Analysis of nucleic acids from preserved samples

Abstract

In some aspects, the present disclosure provides a method for analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) same. The method can comprise providing an FFPE sample comprising cells comprising cross-linked DNA-protein complexes reversing at least a portion of crosslinks in the FFPE sample using a buffer comprising a guanidinium salt, wherein crosslinks in DNA-protein complexes are not disrupted, isolating the DNA-protein complexes, and performing an analysis of nucleic acids of the DNA-protein complexes.

Description

Attorney Docket No. 45269-751.601ANALYSIS OF NUCLEIC ACIDS FROM PRESERVED SAMPLESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 721,287 filed November 15, 2024, which is entirely incorporated herein by reference.BACKGROUND

[0002] Analysis of nucleic acids, for example obtaining genome sequences, phasing information, or other genetic information from preserved samples such as formalin-fixed, paraffin-embedded (FFPE) samples presents particular challenges. FFPE samples are the most common banked clinical and cancer sample type. However, obtaining suitable nucleic acids from FFPE samples remains a challenge.SUMMARY

[0003] In an aspect, provided herein are methods of analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) sample. In some embodiments, the method comprises providing an FFPE sample comprising cells comprising cross-linked DNA-protein complexes. In some embodiments, the method comprises reversing at least a portion of crosslinks in the FFPE sample using a buffer comprising a guanidinium salt, wherein crosslinks in DNA-protein complexes are not disrupted. In some embodiments, the method comprises isolating the DNA-protein complexes. In some embodiments, the method comprises performing an analysis of nucleic acids of the DNA-protein complexes.

[0004] In various aspects of methods provided herein, in some embodiments reversing the crosslinks comprises incubating the FFPE sample in the buffer at a temperature about 70 °C for less than an hour. In some embodiments, reversing the crosslinks comprises incubating the FFPE sample in the buffer at a temperature about 70 °C for about 15 minutes. In some embodiments, reversing the crosslinks comprises incubating the FFPE sample in the buffer at a temperature about 55 °C for less than an hour. In some embodiments, reversing the crosslinks comprises incubating the FFPE sample in the buffer at a temperature about 55 °C for about 15 minutes.

[0005] In various aspects of methods provided herein, in some embodiments, the method comprises cleaving DNA of the DNA-protein complexes to obtain a plurality of DNA segments bound in DNA-protein complexes. In some embodiments, cleaving is effected by a nuclease. In some embodiments, the nuclease is selected from micrococcal nuclease, aAttorney Docket No. 45269-751.601transposase, an integrase, a restriction endonuclease, or a combination thereof. In some embodiments, the nuclease is micrococcal nuclease.

[0006] In various aspects of methods provided herein, in some embodiments, the method comprises lysing cells in the FFPE sample to generate a cell lysate. In some embodiments, the method comprises filtering the cell lysate.

[0007] In various aspects of methods provided herein, in some embodiments, the method comprises ligating at least a first DNA segment of the plurality of DNA segments to a second DNA segment of the plurality of DNA segments to create a plurality of ligated DNA segments bound in DNA-protein complexes. In some embodiments, ligating the first DNA segment and the second DNA segment further comprises ligating a tag oligonucleotide between the first DNA segment and the second DNA segment. In some embodiments, the tag comprises a barcode sequence.

[0008] In various aspects of methods provided herein, in some embodiments, the method comprises isolating the plurality obligated DNA segments from the DNA-protein complexes. In some embodiments, isolating the plurality obligated DNA segments comprises incubating the plurality obligated DNA segments bound in DNA-protein complexes in the buffer at a temperature greater than 60 °C for over an hour. In some embodiments, isolating the plurality obligated DNA segments comprises incubating the plurality obligated DNA segments bound in DNA-protein complexes in the buffer at a temperature about 78 °C overnight.

[0009] In various aspects of methods provided herein, in some embodiments, the method comprises obtaining a sequence of at least a portion of the first DNA segment and a portion of the second DNA segment of the plurality obligated DNA segments. In some embodiments, the method comprises assigning contigs having a sequence common to the sequence of the first DNA segment and the second DNA segment to a common scaffold in a nucleic acid assembly. In some embodiments, the analysis comprises sequencing, immunoprecipitation, nucleic acid hybridization, polymerase chain reaction (PCR), quantitative PCR, mass spectrometry, or a combination thereof. In some embodiments, the analysis comprises deriving genomic structural information indicative of an inversion, a deletion, or a translocation relative to a reference genome. In some embodiments, the analysis comprises deriving information indicative of a phase status for a first segment and a second segment of the nucleic acids.

