at the first base of the contig; the window is shifted until the end of the window would be beyond the end of the contig. S ince the starting position of a contig relative to its chromosome may be an arbitrary value, the chromosome position reported for a given window may not be a nice round number,

[000.109! Approach^: GC normalization process

(0001.10] hi one embodiment, a computer logic (e.g., such as CNV caller 18 in FIG. 1 or a component thereof such as GC correction logic 34) estimates and corrects bias in coverage as follows.

(0001 ϊ 1 ] First. GC content is computed for the 1000-base window centered at every point of the genome (excluding positions less than 500 bases from the ends of contigs). For example, a function isGC ) can be set to 1 if the. base at position / is G or C and 0 otherwise, and the GC content at position i, gc_h can be computed as follows:

V [000112] Positions less than 500 bases from either end of a contig are not considered during the estimation of the GC correction factors,

060113] Next, for each possible GC value % the mean coverage Q is determined for positions with get = I", Letting n_Y be the number of positions in the genome with gc₍— % the mean coverage may be computed as follows:

£ _{w Ci}

^>

[00611.4] In example implementations, the positions with coverage > 500 may he excluded,

[060115] Next, the above two steps are completed for a simulation. Using the superscript to indicate simulation results, the simulated mean coverage may be determined as follows:

T .

; ί γ

000.116] It is noted that fee above result is not entirely flat due to the GC content of ubiquitous repeats, micro-satellite regions, etc., not being the similar to the genome as a whole.

1000117] Next, the ratio of sample coverage to simulation coverage is computed for each GC value, adjusting for the overall average coverage of the sample and the simulatio (i and c* respectively). For example, this ratio may be computed as follows:

[000118) Next, a smoothed coverage ratio is obtained as a function of GC, /I... For example, a locally weighted polynomial regression may be used as follows:

[006119] A a local regression operation, LOESS smoothing is performed except: in numerically unstable regions where LOWESS is performed instead.

Next the GC-eorrecl (single-base) coverage at every position of the genome is computed as follows:

[600120] Near the ends of the contigs, 'missing bases' are filled in with genome- wide average GC content. If the GC content of the window for a given position is too extreme (i.e. < 20% or > 80% GC), the coverage value is treated as unknown (e.g., as missing data). 1000121] Window-smoothing is performed by taking the mean of for each position i within a given window. Windows are tiled (adjacent,, non-overlapping}, with the choice of window boundaries as defined in the section below titled "Window Boundary Definition". That is, for a window corresponding to the interval the average corrected coverage ¾ ,· is computed as

[000122] It is noted that for notational. simplicity, in the sections below the " " subscript is dropped, i.e. s¾ is used in place of as there is at most one window starting at position i.

[0 123| 3. Normalization of covera e by comparison to a baseline sample

[0001 4] In various embodiments, the operations, computations, and method steps described in this section (Section 3) can be performed by computer logic such as, for example, CNV variant caller 18 in FIG. I and/or by a component thereof such as, for example, ploidy-aware correction logic 36.

[000125] In some embodiments, bias in coverage not corrected by the adjustments described above may be taken into account by comparison to a baseline sample. However, in order to obtain coverage proportional to absolute copy number, the baseline sample may be adjusted according to the copy number in said sample.

[000.126] Letting d and p_t be be the coverage and ploidy at position i for the baseline sample, and i be an estimate of the typical diploid coverage for the baseline sample, the bias correction factor ^j ca be determined as follows:

(In one implementation, ά may be taken as the 45% percentile of windows in the autosome. )T he normalized corrected coverage eg is then computed as follows:

If/?,- ^~ 0 (in which case is due to mismappings and not a reliable indicator of coverage

behavior in this location), as treated as missing. This correction of bias performed based o known or hypothesized ploidy and coverage at position(s) in a baseline sample is referred to herein as "ploidy-aware baseline correction." Specifically, the ploidy-aware baseline correction adjusts the coverage value for each position (or locus) in a baseline or reference sample based on the ploidy and coverage detected for that same position in the target polynucleotide sequence of the target sample, as an element of using the baseline values to correct the coverage in the sample to be analyzed.

(000127] In some implementations, the sequences of a group of samples may be used, rather than a single sample, as the baseline, in order to reduce sensitivity to fluctuations due to sampling (statistical noise) or due to library-specific biases. For example, the following for the set of baseline samples S may be used:

where /?,·, the ploidy at window . Ideally, this would be the true ploidy for the baseline sample for this window. However, since it is not known, it needs to be estimated.

[000128] Thus, in one implementation, a baseline generation process includes CNV-calling for each baseline genome, using a simulation where copy number is 2 for autosomes and gender- appropriate for sex chromosomes. Using a simulation as the baseline provides an indirect means of collecting for variation in mapability of the genome, e.g. regions corresponding to high-copy, high-identity repeats that "overflow" during mapping. However, this may not address coverage bias due to biochemistry. In regions of moderate coverage bias, and where the length scale of bias is short relative to the length of the window, the baseline genome(s) will be called al the correct ploidy and consequently the correction factor will appropriately compensate for the bias. However, regions with a sustained bias resulting in coverage being > 50% of the diplo id-average away from the true ploidy will have their copy number miscalled on the baseline genomes; this results in a baseline "correction" that reinforces the tendency to call CNVs at this location, i.e. results in robust/consistent miscalling of abnormal ploidy. In other implementations, estimation of the ploidy of baseline genomes may be based on external information (e.g. chip-based CNV calls), manual curation, or an automated process that attempts to determine population patterns by simultaneous analysis of multiple genomes.

[000129] In other embodiments, d may be determined in various ways. For instance, it may be taken as the median of positions estimated to have ploidy 2 in a previous estimation of ploidy for the baseline sample, as the modal coverage value, or as some fixed percentile of the coverage over the whole genome (perhaps adjusted for male vs female samples). A group of samples may be used, rather than a single sample, as the baseline. In this case, ά\ and p, be might be taken as the sums of coverage and ploidy over all baseline samples at reference position i, and d may be determined as the sum over samples of typical diploid coverage. Alternatively, a mean or median of values computed for each of several baseline samples may be used in order to provide estimates that were less sensitive to differences in coverage among baseline samples.

If no samples are input as baseline, then c" is simply set as follows:

[000130] 4A.HMM segmentation, scoring and output for normal samples

[000131] In various embodiments, the operations, computations, and method steps described in this section (Section 4A) can be performed by computer logic such as, for example, CNV variant caller 18 in FIG. 1 and/or a component thereof such as HMM model logic 20.

[000132] In certain aspects, there are many approaches to segmenting a quantitative time-series that can be applied to calling CNVs— that is, that can be applied to coverage data produced by the above sequence of steps. Hidden Markov Models (HMMs) provide one such approach with certain appealing properties (obvious model fitting methods, flexible models, natural confidence measures, ability to constrain models, ability to incorporate a variety of models of coverage generation), wherein states correspond to copy number levels, emissions are some form of coverage (observed/corrected/relative), and transitions between states are changes in copy number. Emission probabilities may be modeled as Poisson distributions, negative binomial, mixtures of Poisson distributions, piecewise models fit to the data, etc. Choice of model can be made with goodness of fit measures and cross-validation. In one embodiment, it may be desirable to smooth per-position coverage values over longer (sliding) windows, though it is desirable for the window width to be considerably narrower than the desired minimal event size. In one embodiment, it may be desirable to constrain the models in various ways, e.g. require that the expected outputs of each copy number level (e.g. means of emission probabilities of states in an HMM) be consistent multiples of one another, as expected from discrete copy number changes, in one embodiment, it may be desirable to include in the expected coverage

distributions components corresponding to 'contamination' of a tumor sample with normal tissue, or to capture tumor heterogeneity, e.g. with mixture models. [000133] In another aspect, it is possible to integrate other signals (e.g., parameters and values thereof) into CNV detection, or to use other signals (e.g., data values) to confirm or filter output from a coverage-based CNV detector. Such other signals include the presence of anomalous mate pairs at the boundary between two copy number levels, or changes in allele balance in heterozygous positions.

[000134] In yet another aspect, a particular HMM-based method for estimating the copy number may be used based on a function of reference genome position. For example, GC- corrected, window-averaged, normalized coverage data, c", may be input to an HMM whose states correspond to integer ploidy (copy number). Copy number along the genome may be estimated as the ploidy of the sequence of most likely states according to the model. Various scores are computed based on the posterior probabilities generated by the HMM. This aspect is described in more detail below.

[000135) Model definition:

[000136] A fully-connected HMM with states corresponding to ploidy 0, ploidy 1 , ploidy 2, .... ploidy 9, and ploidy "10 or more" is defined by a matrix of transition probabilities, initial state probabilities, and the emission probabilities. (In various embodiments, the exact number of states can be modified.)

[000137} Coverage distributions (i.e. state emission probabilities) are modeled as a negative binomial, which can be parameterized by the mean and variance of the distribution for each state.

[000138] Model estimation:

[000139] In principle, model parameters can all be estimated by estimation-maximization (EM) by the Baum- Welch algorithm; however, in practice, unconstrained estimation (especially of coverage distributions) does not always provide satisfactory results. To address this issue, in one embodiment initial values are chosen and subsequent updates are constrained to reflect the following assumptions: coverage is assumed to depend on the number of copies of a given reference segment in the genome of interest; copy number is assumed to be integer valued;

coverage is assumed to be linearly related to copy number; the majority of the genome is assumed to be diploid, so that the ''typical" value for the autosome can be used to fix the mean coverage for ploidy = 2; for states corresponding to ploidy >= 1 , the standard deviation of a state is assumed to be proportional to the mean of the state; for the state corresponding to ploidy = 0, a separate variance can be used to allow for the impact of mis-mappings and non-unique mappings. Given these constraints and assumptions, there are only two free parameters regarding the- coverage distributions, namely a single value relating coverage to standard deviation for ploidy >- 1 and another variance parameter for ploidy - 0.

[0001401 In one implementation, transition probabilities can be estimated from the data but the default behavior would be to maintain the initial values. The initial values may be set by the user; if not set, initial values may default to i,j - 0.01 . e.g. a one percent chance of being in state j at time t+1 given that the model is in state / at time t for any distinct states i and j. In another implementation, transition probabiHties coold be estimated from the data but ibe risk of over- fitting is high. Consequently, a set of default values may be used, such that the probability of transition from one state to another at any "time" (window) is set to 0.003, and the probability of remaining in a given state is taken as 1-0.003*10 = 0.97.

|0O¾141 j Initial state probabilities are all set to Ί divided by the number of states.

The mean of the emission (coverage) distribution for a state with ploidy n is initialized as

follows, except as noted below: where c*^* is the median of e for all positions at which normalized smoothed corrected coverage has been computed. To allow for the presence of some apparent coverage due to

nnsraappings, in one embodiment μ₍ is set to L i.e., μ_ϋ - 1; in another embodiment μ<, may be set as ½ ~ Cli * ¾f . Initial estimates of the means are not updated during subsequen model fitting.

[000142] The initial variance of the ploidy 2 state is set to;

|0Οβ143^'] In some implementations, variance for other states is set so that standard deviations will be proportional to the means;

|!000144j In other implementations,, the initial variance of the negative binomial may be set as follows [000145] The variance-determining parameters are updated by EM until the model has 'converged¹, e.g. the change in log likelihood of the data given the model between successive iterations is sufficiently small, e.g., below a certain threshold.

[000146] In another aspect, the initial variance estimates can be updated during model fitting (using EM with the modifications to constrain the mean), but are constrained to never be smaller than the above. This model operates under the assumption that the majority of the genome is diploid, that the median of the entire distribution will be near the median, and thus the mean, of the diploid portion of the genome, and that copy numbers are strictly integer values. In this aspect, adjustments may need to be made over time to estimate copy number for highly aneuploid samples, for tumors with substantial 'normal contamination', and for regions that are not unique in the reference.

[000147] The updating procedure is allowed to iterate until it has 'converged', e.g. the change in log likelihood of the data given the model changes less than 0.001 between successive iterations.

[000148] Ploidy inference, segmentation and scoring:

[000149] In a further embodiment, after convergence of the estimation procedure, tlie usual HMM inference computations are performed. The final result is based on the most likely state at each position. (A standard alternative is to assign ploidy corresponding to the states of the most likely single path.)

[000150J In one embodiment, the "calledPloidy" of each position in the input is taken to be that of the most likely state at that position. The "ploidy Score" is given as a phred-like score (e.g., a logarithm-based score measured in decibels dB) reflecting the confidence that the called ploidy is correct. The "CNVTypeScore" is given as a phred-like score reflecting the confidence that the called ploidy correctly indicates whether the position has decreased ploidy, expected ploidy, or increased ploidy relative to the nominal expectation (diploid except that sex chromosomes in males are expected haploid). Additional scores at each position ("scorePIoidy=0",

"scorePloidy^l^" etc) reflect the probability of each possible pioidy; the score at each state is int(101o l 0 (Lis)) where Lj_S is the likelihood of being in state s at position /.

[000151] In another embodiment, 'segment' is a sequence of adjacent positions that have the same called ploidy. The 'begin' and 'end¹ positions of the segment are taken as outside the midpoints of the beginning and ending windows. Each segment is given a ploidyScore equal to the average of the ploidyScores for the positions in the segment, and a C VTypeScore that is the mean of the CNVTypeScores for the positions in the segment.

[000152] Precise definitions and justification of the above scores are given the section below titled "Score Computation".

[GG0153J 4B. Modifications to HMM segmentation, scoring and output for tumor samples (Tumor CNV approach)

[000154] In various embodiments, the operations, computations, and method steps described in this section (Section 4B) can be performed by computer logic such as, for example, CNV variant caller 18 in FIG. l and/or a component thereof such as HMM model logic 20.

[000155] In certain aspects, copy-number catling in tu or samples poses several challenges to the methods described so far. Due to the possibility of high average copy number, it is not advisable to assume that diploid ('normal') regions of the genome will have coverage near the sample median. Even if the typical coverage for a diploid region could be determined (e.g. by analysis of minor allele frequency), the expected change in coverage for an increase or decrease of a single copy is not necessarily 50% of this value, due to the possibility of an unknown amount of contamination from adjacent or mixed-in normal cells ('normal contamination^"). And even among tumor cells, a segment of the genome may not be characterized by an integer copy number, due to tumor heterogeneity.

[000156] Consequently, it is useful to relax the assumptions that constrain the coverage levels of the states of the model, allowing the ratios of coverage to be continuously valued. This increases the challenge of finding the correct values and also introduces the problem of deciding how many states to include, leading the analysis to include a model selection component.

Consequently, the goal of the analysis is modified to be to segment the genome into regions of uniform 'abundance class', without forcing an interpretation of a given class as being an integer copy number.

[000157] In theory, the HMM could simply be fit with varying numbers of states, using EM to determine the expected coverage level for each state, and choose the number of states that gives the best fit. In practice, unconstrained estimation of model parameters for any given number of states is not a robust process. Consequently, to address this, in another aspect an additional initial step or module is introduced that estimates the number of states and their means based on the overall coverage distribution, and another step is introduced which optimizes an initial model by sequentially adding states to the model and then sequentially removing states from the model.

{000.158] initial model generation;.

[000159] In one embodiment, the distribution of (corrected, normalized, window-averaged) coverage over the whole genome to be segmented is a mixture of the distributions of the different abundance classes. One approach to identifying distinct abundance classes is to use input data representing initial states (or peaks) that is generated by computer logic that performs a model for interpretation of absolute copy number of complex tumors (as described heretofore). The resulting peak locations, P, are used as states in an initial model, with expected coverage values equal to the center of each peak. The variances may be estimated using EM (the same constrained model fitting as described above in connection with normal sample segmentation).

[000160]: One approach to identifying distinct abundance classes is to look for peaks in the smoothed whole genome coverage distribution. In another embodiment, another approach is to identify a mixture model which closely fits the observed coverage distribution. An improvement over direct identification of peaks is realized by applying the quantile function for a normal distribution to the cumulative distribution function (edf), and then take the difference between successive values, prior to smoothing and peak detection. ^'This latter approach may provide better sensitivity to identifying small peaks outside the central abundance classes.

000161] For example, given a histogram of coverage H ~ ¾<_¾ hi, ½, .... If„ where h_t is the. number of positions with coverage i and n is the small est value such thai less than 0.001 of the foil histogram is truncated, and letting Oip) be the quantile function for a normal distribution, the resulting peak locations, P, can be computed as follows:

1000162] The resulting peak locations, P. are used as states in an initial model, with expected coverage values equal to the center of each peak. The variances may be estimated usin EM (the same constrained model fitting as described above in connection, with normal sample

segmentation).

900163] Model refinement:

[900164] In a further embodiment, once an initial model has been inferred in this .fashion, the mode! is iteratively refined. First, additional states are evaluated. The addition of a state is evaluated between each successi ve pair of states (abundance classes, ordered by expected coverage), acceptin the addition if the improvement in likelihood (Pr(daiajmodel)) exceeds some threshold. That is. between each successive pair of states / and,;^' with expected coverage e₍ and Cj , an attempt is made t add a slate i ' with initial coverage ογ=(¾+*¼)/2, The c is optimized using EM, holding the expected coverage levels of all other (pre-existing) states fixed, if the optimizatio results in a value outside the interval (a ,■£>^■), or if the reduction Pr(data]model ) does not exceed an acceptance threshold, the addition is rejected; otherwise, the addition is accepted. If an addition is accepted, addition of a further state between i and / ^* is attempted, with recursion until the further addition is not accepted. Once addition between all pairs of successive states is rejected, the addition process is terminated. Second, removal of states is evaluated. States are removed from the model one at a time and the resulting model is optimized using EM; if the resulting model is not sufficiently worse than the previous model, then the state removal is accepted,

[090165] In certain embodiments, the segment ation further comprises generation of an ini tial model that estimates the number of states and their means based on the overall coverage distri bution, in certain embodiments, the method includes optimization of an initial model by various means known to those versed in modeling of quantitative data, including modifications to the number of states in the model as well as optimization of the parameters of each state. For example, modifications to the number of states in the model can be performed by sequentially adding states to the model and. then sequentially removing stales, or a combination of the two; similar procedures are employed in model selection methods used in multivariate regression. Optimization of the parameters of each state may be performed by estimation-maximization or many other approaches to optimizing a multivariate model.