[0010] In various aspects of methods provided herein, in some embodiments, the method comprises treating the FFPE sample with ethanol and / or xylene. In some embodiments,Attorney Docket No. 45269-751.601treating the FFPE samples with ethanol comprises treating the FFPE samples with 100% ethanol, 70% ethanol, 50% ethanol, and 20% ethanol each for about 10 minutes. In some embodiments, treating the FFPE samples with ethanol comprises treating the FFPE samples with 100% ethanol for about 10 minutes.

[0011] In various aspects of methods provided herein, in some embodiments, the FFPE sample comprises 200 ng to 1000 ng of DNA. In some embodiments, the FFPE sample comprises 200 ng to 500 ng of DNA. In some embodiments, the FFPE sample comprises a 1x10 pm scroll, a 5x10 pm scroll, a 10x10 pm scroll, or a 20x10 pm scroll. In some embodiments, the buffer further comprises at least one of a buffering agent and a detergent. In some embodiments, the detergent is Tween 20, Triton X-100, or a combination thereof.INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in its entirety as well as any references cited therein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A depicts an example schematic of a formalin fixed, paraffin embedded (FFPE) tissue sample.

[0014] FIG. IB depicts an example schematic of a protocol for chromatin-based next generation sequencing (NGS) library preparation.

[0015] FIG. 1C shows an example schematic of a workflow for chromatin extraction and library preparation (e.g., Chicago library preparation) from a preserved sample (e.g., an FFPE sample).

[0016] FIG. 2 illustrates results of optimal versus non-optimal crosslink reversal of FFPE samples.

[0017] FIG. 3 illustrates an optimal workflow of library conversion from FFPE tissue samples.

[0018] FIG. 4 shows an example computer system that is programmed or otherwise configured to implement the methods provided herein.DETAILED DESCRIPTION

[0019] A large repository of biological information is stored in preserved samples, such as formalin-fixed paraffin embedded (FFPE) tissue samples, such samples are routinely obtained during surgery such as surgery to excise a diseased or damaged tissue from a patient. However,Attorney Docket No. 45269-751.601crosslinking that occurs during preservation of such samples was thought to prohibit DNA extraction from these samples. Preservation and storage are technically straightforward and economical, and as a result large numbers of patient samples have been stored using this approach. As a result, obtaining and preserving samples from, for example, tumor tissue of patients undergoing a cancer therapeutic trial has long been routine.

[0020] One challenge with analysis of nucleic acids from FFPE samples, especially in a proximity ligation based analysis where a certain amount of crosslinking of the nucleic acids to the nucleic acid binding proteins (e.g., chromatin proteins) is needed for the sample preparation, is reversing the crosslinks in the FFPE sample enough to manipulate the protein-DNA complexes of the chromatin present in the sample but not so much that the complexes are degraded and useless for downstream proximity ligation and sequencing analysis. As such it is necessary and desired to optimize the crosslink reversal and enzymatic treatment of the sample prior to proximity ligation. Provided herein are methods of processing FFPE samples for proximity ligation and downstream analysis.

[0021] Until recently these samples were useful only for accessing structural information of the tissue. Three-dimensional tissue sections were well-preserved and available for morphological analysis, but the process of tissue preservation prohibited accessing genome-level information from the preserved samples. For example, FIG. 1A depicts an example schematic of a preserved sample (e.g., an FFPE sample). Cells 101 are depicted as spatially distributed within the tissue 102 of the fixed sample, such that their three-dimensional distribution is preserved. Nucleic acids 103 are present within cells.

[0022] Genome-scale rearrangements have been implicated in a number of diseases and disorders. Gene fusions, particularly those resulting from genome rearrangements, are particularly common in some cancers, and are often indicative of disease outcome in response to therapy. Generally, these rearrangement patterns do not reliably correlate to one or another morphological structure in a preserved sample. Rather, they must be genotyped directly. As a result, this information was unavailable despite tumor samples themselves being preserved, and data regarding the tumors’ response to chemotherapy or other therapy being readily available.

[0023] Methods and compositions herein relate to the determination of genomic information from preserved samples, such as the samples contemplated above. Some methods herein rely upon approaches that utilize extraction approaches so as to access genomic structural information contained in preserved samples. Protein DNA complexes that are extracted from the samples without destroying or disrupting these complexes utilize the fact that a first segment and a second segment of nucleic acid are held together independent of their phosphodi ester backbones. TheAttorney Docket No. 45269-751.601segments are tagged, either using oligos or by ligating the segments to one another, and sequence information is obtained allowing one to assign contigs to which the sequence information maps to a common scaffold. By assessing the frequency and types of read pairs generated by evaluating ligated segments, one may infer both physical linkage or phase information, and determine the presence of particular genomic structural rearrangements, such as structural rearrangements implicated in a disorder.