[000166] Variations on the preceding process are well-known to those of skill in the art. For example, one might try removing each state from the maximal model to detennine which has the least impact, remove that state and recurse. Such alternatives will be known to those versed in approaches to multivariate model selection. In another example, modifications to the number of states in the model can be performed by sequentially adding states to the model and then sequentially removing states, or a combination of the two; similar procedures are employed in model selection methods used in multivariate regression. Optimization of the parameters of each state may be performed by estimation-maximization or many other approaches to optimizing a multivariate model.

[000167] Segmentation and segment scoring'.

[000168] Once the model is selected and parameters optimized, segmentation and segment scoring are determined as described for normal samples. Γη brief, continuous segments of positions with the same most-likely state are reported, with scores indicating the average over positions in the segment of the probability of a classification error.

[000169] The instant disclosure differs from many known approaches in that the key difference is that in place of intensity measurements at a large but specific set of locations on the genome (e.g. microarray data), the described approach is relevant to sequencing-based coverage depth measurements at every position on the genome (e.g.. next gen sequencing data). Some of the additional differences are as follows:

[000170] 1 ) Use of fractional counts for measuring coverage, in yet another embodiment, when a paired read (e.g., corresponding to a full DNB) maps to more than one location, measures of confidence are used to fractionally attribute the mapping to each location. The consequence is that this allows for assessing coverage in segmental duplications to a greater degree than other approaches.

[000171] 2) One of the described approaches to correcting coverage bias. In yet another embodiment, the approach that weights each DNB (one specific implementation being to use logistic regression) provides the ability model the impact of multiple biasing factors, arguably giving better bias correction than previous approaches. [000172] 3) The use of estimates of copy number in each of the baseline/matched samples. In yet another embodiment, by estimating the copy number of each sample in a general baseline, or in a matched baseline, one of the challenges for previous methods, which involves the computation of relative intensity (microarrays) or relative coverage (sequencing-based CNV)), is avoided, namely the fact that the sample(s) used as the baseline can themselves have CNVs. When a baseline sample has a CNV, the intensity/coverage measured within the CNV locus will not provide an estimate of the intensity for the normal (typically diploid) copy number, leading the relative coverage of the sample of interest to have a different relationship to absolute copy number than in most of the genome. By adjusting the baseline samples tliemselves according to estimates of copy number, an expected linear relationship is preserved between copy number and relative coverage, allowing to more accurately infer absolute copy number.

[000173] 4) Within the HMM. two features are distinctive. In yet another embodiment, these features permit more robust modeling of the data (more accurate CNV calling);

[000174] a) the methods by which the mean of each state is determined; these methods provide an alternative to using the usual HMM training methods (EM), which do not seem to reliably converge on useful values.

i) for normal samples, the median of the coverage in the expected-diploid portion of the sample is used to determine the mean of the diploid state, and fix other states (copy numbers) at 50% increments or decrements from the diploid state. (The 0-copy state is special, being given a value slightly above 0 to allow for mis-mapping.)

ii) for tumor samples, a separate process is used to infer an initial set of levels; this process can be based on analysis of a histogram of coverage data; once initial levels are chosen, further calculations are applied to refine the set of levels.

[000175] b) the methods by which the variances of the states are estimated (constraint); at least in some implementations, the variances are constraint to be linearly related to the means of the states, reflecting the fact that most of the variance is a result of bias rather than sampling noise; thus, within a given sample, a state (coverage level) with twice the mean as a second state will typically have twice the spread (standard deviation) of observed coverages as the second state.

[000176] 5) The use of coverage data from a large (e.g. 50 sample) baseline to determine locations where some aspect of the sequencing process leads to highly variable coverage levels. [000177} In yet another embodiment, such locations would lead to spurious CNV calls if they are not identified as problematic. Once identified, such locations are marked as of unknown copy number rather than being assigned spurious changes.

[000178] Window Boundary Definition (for performing window-smoothing)

[000179] When choosing window boundaries for performing window-smoothing, in an example embodiment, for the most part windows are defined so that their chromosome coordinates are even multiples of the window length, so that for 2k windows, e.g., the chromosome positions of window boundaries will end with 'χΟΟΟ', where x is an even digit. The boundaries of these windows are called the 'default boundaries'. Exceptions to these default boundaries will be windows at the ends of contigs. Windows will never span bases taken from more than one contig, even if the gap between contigs is small enough to permit this. Moreover, there will be special treatment of the bases outside the outermost full default windows for each contig. These 'outside bases' will either be added to the first full window towards the center of the contig or be placed in their own window, depending on whether the number of bases is larger than ½ the window width or not. For example, for a contig running from position 17891 to position 25336, and window width of 2000, the following list of window intervals may be used

(17891 ,20000), (20000,22000), (22000,24000), (24000,25336)

[000180] It is noted that the first 109 bases of the contig are added to the 2k interval immediately to their right, while the last 1336 bases are placed in their own window. A contig that is smaller than the window width (e.g., chrM for 100k windows) will be made into a single window that includes the entire contig. No windows will be reported for inter-contig gaps. To illustrate, suppose a chromosome consists of three contigs as illustrated in Table 1.

[000181] Table 1. Chromosome contigs example

[000182} This would result in the following windows being used/reported; contig Id is shown only for clarity of presentation here:

Contig 1 : (17891,20000), (20000,22000), (22000,24000), (24000,25336)

Contig 2: (25836,2800), (28000,29277) Contig 3 : (33634,3421 1)

[000183] The Consequences of this approach are:

• all non-gap bases of the genome are included in a window (and only one window);

• windows are restricted to a single contig;

• windows are between 0.5 and 1.5 times the nominal window width;

• window boundaries are mostly round numbers, making it more obvious that segment boundaries correspond to window boundaries, with less chance of over- interpreting the precision of the CNV call boundaries.

[000184} 5. Population-based no-calHng/identification of lo -confidence regions

[000185] In various embodiments, the computations and steps described in this section (Section 5) can be performed by computer logic such as, for example, CNV variant caller 8 in FIG. 1 and/or by a component thereof such as popuiation-based no-calling logic 38.

[000186] In one aspect, the HMM-based calls described above typically contain a variety of inferred CNVs that are either artifacts or of lesser interest. Primai'iiy, these arise in one of two situations: A) The reference genome sequence does not provide an explanation for coverage patterns in most or all sample genomes, with most or all sample genomes matching one another. B) There is more variation in coverage than can be explained by a small number of discrete ploidy levels. The utility of CNV inference may be increased by identifying and annotating such regions. Below, regions so-annotated are considered to be 'no-called^; in the sense that a discrete estimate of ploidy may not be given for these regions.

[000187] Such behaviors may result from multiple causes; some of the possible mechanisms include:

• Errors in the reference genome. For instance, two contigs may in fact overlap one another, i.e. correspond to a single genomic interval, in most or all genomes. In this case, the two contig ends may consist in part of highly similar sequence that is otherwise unique, causing DNBs to map to both locations. Observed measured coverage will be reduced, leading to an apparent copy number reduction.

Alternatively, most or all sample genomes may contain a duplication that is not present in the reference. In this case, observed coverage will be elevated over the portion of the reference corresponding to the duplicated segment, leading to a copy number increase relative to the reference but not a true polymorphism. Uncorrected coverage bias. In one aspect, a region mat is substantially over or under represented in the sequencing results may appear to be a CNV relative to the reference. In order to retain the ability to make absolute copy number inferences, baseline correction as described above is done taking into account an initial copy number inference for the baseline genome(s). A consequence of this may be that regions that are severely biased in the baseline as well as the sample of interest may be interpreted as true CNVs. The signature of this sort of event will be that most or all samples will show similar elevated or suppressed coverage patterns.

Analysis artifacts. Though rare, there are occasional mapping artifacts that can result in a large number of spurious mappings at a given location. Such artifacts may result from particular arrangements of variations from the reference in repeated segments, such that the wrong reference copy of a repeat is more similar to the sequence of the sample of interest. These can result in a very substantial spike in coverage at certain locations on the reference, in a manner that is dependent on the variations present in a given sample.

Segmental duplications and tandem repeats. A segment that is present in duplicated form in the reference and subject to population variation may result in changes in coverage among samples that are smaller than typical of a copy number gain or loss in unique sequence. In the limit, sufficient variability in the population regarding a high-copy sequence type may result in an essentially continuous range of coverage values across a large number of samples.

Unstable estimation due to extreme correction factors or very low raw coverage. Examples include: 1) regions where coverage is very low due to GC correction, and the GC correction factors are correspondingly large, so that noise in the coverage estimate is amplified by the correction factors; 2) regions where coverage is very low due to mapping overflow, in simulations as well as real data, leading to large correction terms in the baseline bias correction factors; 3) regions where nearly all baseline genomes have ploidy 0. 1000188] Identification of such regions could be conducted in various ways. Ultimately, manual duration of coverage patterns at individual locations ma be highly effective, but may be prohibitive in some circumstances due to lack of data, degree of effort, and/or process instability. Use of sequence similarity and/or structural annotations has some promise, as a large fraction of problematic regions in practice correspond to known repetitive portions of the reference genome (segmental duplications, self-chains, STRs, repeat-masker elements); however, since many real copy number polymorphisms occur in such regions, it is unappealing to overly-broadly exclude such segments and challenging to find criteria that are more selecti ve. Thus, in yet another aspect, it is desirable to be able to identify problematic regions directly from coverage data. 1000189] Two sorts of coverage patterns typify several of the above circumstances. The first involves regions where coverage is more variable than can be explained^* by a small number of discrete ploidy levels ("hypervariable")- The- second involves regions where coverage is not as expected of a euploid region matching the reference but it is similar among all samples

("invariant").

[000.190] Given a substantial number of genomes (e.g. 50 or more), the "background set". summary statistics on bias-corrected and smoothed but unnormalized coverage dat are sufficient to usefully (ifheuristically/imperfectiy) separate the genome into well-behaved regions,

hypervariable regions and invariant regions. The following summary statistics computed for every genomic position i over a set G of n genomes can be used in this way. Let ft _:i; for

1 s x≤ n be- the x ^'in order statistic of e< (g)_s g€ 6, i.e. the x h smallest corrected and.

smoothed coverage at position i among the genomes in the background set. .

Median ¾ :

Spread s_s :

Clustering coefficient :

where SSlt ^is the sum. of squared error for £¾_Csr ._{1 y} ~.,«¾_<v>» i.e. for

Given these summary statistics, criteria may be defined for labeling positions as hypervariahie or invariant.

[0001 1] Annotation of hypervariahie regio

[0001 21 A position thai satisfies all of the following four criteria may be labeled

"hypervariahie" (rather than being marked as a CNV or classified as euptoid):

(i) The position would be called a CNV/aneuploid by the HMM inference process described above.

(ii) Coverage values in the background set are not clustered in ways suggesting simple

polymorphism in the population. Formally, for Q a value that can be chosen empirically as described below:

¾ > Q

(in) The range of co verage values at this position in the background set is wider than is seen at most of the (euploid) genome, formally, for S a value that can be chosen empirically as described below:

(iv ) The observed coverage for the sample of interest falls within the range of values seen in the background set, or outside the observed range by a small absolute amount (e.g. an amount that could readily be explained by sampling or process variation). Formally, for R and X values that can be chosen empirically as described below:

l i— m.; I < mhi(s< * R_< .X^'}

[900193] A noiati on of invariant regiom

A position that sa tisfies all of the following criteria may be labeled "invariant" (rather ^'than being marked as a CNV):

(i) The position would be called a CNV/aneuploid by the HMM segmentation process described above.

(ii) Coverage values in the background set are not clustered in ways suggesting simple

polymorphism in the population. Formally, for Q a value that can be chosen empirically as described below: (iii) Coverage at this position, across the background savnples shows low variability, suggesting both absence of a high-minor-alleie-frequency polymorphism in the population and. low process variation, (artifact). Formally, tor S a value that can be chosen empirically as described below:

¾ ¾ < S

(iv) The observed coverage for the sample of interest falis within the range of values seen in the background set or outside the background range by a small absolute amount (e.g. an amount that could readily be explained by sampling or process variation). Formally, for R and X values thai can be chosen empiricall as described below:

>— iii_; mm.(¾ * /?)

[ΘΘΘ1 4] Refinement of annotations

[0001951 In one aspect, the above criteria may cause CNV calls to be overly fragmented into alternating called and no-called segments. It may be desirable to permit short intervals that would be "no-called" (i.e. annotated as "hypervariable" or "invariant") based on the criteria above to be allowed to be called (left unannotated) if the observed coverage is sufficiently similar to a Hanking interval that is not annotated. Concretely, the ''hypervariable" or 'Invariant'* labeling of intervals, which are less than L bases that satisfy the above criteria but are part of longer segments in the HMM output, may be suppressed.

10Mi5⁾6] Selection of ^'cutoff ^'values

0001 7] In one aspect, cutoffs O, S, R, Xmd L in the above criteria may be selected based on analysis of a subset of initial CNV calls and comparison to genome-wide distributions on the background coverage summary statistics. Given a classification of an initial set of CNV calls (the "training set")- into those that are suspect (to be labeled "hypervariable" or "invariant*) and those that are believed to be true CNVs. as well as summary statistics for the entire genome (that is, of selected positions spaced along the genome, e.g. those resulting from the windows described above), the -cutoffs that are near optimal may be identified with regard to the following criteria:

• most of the genome is called either euploid or CNV/aneuploid (e.g., only a small fraction of the genome is no-called/annotated as hypervariable or in variant);

» most of the problematic regions in the 'training set' are no-called; * most of the trusted regions in the training set are called (not annotated).

[000198} The traini ng set can be deri ved based on manual curation of a collection of

preliminary CNV calls. Said coraiion may involve manual inspection of coverage profiles to identify calls and comparison to external datasets of putative CNVs identified by independent means.

{0001.99] Candidate vaiues of Q, R, S and L may be evaluated by determining concordance with the training set or a separate test set, as well as the fraction of the genome that is no-called. The final choice of cutoffs may involve a tradeoff between completeness of calling (fraction of the genome called) and the amount of problematic CNV calling,

10002001 Score computation

[000201] The CNV segmentation scores described above are more explicitly described in this section,

O002O2[ The probability of a given

of length t occurring as the result of a specific sequence of states er=si, sr_? can be computed on a given HM consisting of n states defined by initial state probabilities P *pi, transition probabilities T— { _iSj and emission probabilities £ -^: {e_s as fol lows:

[000203] The probability of the data given the model is the sum over all possible sequences of quences of states of length t:

[000204} This and other equations involving sums over subsets of S can be efficiently

computed using the Forward/Backward algorithm. Application of^'Bayes' Rule allows the determination of th a given path given the data and the model:

1000205] From this, it can be seen that the most probable path, given the data and model is the path which maximizes Pr(P_< s?|F f _s £ ^' }■ The path which maximizes this equation can efficiently be determined using the Viterbi algorithm. £600206] However, the probability of partial paths can also be computed. For example probabii ity the pa th through the model that actually led to an observed sequence of data

as follows:

[00O207| The denominator is discussed above, and the numerator can be obtained by summing the probabiiity of the data and a particular path over all paths for which ½ = denoted S_{fcs =g}:

{000208] State assignment ("called ploidy") is done as follows; the state (ploidy) inferred at position u , ¾ is that state with maximal probability:

[000209] The bounds on the summation, and b, are as follows. For a region expected to be diploid, if ¾<2_i 0 - ----- 1; if s¾<2, a-b^{' ~}2; if ¾ 2₅ ^-m ximum ploidy (typically, 10).

For a region expected to be haploid (male sex chromosomes), if ^ <1 , -0_tb=Q; if , =b-l; if >1 , a~2.

ploidy (typieally, 10).

[600210] A segment is deimed as a maximal run of Hke-ploidy positions. For a segment from position / to position r, t he ploidyScore is taken to be the mean of the ploidySeores for the constituent positions:

[00021 ϊ{ And similarly the CNVTypeScore of a segment π^, is the mean of the CNVTypeSeores fe ile constituen ositions;

H2] An Alternative Approach For Scoring;

)213] An alternative set of scores for segments can be computed based on the likelihoods of partial pat hs. For instance, the probability of the true path being in state q from position I to position r can be computed as follows:!

[0002141 Another statisiic that may be relevant to computing confidence in segment boundaries is the probability of being in state q at position ?/, but not at position u-l (or, analogously, at

f 000215] Finally, an alternative to the DEI Scores defined above can be computed for

example, the probability of being in states with ploidy greater than 2 from position / to position r is;

[000216] As noted earlier, all the summations over paths can be efficiently computed via the Forward-Backward algorithm.