[0024] Also preserved in these samples is the three-dimensional configuration of the preserved tissue. Cancerous tumors are generally heterogeneous as to their genomic structure. Tumors are often characterized by separate mutations relating to DNA repair defects, cell death suppression, tumor growth, and metastasis. Tumors generally involve multiple cell sub-populations having various combinations of mutations and having various degrees of health risk. Often, these risks are correlated with local morphology. Tumor cell populations range from quiescent, to benign locally replicating cell populations, to metastasizing cell populations representing relatively high health risks. Thus, identifying not only the presence of a given genome architecture generally in a tumor but the local genome architecture of spatially separated subpopulations within a tumor sample is of value to researchers and practitioners trying to assess the relative efficacy of a prior drug treatment or trying to select an appropriate drug for a patient presenting a tumor of unknown risk. In particular, correlating a genome architecture with a position in a tumor and with a known cell morphology within the tumor is valuable for determining which genome architectures correspond most closely to tumor positions and local cell morphologies of highest risk.

[0025] It is thought that DNA extracted from preserved samples, such as FFPE samples, using approaches in the art are often less than 300 base pairs in length. Some nicking and damage may occur during the preservation (e.g., FFPE) process and subsequent dehydration and long-term storage. A significant amount of fragmentation can also occur during the extraction process, which historically involves overnight proteinase K treatment followed by boiling in order to reverse crosslinking and release the DNA. Nonetheless, through the approaches herein, such nucleic acid molecules, in combination with structural information preserved in DNA protein complexes excised without destruction or disruption of DNA protein complexes, yield information informative as to genome structural rearrangements.Crosslink Reversal of Preserved Samples

[0026] Another feature of preserved samples, such as FFPE samples, is the degree of crosslinking present in the preserved samples. In some cases, it is necessary to partially reverse the crosslinking in order to isolate DNA protein complexes prior to further sample preparation steps, such as proximity ligation for genomic analysis. For example, a certain amount of crosslinking isAttorney Docket No. 45269-751.601needed to keep DNA protein complexes intact, however, too much crosslinking can inhibit or make further sample preparation steps inefficient. In particular, crosslinks may need to be reversed enough to make chromatin accessible without further degrading the DNA.

[0027] In an aspect, disclosed herein, is a method of analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) sample comprising providing an FFPE sample comprising cells comprising cross-linked DNA-protein complexes. In some embodiments, the method comprises reversing at least a portion of crosslinks in the FFPE sample using a buffer comprising a guanidium salt. In some embodiments, crosslinks in DNA-protein complexes are not disrupted. In some embodiments, the method comprises isolating the DNA-protein complexes. In some embodiments, the method comprises performing an analysis of nucleic acids of the DNA-protein complexes.FPPE Tissue Deparaffinization and Rehydration

[0028] FFPE tissue can be processed to optimize crosslink reversal and DNA proximity ligation. For example, FFPE tissue can be provided a solvent to dissolve paraffin (deparaffinization). In some cases, the solvent comprises benzene, xylene, toluene, acetone, mineral oil, or a combination thereof. In some cases, the solvent can comprise xylene to dissolve paraffin. In some cases, the paraffin can be dissolved by heat at about 70 °C.

[0029] In addition to deparaffination, tissue rehydration can optimize crosslink reversal and DNA proximity ligation. For example, FFPE tissue can be provided with ethanol (EtOH), EtOH diluted in water (H2O), and water to restore water to the FFPE tissue. In some cases, FFPE tissue can be provided with about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, and 0% EtOH. In some cases, FFPE tissue can be provided with about 100%, 70%, 50%, 20%, and 0% EtOH. In some cases, rehydration with EtOH can be about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. In some cases, FFPE can be rehydrated by providing increasing dilutions of EtOH in water. In some cases, FFPE can be rehydrated by providing about 100% EtOH for about 10 minutes, about 70% EtOH in water for about 10 minutes, about 20% EtOH in water for about 10 minutes, and water for about 10 minutes. In some cases, FFPE can be rehydrated by providing about 100% EtOH for about 10 minutes, about 50% EtOH in water for about 10 minutes, and water for about 10 minutes.Crosslink Reversal Buffer