100021?] The application of HMM model is well known in the art and is discussed i for example, Rabiner, L.R. A Tutorial on Hidden Markov Models and Selected Applications in Speech Recognition. Proceedings of the IEEE, 1989, 77.2:257-286:

|^'000218| Exemplar Implementation Mechanisms for CNV Calling

(000219] Computer system

[000220] An exemplary computer system which can be used in accordance with the

embodiments of the instant disclosure can be implemented in software and the results may be presented to a user on a monitor or other display device, in some embodiments, the exemplary computer system configured to estimate. copy number variation in a target sequence in a sample can present results to a user as a graphical user interface (GUI) on a display device such as computer monitor. FIG. 3 illustrates one example of an architecture of a computer system 400 configured to implement the estimate of copy number variation in accordance with the present disclosure. As illustrated in FIG. 3, computer system 400 may include one or more processors 402 (e.g., such as CPUs). The processor 402 is connected to a communication infrastructure 406 (e.g., a communications bus, cross-over bar, or network). Computer system 400 may include a display interface 422 that forwards graphics, text, and other data from the communication infrastructure 406 (or from a frame buffer not shown) for display on the display unit 424.

[000221] Computer system 400 also includes a main memory 404, such as a random access memory (RAM), and a secondary memory 408. The secondary memory 408 may include, for example, a hard disk drive (HDD) 410 and/or removable storage drive 412, which may represent a floppy disk drive, a magnetic tape drive, an optical disk drive, or the like. The removable storage drive 412 reads from and/or writes to a removable storage unit 416. Removable storage unit 416 may be a floppy disk, magnetic tape, optical disk, or the like. As will be understood, the removable storage unit 416 may include a computer readable storage medium having stored therein computer software and/or data.

[000222] In alternative embodiments, secondary memory 408 may include other similar devices for allowing computer programs, computer logic, or other instructions to be loaded into computer system 400. Secondary memory 408 may include a removable storage unit 418 and a corresponding interface 514. Examples of such removable storage units include, but are not limited to, USB or flash drives, which allow software and data to be transferred from the removable storage unit 418 to computer system 400.

[000223J Computer system 400 may also include a communications interface 420.

Communications interface 420 allows software and data to be transferred between computer system 400 and external devices. Examples of communications interface 420 may include a modem, Etliemet card, wireless network card, a Personal Computer Memory Card International Association (PCMCIA) slot and card, or the like. Software and data transferred via

communications interface 420 may be in the form of signals, which may be electronic, electromagnetic, optical, or the like that are capable of being received by communications interface 420. These signals may be provided to communications interface 420 via a

communications path (e.g., channel), which may be implemented using wire, cable, fiber optics, a telephone line, a cellular link, a radio frequency (RF) link and other communication channels. [000224] In this document, the terms "computer-program medium" and "computer-readable storage medium¹' refer to non-transitory media such as main memory 404, removable storage drive 412, and a hard disk installed in hard disk drive 410. These computer program products provide software or other logic to computer system 400. Computer programs (also referred to as computer control logic) are stored in main memory 404 and/or secondary memory 408.

Computer programs or other software logic may also be received via communications interface 420. Such computer programs or logic, when executed by a processor, enable the computer system 400 to perform the features of the method discussed herein. For example, main memory 404_; secondary memory 408, or removable storage units 416 or 418 may be encoded with computer program code (instructions) for performing operations corresponding to the process shown in FIG. 3.

[000225] In an embodiment implemented using software logic, the software instructions may be stored in a computer program product and loaded into computer system 400 using removable storage drive 412, hard drive 410, or communications interface 420. In other words, the computer program product, which may be a computer readable storage medium, may have instructions tangibly embodied thereon. The software instructions, when executed by a processor 402, cause the processor 402 to perform the functions of (operations o±) methods described herein. In another embodiment, the method may be implemented primarily in hardware using, for example, hardware components such as a digital signal processor comprising application specific integrated circuits (ASICs). In yet another embodiment, the method is implemented using a combination of both hardware and software.

[000226] Exemplary system for CNV calling in accordance with an embodiment of the present disclosure. FIG. 1 is a block diagram illustrating a system for calling variations in a sample polynucleotide sequence according to one exemplary embodiment. In this embodiment, the system may include a computer cluster 10 of one or more computing devices such as computers 12 and a data repository 14. The computers 12 may be connected to the data repository 14 via a high-speed local area network (LAN) 16. At least a portion of the computers 12 may execute instances of a CN V caller 18. (In some embodiments, a CNV caller such as CNV caller 18 may be included as part of an assembly pipeline logic that is configured and operable to assemble raw reads into mapped and sequenced genomes that include the detected variations from a reference genome; examples of such embodiments are described in U.S. Application Serial No. 12/770,089 filed on April 29, 2010, the entire contents of which are hereby incorporated by reference as if fully set forth herein.) The CNV caller 18 may include HMM model logic 20, coverage calculation logic 22, GC correction logic 34, ploidy-aware correction logic 36, and population- based no-calling logic 38.

[000227] The data repository 14 may store several databases including one or more databases that store a reference polynucleotide sequence 24, mated reads 26 obtained by sequencing a sample polynucleotide sequence using biochemical processes, and mapped mated reads 28 that are generated from the mated reads 26.

{000228] The reference polynucleotide sequence 24 (hereinafter referred to as simply the reference) refers to a known sequence of nucleotides of a reference organism (e.g., a known genome). This includes references comprising sequences having known variations at one or more location within the genome. A polynucleotide molecule is an organic polymer molecule composed of nucleotide monomers covalently bonded in a chain. Deoxyribonucleic acid (DNA) and ribonucleic acid (R A) are examples of polynucleotides with distinct biological function. The genome of an organism (e.g., such as a hitman) is the entirety (or the substantial entirety) of an organism's hereditary information, which is encoded as DNA or RNA. A haploid genome contains one copy of each hereditary unit of the organism. In diploid organisms such as mammals, the genome is a series of complementary polynucleotides comprising two copies of the majority of the hereditary information organized as sets of chromosomes having discrete genetic units, or alleles. Each copy of the allele is provided at a specific position on an individual chromosome, and the genotype for each allele in a genome comprises the pair of alleles present at particular positions on homologous chromosomes that detennine a specific characteristic or trait. Where a genome comprises two identical copies of an allele it is homozygous for that allele, and when the genome comprises two different alleles it is heterozygous for that locus. The DNA itself is organized as two strands of complementary polynucleotides.

[000229] The reference 24 may be an entire genome sequence, a portion of a reference genome, a consensus sequence of many reference organisms, a compilation sequence based on different components of different organisms, or any other appropriate sequence. The reference 24 may also include information regarding variations of the reference known to be found in a population of organisms. [000230] The mated reads 26 may be obtained during a sequencing process performed on polynucleotide sequences derived from a biological sample of an organism, e.g., nucleic acid sequences from a gene, genomic DNA, RNA, or fragments thereof, that is to be analyzed. The mated reads 26 can be obtained from a sample comprising an entire genome, such as an entire mammalian genome, more specifically an entire human genome. In another embodiment, the mated reads 26 may be specific fragments from a complete genome. In one embodiment, the mated reads 26 may be obtained by performing sequencing on amplified nucleic acid constructs, such as amplimers created using polymerase chain reaction (PC ) or rolling circle replication. Examples of amplimers that may be used are described, for example, in U.S. Pat. Publication Nos. 20090111705, 200901 11706 and 20090075343, which are incorporated by reference in their entirety.

[000231] The mapped mated reads 28 refer to the mated reads 26 that have been mapped to locations in the reference 24. Exemplary mapping methods are described in the following patent applications: U.S. Patent Application Serial No. 12/698,965 filed on February 2, 2010, the entire contents of which is hereby incorporated by reference; U.S. Patent Application Serial No.

12/698,986 filed on February 2, 2010, the entire contents of which is hereby incorporated by reference; U.S. Patent Application Serial No. 12/698,994 filed on February 2, 2010, the entire contents of which is hereby incorporated by reference.

{000232) The copy number variation CNV caller 18 generates and scores sequences for the purpose of identifying and calling the copy number variations or differences detected in a sequence of the mapped mated reads 28 in relation to the reference 24.

[000233] The CNV caller 18 may output a CNV calls file(s) 32, list or other data structure containing the identified variations, each describing a manner in which pails of the sequence of mapped mated reads 28 are observed to differ from the reference 24 at or near specific locations.

[000234} The computer cluster 10 may be configured such that instances of the CNV caller 18 executing on different computers 12 operate on different portions of the reference 24 and the mapped mated reads 26 in parallel. The job scheduler 30 is responsible for assignment of jobs or packets of work across the various computers 12 in the computer cluster 10.

[000235] The computers 12 may include typical hardware components (not shown) including, one or more processors, input devices (e.g., keyboard, pointing device, etc.), and output devices (e.g., a display device and the like). One example of a computer 12 is computer system 400 and/or computing device 2500, illustrated in Fig. 3. The computers 12 may include computer- readable/writable media, e.g., memory and storage devices (e.g., flash memory, a hard drive, an optical disk drive, a magnetic disk drive, and the like) containing cojnputer instructions that implement the functionality disclosed when executed by the processor. The computers 12 may further include computer writeable media for implementing the data repository 14 and for storing the CNV call file(s) 32. The computers 12 may further include wired or wireless network communication interfaces for communication.

[000236] Data Generation

[0002371 In some embodiments, a sequencing machine (e.g., such as a sequencing machine illustrated in FIGs. 4A and 4B) may be used to generate the mated reads 26 obtained from the sample polynucleotides of an organism to be analyzed. In one embodiment, the sequencing machine provides the data in discrete but related sets, such that contents of the mated reads 26 may include predicted spatial relationships and/or separation variations. The relationships may be determined based on existing knowledge about the biochemical process used to generate the mated reads 26 (e.g., based on sequences that would be expected to be obtained if the biochemical process were applied to a sample), empirical estimates based on preliminary analysis of tile sequence data of the mated reads 26 or subsets thereof, estimation by experts, or other appropriate techniques.

[000238] Numerous biochemical processes can be used to facilitate the generation, by a sequencing machine, of the mated reads 26 for use with the present CNV calling methods. These include, but are not limited to hybridization methods as disclosed in U.S. Pat. Nos. 6,864.052; 6.309,824; 6,401 ,267; sequencing-by-synthesis methods as disclosed in U.S. Pat. Nos.

6,210,891; 6,828,100, 6,833,246; 6,911 ,345; 7,329,496 and Margulies, et al. (2005), Nature 437:376-380 and Ronaghi, et al. (1996), Anal. Biochem. 242:84-89: ligation-based methods as disclosed in U.S. Pat. No. 6,306,597, WO2006073504, WO2007120208, nanopore sequencing technology as disclosed in U.S. Pat. Nos. 5,795,782, 6,015,714, 6,627,067, 7,238,485 and 7,258,838 and U.S. Pat Appb Nos. 2006003171 and 20090029477, and nanochannel sequencing technology as disclosed in US Pat. App. Pub. No. 200901 11 1 15, all of which are incorporated by reference in their entirety. In a specific implementation, a Combinatorial Probe Anchor Ligation (cPAL) process is used in some embodiments (see US Pat. App. Publication Nos. 20080234136 and 20070099208, which are incorporated herein by reference in their entirety). [000239] Once the primary mapped mated read data is generated, the information is processed in accordance with the CNV calling methods of the instant disclosure as illustrated in FIG. 2. which depicts an exemplary method for determining the copy number of a genomic region at a detection position of a target polynucleotide sequence in a sample, mapped read data is obtained to measure the sequence coverage for said sample 202; the sequence coverage bias is corrected wherein the sequence coverage bias correction comprises performing ploidy-aware baseline correction 204; total copy number value and region-specific copy number value for a plurality of genomic regions are estimated 210 after population based no calling / identification of low confidence regions is performed 206 and HMM segmentation, scoring, and output is performed 208.

(000240] Examples of output of the CNV calling process (e.g., as may be provided in variation call file(s) 32 in FIG. 1) for diploid/non-tumor/non-aneuploid samples generated in accordance with an exemplary embodiment of the instant disclosure are illustrated in Table 2.

[000241 ] Table 2.

[000242] In Table 2, column "Chromosome" identifies the chromosome number, columns "Begin" and "End" identify the starting and ending locus of a given region, column "Ploidy^" indicates the ploidy (e.g., such as copy number) for a region, column "Ploidy Score" indicates a score for a given region (where the score is a logarithm-based value expressed in decibels dB), column "Type^"' indicates the type of the ploidy observed for a region (e.g., "=" indicates the normal ploidy of 2, "+" indicates ploidy higher than the normal, indicates ploidy lower than normal, "hypervariable" indicates that the ploidy could not be called, and "invariant" indicates that the ploidy is different than normal but is the same as observed in a baseline that is a collection of at least several reference genomes), and column "TypeScore" indicates a confidence score for the type called in the same row in column "Type". For example, the second row in Table 2 indicates that: the region, beginning at locus 5100001 and ending at locus 5800000 on chromosome 1 , has a ploidy of 3 that has a score of 15 dB and has an "increased" Type having a score of 40.

[000243] Examples of output of the CNV calling process output (e.g., as may be provided in variation call fije(s) 32 in FIG. 1) for non-diploid/tumor/aneuploid samples generated in accordance with an exemplary embodiment of the instant disclosure are illustrated in Table 3.

[000244] Table S.

[000245] In Table 3, column "Chromosome" identifies the chromosome number, columns "Begin" and "End" identify the starting and ending locus of a given region, column "Level" indicates the coverage level for a region output by the HMM model (where the coverage level is computed without making the assumption of a normal ploidy of 2 because of the aneuploidy and other characteristics of the tumor sample), and column "LevelScore" indicates a confidence score for the level called in the same row in column "Level'^". For example, the second row in Table 3 indicates thai: the region, beginning at locus 10001 and ending at locus 243189373 on chromosome 2, has a coverage level of 1.05 that has a score of 38.

[000246] Techniques for Graphical Interpretation of Absolute Copy Number In an example embodiment, bias-corrected windowed average coverage and allele specific read counts are inputs to OptSeg logic, which determines a segmentation into regions of uniform coverage and lesser allele fraction (LAF). The effect of segmentation is conceptually similar to circular binary segmentation models in not having a fixed set of modeled states, but provides a globally optimal solution. Overly short segments are suppressed to reduce noise.

[000247] The product of LAF and total coverage provides an estimate of lesser allele coverage. The two-dimensional space defined by total coverage and lesser allele coverage can be used in a graphical representation of a tumor where most states are expected to lie on the vertices of a rectangular grid. Density in this space may be tabulated and kernel-smoothed by computer logic prior to visualization. Peaks in the smoothed distribution of sufficient height determine an initial set of states.

[000248] A rule-based logic finds a constrained (one tumor component) model that attempts to capture as much of the density under the initial states as possible, subject to a limit on maximum average ploidy. The support of the data for a given model can be evaluated visually, and various properties of the model can be directly observed in the graph.

[000249] The constrained model provides an estimate of the stromal contamination fraction, and peaks matching the model receive an interpretation in terms of integer total and minor copy numbers. Initial states that are not accounted for by the model are interpreted as the result of tumor heterogeneity; they may be included in a final model and receive non-integer total and/or minor copy numbers.

[000250] The final model may be input to logic that implements a separate model-based segmentation process (e.g. an HMM), with the state interpretations used to annotate the final set of segments. Due to the separation of the modeling process and the final segmentation, computer logic can generate a visualized representation of the tumor; if problematic, an alternative model can be substituted for the automatically derived model.

[000251] LAF Estimation

[000252] Lesser allele fraction (also referred to as "minor copy proportion'^'') is the fraction of copies of a given region of the sample that contain the less-abundant allele. LAF may be estimated based on read counts from a tumor sample at heterozygous variant loci in the matched normal sample. Estimation allows for uncertainty of phasing (e.g., either allele at a given locus may be the 'lesser allele', independent of read counts) to avoid biased estimation. Deviation from binomial sampling can be handled via a beta binomial model.

[000253} Conceptual Modeling

[000254] Exemplary Standard oiw-component-phis-siromal-contaminalioti model

[000255] In certain embodiments, the following assumptions are made:

* Cells of the sample are either tumor cells, with probability t, or normal, with probability 1-/.

* AH tumor cells have identical genomes.

* All portions of the normal cells are diploid.

[000256] For a given region of the genome, let:

* a,- - number of major allele copies per tumor cell in /

* bj— number of minor allele copies per tumor cell in (a, > &,·)

* Cj = average copy number in /

* /, = lesser allele fraction in

[000257] Then:

ct = 2(1-/)+ (cii + bdt

[000258] The allowed copy number states (c,-, ¾ in this model lie on a square grid in the graphical representation (see Figure 6).

[000259] Multi-component models

The 1 -component tumor model (FIG. 6) of possible states can easily be extended to more complex model involving two or more components (subclones) of the tumor portion of a sample. However, even two-component models lead to great expansion of possible states (see FIG. 7). Both the overall model (relative fraction of each component, coverage per full copy, etc.) and the interpretation of an individual state are typically under-determined. (See Figures 7-9)

[000260] Illustration of Results

[000261] Robustness of the process to varying degrees of stromal contamination can be seen in an artificial (in siiico) titration of stromal content using matched tumor and normal cell lines. Figures ί OA - 10C showed three datasets, the first containing pure cell line tumor, the second 50% tumor/50% matched normal, and the third 25% tumor/75% normal. [000262] Although all regions of the genome have significant minor allele content in the contaminated samples, the lowest states can be interpreted as loss of heterozygosity (LOH) states.

[000263] Tumors with high average copy number and high variability in copy number can have a compelling fit, while highlighting regions where the tumor is heterogeneous (as illustrated in Figure 1 1 ).

[000264] Some samples are hard: interpretation is elusive even manually. At least visualization can easily identify poor model fit (as illustrated in Figure 12).