[0030] In some embodiments, reversing crosslinks in methods provided herein using a crosslink reversal buffer can comprise a detergent, a salt, and a protease. In some cases, the crosslinkAttorney Docket No. 45269-751.601reversal buffer comprises a Tris buffer, a phosphate buffer, a glycine buffer, a TAPS buffer, a Bicine buffer, a Tricine buffer, a TAPSO buffer, a HEPES buffer, a TES buffer, a MOPS buffer, a PIPES buffer, a Cacodylate buffer, a MES buffer, a citrate buffer, an acetate buffer, a CHES buffer, a Borate buffer, or a combination thereof. In some cases, the detergent comprises an ionic detergent or a non-ionic detergent. In some cases, the detergent comprises an ammonium lauryl sulfate, a sodium lauryl sulfate, a sodium laureth sulfate, a sodium myreth sulfate, a sodium dodecyl sulfate, an alkylbenzene sulfonate, a dioctyl sodium sulfosuccinate, a perfluorooctane sulfonate, a perfluorobutane sulfonate, an alkyl -aryl ether phosphate, an alkyl ether phosphate, an ethoxylate, an octaethylene glycol monododecyl ether, a pentaethylene glycol monododecyl ether, a nonoxynol, a Triton X-100, a Poloxamer, a glycerol monosterate, a glycerol monolaurate, a sorbitan monolaurate, a sorbitan monostearate, a sorbitan tristearate, a Tween 20, a Tween 40, a Tween 60, a Tween 60, a Tween 80, a decyl glucoside, a lauryl glucoside, an octyl glucoside, a cocamidopropyl betaine, or a combination thereof. In some cases, the salt comprises a sodium salt, a potassium salt, a magnesium salt, a calcium salt, a guanidinium salt, or a combination thereof. In some cases, the solution has a pH of about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0031] In some cases, the protease comprises proteinase K, endoproteinase trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C, endoproteinase Lys-C, thermolysin, papain, subtilisin, clostripain, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acylamino-acid-releasing enzyme, pyroglutamate aminopeptidase, or a combination thereof. Proteinase enzymes can be serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, asparagine peptide lyases, or a combination thereof. In some cases, the crosslink reversal buffer comprises Tris, sodium dodecyl sulfate, Proteinase K, and guanidium salt.

[0032] In some cases, the sample is treated in the solution for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, overnight, or longer. In some cases, the sample is treated in the solution at a temperature of about 25 °C, about 30 °C, about 35 °C, about 37 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, or about 70 °C.Attorney Docket No. 45269-751.601Reversing Crosslinks

[0033] In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer at a temperature about 70 °C for less than an hour. In some embodiments, reversing in (b) comprises incubating the FFPE sample in the crosslink reversal buffer at a temperature about 70 °C for about 15 minutes. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer further comprising a tissue dissociation enzyme. In some embodiments, the tissue dissociation enzyme can comprise Liberase™ or collagenase, or any combination thereof. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer at about 70 °C for about 15 minutes and incubating the FFPE sample in Liberase™ at about 37 °C overnight. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer at about 55 °C for about 15 minutes and incubating the FFPE sample in Liberase™ at about 37 °C overnight. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in Liberase™ at about 37 °C, without incubating the FFPE sample in the crosslink reversal buffer. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in a buffer comprising about 20 mM tris(hydroxymethyl)aminomethane (Tris) and 0.3% sodium dodecyl sulfate (SDS) at about 62 °C overnight. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer at about 70 °C for about 15 minutes and incubating the sample in about 20 mM Tris and 0.3% SDS overnight. In some embodiments, reversing crosslinks comprises incubating the FFPE sample in the crosslink reversal buffer at about 55 °C for about 15 minutes and incubating the sample in about 20 mM Tris and 0.3% SDS at about 62 °C overnight. In some embodiments, overnight is at least 12, 13, 14, 15, or 16 hours. In some embodiments, overnight is at most 12, 13, 14, 15, or 16 hours.Isolating DNA-Protein Complexes

[0034] Providing the FFPE sample with crosslink reversal buffer can reverse at least a portion of crosslinks without disrupting crosslinks in DNA-protein complexes. In some embodiments, nucleic acids isolated in nucleic acid-protein or DNA-protein complexes are cleaved prior to proximity ligation. In some embodiments, isolating the DNA-protein complexes in (c) further comprises cleaving DNA of the DNA-protein complexes to obtain a plurality of DNA segments bound in DNA-protein complexes. In some embodiments, the cleaving DNA of the DNA-protein complexes is effected by a nuclease. Any suitable nuclease is contemplated for cleavage. In some embodiments, the nuclease is selected from Micrococcal nuclease (MNase), a transposase, an integrase, a restriction endonuclease, or a combination thereof. In a preferred embodiment, the cleaving DNA of the DNA-protein complex is effected by MNase.Attorney Docket No. 45269-751.601Cell Lysis and Lysate Filtration