[000265] In certain embodiments, regions that are heterogeneous within the tumor are not decomposed into a combination of homogeneous components but rather reported in terms of average behavior. In certain other embodiments, where the sample peaks clearly identify a grid, there are certain degeneracies in model selection which can lead to a state with major and minor copy numbers ( ,b) that cannot be distinguished from (a+n, b+n) with less stromal

contamination. In those embodiments, the model with lowest possible ploidies is preferred.

[000266] Exemplary Model-Based Segmentation Process for CNV Calling

In an exemplary embodiment, a method for determining the copy number of a genomic region, at a detection position of a target polynucleotide sequence in a sample, said method comprises: obtaining measurement data for the sequence coverage for said sample; correcting the measurement data for sequence coverage bias, where the sequence coverage bias correction comprises performing ploidy-aware baseline correction; performing Hidden Markov Model (HMM) segmentation process, scoring, and output; and estimating a total copy number value and region-specific copy number value for a plurality of genomic regions.

[000267] For example, in one embodiment, the method comprises generating plural states of an HMM model that correspond to respective copy numbers, where the sample is a tumor sample and performing HMM segmentation, scoring, and output, includes: using input data representing initial states determined by a model for interpretation of absolute copy number of complex tumors (as described heretofore) to generate an initial model for the HMM; optimizing the initial model by modifying the number of states in the model as well as optimizing the parameters of each state; and modifying the number of states in the model by sequentially adding states to the model and then sequentially removing states, or a combination thereof. [000268] in one embodiment, the method further comprises annotating a total copy number value and a region-specific copy number value for a plurality of genomic regions based on the initial states that are determined by the model for interpretation of absolute copy number of complex tumors.

[000269] Example Sequencing Machines and Computing Devices

[000270] In some embodiments, sequencing of DNA samples (e.g., such as samples representing whole human genomes) may be performed by a sequencing system. Two examples of sequencing systems are illustrated in FIGs. 4A and 4B.

[000271] FIGs. 4A and 4B are block diagrams of example sequencing systems 2490 that are configured to perform the techniques and/or methods for interpreting absolute copy number of complex tumors and for CNV calling according to the example embodiments described herein. A sequencing system 2490 can include or be associated with multiple subsystems such as, for example, one or more sequencing machines such as sequencing machine 2491, one or more computer systems such as computer system 2497, and one or more data repositories such as data repository 2495. In the embodiment illustrated in FIG. 4A, the various subsystems of system 2490 may be communicatively connected over one or more networks 2493. which may include packet-switching or other types of network infrastructure devices (e.g., routers, switches, etc.) thai are configured to facilitate information exchange between remote systems. In the embodiment illustrated in FIG. 4B, sequencing system 2490 is a sequencing device in which the various subsystems (e.g.. such as sequencing machine(s) 2491 , computer system(s) 2497, and possibly a data repository 2495) are components that are communicatively and/or operatively coupled and integrated within the sequencing device.

[000272] In some operational contexts, data repository 2495 and/or computer system(s) 2497 of the embodiments illustrated in FIGs. 4A and 4B may be configured within a cloud computing environment 2496. In a cloud computing environment, the storage devices comprising a data repository and/or the computing devices comprising a computer system may be allocated and instantiated for use as a utility and on-demand; thus, the cloud computing environment provides as services the infrastructure (e.g., physical and virtual machines, raw/block storage, firewalls, load-balancers, aggregators, networks, storage clusters, etc.), the platforms (e.g., a computing device and/or a solution stack that may include an operating system, a programming language execution environment, a database server, a web server, an application server, etc.), and the software (e.g., applications, application programming interfaces or APIs, etc. ) necessary to perform any storage-related and/or computing tasks.

[000273] It is noted that in various embodiments, the techniques described herein can be performed by various systems and devices that include some or all of the above subsystems and components (e.g., such as sequencing machines, computer systems, and data repositories) in various configurations and form factors; duis, the example embodiments and configurations illustrated in FIGs. 4A and 4B are to be regarded in an illustrative rather than a restrictive sense.

[000274J Sequencing machine 2491 is configured and operable to receive target nucleic acids 2492 derived from fragments of a biological sample, and to perform sequencing on the target nucleic acids. Any suitable machine that can perform sequencing may be used, where such machine may use various sequencing techniques that include, without limitation, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, single-molecule sequencing, optica! sequence detection, electro-magnetic sequence detection, voltage-change sequence detection, and any other now-known or later-developed technique that is suitable for generating sequencing reads from DNA. In various embodiments, a sequencing machine can sequence the target nucleic acids and can generate sequencing reads that may or may not include gaps and that may or may not be mate-pair (or paired-end) reads. As illustrated in FIGs. 4A and 4B, sequencing machine 2491 sequences target nucleic acids 2492 and obtains sequencing reads 2494, which are transmitted for (temporary and/or persistent) storage to one or more data repositories 2495 and/or for processing by one or more computer systems 2497.

[000275} Data repository 2495 may be implemented on one or more storage devices (e.g., hard disk drives, optical disks, solid-state drives, etc.) that may be configured as an array of disks (e.g., such as a SCSI array), a storage cluster, or any other suitable storage device organization. The storage device(s) of a data repository can be configured as internal/integral components of system 2490 or as external components (e.g., such as external hard drives or disk arrays) attachable to system 2490 (e.g., as illustrated in FIG. 4B), and/or may be communicatively interconnected in a suitable manner such as, for example, a grid, a storage cluster, a storage area network (SAN), and/or a network attached storage (NAS) (e.g., as illustrated in FIG. 4A). In various embodiments and implementations, a data repository may be implemented on the storage devices as one or more file systems that store information as files, as one or more databases that store information in data records, and/or as any other suitable data storage organization. [000276] Computer system 2497 may include one or more computing devices that comprise general purpose processors (e.g., Central Processing Units, or CPUs), memory, and computer logic 2499 which, along with configuration data and/or operating system (OS) software, can perform some or ail of the techniques and methods described herein, and/or can control the operation of sequencing machine 2491. For example, any of the data processing and data analysis methods described herein can be totally or partially performed by a computing device including a processor that can be configured to execute logic 2499 for performing various steps of the methods. Further, although method steps may be presented as numbered steps, it is understood that steps of the methods described herein can be performed at the same time (e.g., in parallel by a cluster of computing devices) or in a different order. The functionalities of computer logic 2499 may be implemented as a single integrated module (e.g., in an integrated logic) or may be combined in two or more software modules that may provide some additional functionalities.

[000277] In some embodiments, computer system 2497 may be a single computing device. In other embodiments, computer system 2497 may comprise multiple computing devices that may be communicatively and/or operatively interconnected in a grid, a cluster, or in a cloud computing environment. Such multiple computing devices may be configured in different form factors such as computing nodes, blades, or any other suitable Iiardware configuration. For these reasons, Computer system 2497 in FIGs. 4A and 4B is to be regarded in an illustrative rather than a restrictive sense.

[000278] FIG. 5 is a block diagram of an example computing device 2500 thai can be configured to execute instructions for performing the techniques and/or methods described herein as part of sequencing machine(s) and/or computer system(s).

[000279] In FIG. 5, computing device 2500 comprises several components that are

interconnected directly or indirectly via one or more system buses such as bus 2575. Such components may include, but are not limited to, keyboard 2578, persistent storage device(s) 2579 (e.g., such as fixed disks, solid-state disks, optical disks, and the like), and display adapter 2582 to which one or more display devices (e.g., such as LCD monitors, flat-panel monitors, plasma screens, and the like) may be coupled. Peripherals and input/output (I/O) devices, which couple to I/O controller 2571 , can be comiected to computing device 2500 by any number of means known in the art including, but not limited to. one or more serial ports, one or more parallel ports, and one or more universal serial buses (USBs). External interface(s) 2581 (which may include a network interface card and/or serial ports) can be used to connect computing device 2500 to a network (e.g., such as the Internet or a local area network (LAN)). External interface(s) 2581 may also include a number of input interfaces that can receive information from various external devices such as, for example, a sequencing machine or any component thereof. The interconnection via system bus 2575 allows one or more processors (e.g., CPUs) 2573 to communicate with each connected component and to execute (and control the execution of) instructions from system memory 2572 and/or from storage device(s) 2579, as well as the exchange of information between various components. System memory 2572 and/or storage device(s) 2579 may be embodied as one or more computer-readable non-transitory storage media that store the sequences of instructions executed by processors) 2573, as well as other data. Such computer-readable non-transitory storage media include, but is not limited to, random access memory (RAM), read-only memory (ROM), an electro-magnetic medium (e.g., such as a hard disk drive, solid-state drive, thumb drive, floppy disk, etc.)^ an optical medium such as a compact disk (CD) or digital versatile disk (DVD), flash memory, and the like. Various data values and other structured or unstructured information can be output from one component or subsystem to another component or subsystem, can be presented to a user via display adapter 2582 and a suitable display device, can be sent tlirough external interface(s) 2581 over a network to a remote device or a remote data repository, or can be (temporarily and/or permanently) stored on storage device(s) 2579.

[000280] It should be understood that any of the methods and functionalities performed by computing device 2500 can be implemented in the fonn of logic using hardware and/or computer software in a modular or integrated manner.

[000281] While present invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention.

Claims

What is claimed is:

1. A method for determining the copy number of a genomic region at a detection position of a target polynucleotide sequence in a sample, said method comprising:

obtaining measurement data for the sequence coverage for said sample using data

generated from mate-pair mappings;

correcting the measurement data for sequence coverage bias, wherein correcting the

measurement data comprises performing pioidy-aware baseline correction; and based at least on the corrected measurement data, estimating a total copy number value and region-specific copy number value for each of a plurality of genomic regions; wherein the method is performed by one or more computing devices.

2. The method of claim 1, wherein the method further comprises performing Hidden Markov Model (HMM) segmentation, scoring, and output based on the corrected measurement data.

3. The method of claim 1 , wherein the method further comprises performing population- based no-calling and identification of low-confidence regions.

4. The method of claim 1 wherein said method further comprises normalizing the measurement data for the sequence coverage by comparison to sequence data obtained from a baseline sample.

5. The method of claim 1 wherein obtaining the measurement data for the sequence coverage comprises measuring sequence coverage depth at every position of the genome.

6. The method of claim 1 wherein correcting the measurement data for the sequence coverage bias comprises calculating window-averaged coverage.

7. The method of claim 1 wherein correcting the measurement data for the sequence coverage bias comprises performing adjustments to account for GC bias in the library construction and sequencing process.

8. The method of claim 1 wherein correcting the measurement data for the sequence coverage bias comprises performing adjustments based on additional weighting factor associated with individual mappings to compensate for bias.

9. method of claim 1 wherein the sequence coverage, c_h is determined by

10. The method of claim 1 wherein obtaining the measurement data for the sequence coverage comprises:

a) determining reads that represent the sequences of a plurality of approximately random fragments of the genome of the sample, wherein said plurality provides a sampling of the genome whereby on average a base position of the genome is sampled one or more times;

b) obtaining mapping data by mapping said reads to the reference genome, or by mapping said reads to an assembled sequence; and

c) obtaining coverage data by measuring the intensity of sampled sequences along the reference genome or along the assembled sequence;

wherein the measurement data comprises the mapping data and the coverage data.

1 Ϊ . The method of claim 10 wherein determining the reads further comprises steps of:

a) providing a plurality of amplicons, wherein:

i) each amplicon comprises multiple copies of a fragment of the target nucleic acid,

ii) each amplicon comprises a plurality of interspersed adaptors at predetermined sites within the fragment, each adaptor comprising at least one anchor probe hybridization site.

iii) said plurality of amplicons comprise fragments thai substantially cover the target nucleic acid;

b) providing a random array of said amplicons fixed to a surface at a density such that at least a majority of said ampHcons are optically resolvable;

c) hybridizing one or more anchor probes to said random array;

d) hybridizing one or more sequencing probes to said random array to form perfectly matched duplexes between said one or more sequencing probes and fragments of target nucleic acid;

e) ligating the anchor probes to the sequencing probes;

f) identifying at least one nucleotide adjacent to at least one interspersed adaptor; and g) repeating steps (c) through (f) until a nucleotide sequence of said target nucleic acid is identified;

wherein steps (a)-(g) are performed by a sequencing machine.

12. The method of claim 2 wherein performing the HMM segmentation farther comprises generating an initial model that estimates the number of states and their means based on the overall coverage distribution.

13. The method of claim 12 wherein performing the ITMM segmentation comprises optimizing the initial model by performing one or more of modifying the number of states in the model and optimizing the parameters of each state.

14. The method of claim 12 wherein the corrected coverage at position is:

15. The method of claim 4 wherein normalizing the measurement data comprises

determining normalized corrected coverage by using the equation:

16. The method of claim 1 further comprising using sequence coverage estimation to generate mappings of a sequenced fragment to more than one location on the genome, and using confidence measurements on each of the mappings to fractionally attribute said each mapping to each detection location.

17. The method of claim 1 further comprising performing HMM calculations to determine a ploidy number at each detection position.

I S. The method of claim 1 further comprising performing HMM calculations to determine a ploidy score at each detection position, said ploidy score representing a confidence that the determined ploidy number at said detection position is correct

19. The method of claim 1 further comprising performing HMM calculations to determine a CNV-Type-Score at each detection position, the CNV-Type-Score representing a confidence that said determined ploidy number at said detection position correctly indicates decreased ploidy, expected ploidy. or increased ploidy at said detection position.

20. The method of claim 2, wherein a plurality of states of the HMM correspond to respective copy numbers, and wherein if the sample is a normal sample, performing the HMM segmentation, scoring, and output includes:

initializing a mean of an emission distribution of the HMM for each state with copy number N greater than zero to N/2 multiplied by the median of the coverage in a portion of the sample expected to be diploid; and

initializing the mean of the emission distribution for the state with copy number 0 to a positive value smaller than that used for the state with copy number 1.

21. The method of claim 2, wherein plural states of the HMM correspond to respective copy numbers, and wherein if the sample is a tumor sample, performing the HMM segmentation, scoring, and output includes:

estimating the number of states and a mean of each state based on a distribution of the coverage to generate an initial model for the HMM;

optimizing the initial model by modifying the number of states in the model as well as optimizing the parameters of each state; and

modifying the number of states in the model by sequentially adding states to the model and then sequentially removing states_f or a combination thereof.

22. The method of claim 21 wherein modifying the initial model comprises:

a) adding a new state between a pair of states if adding said new state improves a likelihood associated with the HMM beyond a first predetermined threshold;

b) repeating step (a) recursively between each pair of states until no more additions are possible;

c) removing a state from the HMM if removal of said state does not decrease the likelihood beyond a second predetermined threshold; and

d) repeating step (c) iteratively for all the states.

23. The method of claim 2, wherein plural states of the HMM correspond to respective copy numbers, and wherein performing the HMM segmentation, scoring, and output includes initializing a variance of an emission distribution of the HMM for each state with copy number N to a constant multiplied by a mean of the emission distribution for said state.

24. A computer-readable storage medium comprising instructions tangibly embodied thereon, the instructions when executed by a computer processor cause the processor to perform the operations of:

obtaining measurement data for the sequence coverage for a biological sample using data generated from mate-pair mappings;

correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction; and based at least on the corrected measurement data, estimating a total copy number value and region-specific copy number value for each of a plurality of genomic regions.

25. A computer-readable storage medium comprising instructions tangibly embodied thereon, the instructions when executed by a computer processor cause the processor to perform the operations of:

obtaining measurement data for sequence coverage for a sample comprising a target polynucleotide sequence;

correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction;

performing Hidden Markov Model (HMM) segmentation, scoring, and output based on the corrected measurement data;

based on the HMM scoring and output, performing popu!ation-based no-calling and identification of low-confidence regions; and

based on the HMM scoring and output, estimating a total copy number value and region- specific copy number value for a plurality of regions.

26. A system for determining copy number variation of a genomic region at a detection position of a target polynucleotide sequence, comprising:

a. a computer processor; and

b. a computer-readable storage medium coupled to said processor, the storage medium having instructions tangibly embodied thereon, the instructions when executed by said processor causing said processor to perform the operations of:

obtaining measurement data for the sequence coverage for said sample using data

generated from mate-pair mappings;

correcting the measurement data for sequence coverage bias, wherein correcting the measurement data comprises performing ploidy-aware baseline correction; and based at least on the corrected measurement data, estimating a total copy number value and region-specific copy number value for each of a plurality of genomic regions.

27. A method for detemiining the copy number of a genomic region at a detection position of a target polynucleotide sequence in a sample, said method comprising:

obtaining measurement data for the sequence coverage for said sample using data

generated from mate-pair mappings;

correcting the measurement data for sequence coverage bias, wherein correcting the

measurement data comprises performing ploidy-aware baseline correction;

performing Hidden Markov Model (HMM) segmentation, scoring; and output based on the corrected measurement data; and

estimating a total copy number value and a region-specific copy number value for each of a plurality of genomic regions;

wherein the method is performed by one or more computing devices.

28. The method of Claim 27, further comprising: a computer logic generating input data that represents initial states by perfomiing a model for interpretation of absolute copy number of complex tumors;

wherein performing the HMM segmentation further comprises generating an initial

model based on the input data.

The method of Claim 27, further comprising annotating the plurality of genomic regions with the total copy number value and the region-specific copy number value based on state interpretation data that is generated by performing a model for interpretation of absolute copy number of complex tumors.

A computer-readable storage medium comprising instructions tangibly embodied thereon, the insiruciions when executed by a computer processor cause the processor to perform the method in any of Claims 27-29.

An apparatus for determining copy number variation of a genomic region at a detection position of a target polynucleotide sequence, comprising:

a computer processor; and

a computer-readable storage medium coupled to said processor, the storage medium having instructions tangibly embodied thereon, the instructions when executed by said processor causing said processor to perform the method in any of Claims 27-29.