[0035] Methods for the extraction and purification of nucleic acids are well known in the art. For example, nucleic acids can be purified by organic extraction with phenol, phenol / chloroform / isoamyl alcohol, or similar formulations, including TRIzol® and TriReagent™. Other non-limiting examples of extraction techniques include: (1) organic extraction followed by ethanol precipitation, e.g, using a phenol / chloroform organic reagent (Ausubel et al., 1993), with or without the use of an automated nucleic acid extractor, e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif.); (2) stationary phase adsorption methods (U.S. Pat. No. 5,234,809; Walsh etal., 1991); and (3) salt-induced nucleic acid precipitation methods (Miller et al., (1988), such precipitation methods being typically referred to as “salting-out” methods. Another example of nucleic acid isolation and / or purification includes the use of magnetic particles to which nucleic acids can specifically or non-specifically bind, followed by isolation of the beads using a magnet, and washing and eluting the nucleic acids from the beads (see e.g., U.S. Pat. No. 5,705,628). FIG. 2 provides an illustration of isolating nucleic acid in DNA-protein complexes to beads. In some embodiments, the above isolation methods may be preceded by an enzyme digestion step to help eliminate unwanted protein from the sample, e.g., digestion with proteinase K, or other like proteases. See, e.g., U.S. Pat. No. 7,001,724. If desired, RNase inhibitors may be added to the lysis buffer. For certain cell or sample types, it may be acceptable to add a protein denaturation / digestion step to the protocol. Purification methods may be directed to isolate DNA, RNA, or both. When both DNA and RNA are isolated together during or subsequent to an extraction procedure, further steps may be employed to purify one or both separately from the other. Sub-fractions of extracted nucleic acids can also be generated, for example, purification by size, sequence, or other physical or chemical characteristic. In addition to an initial nucleic isolation step, purification of nucleic acids can be performed after any step in the methods of the disclosure, such as to remove excess or unwanted reagents, reactants, or products.Nucleic acid template molecules can be obtained as described in U.S. Patent Application Publication Number US2002 / 0190663 Al, published Oct. 9, 2003. Generally, nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Maniatis, etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). In some cases, the nucleic acids can be first extract from the biological samples and then crosslinked in vitro. In some cases, native association proteins (e.g, histones) can be further removed from the nucleic acids.Attorney Docket No. 45269-751.601Preserving physical linkage

[0036] Preserved samples, such as formalin-fixed, paraffin embedded samples, often pose challenges in determining physical linkage information of nucleic acids from the preserved sample. A number of downstream analyses can be used to obtain physical linkage information from a sample and are thus harmed or complicated by loss of such information during FFPE-sample DNA extraction. Nucleic acid samples are often intended as templates for amplification of large fragments, for example via polymerase chain reaction (“PCR”) using primers known to anneal adjacent to a region of interest. PCR relies upon the presence of a template from which one generates multiple amplicon nucleic acid molecules. Amplification relies upon two annealing sites (or an annealing site and the reverse complement of a second annealing site) being physically linked to one another on a single molecule. Accordingly, loss of physical linkage between primer annealing sites complicates analyses comprising PCR amplification.

[0037] Additionally, DNA segments within close proximity within FFPE tissue can be ligated together to retrieve more complete genomic DNA segments. In an embodiment disclosed herein, the method can further comprise ligating at least a first DNA segment of the plurality of DNA segments to a second DNA segment of the plurality of DNA segments to create a plurality of ligated DNA segments bound in DNA-protein complexes. In an embodiment, DNA segments can be ligated by polymerase chain reaction (PCR), as disclosed herein. In an embodiment, ligating the first DNA segment and the second DNA segment further comprises ligating a tag oligonucleotide between the first DNA segment and the second DNA segment. In an embodiment, the tag can comprise a barcode sequence.

[0038] Similarly, cloning a fragment into a cellular host so that it may be replicated, amplified, expressed or manipulated transgenically, is greatly facilitated by having a single molecule as a starting material. Loss of physical linkage for a fragment (that is, cleavage of that fragment) complicates cloning and necessitates multiple additional steps in fragment assembly.

[0039] Alternately, some analysis approaches require the preservation of physical proximity but do not require that a first segment and a second segment of a nucleic acid remain physically linked by their phosphodiester backbone. For example, one may assay for co-localization of probes to a first nucleic acid segment and a second nucleic acid segment so as to determine whether they exist on a common molecule in an un-degraded sample. Preservation of physical linkage facilitates this analysis but is not necessary for such analysis. Assembling the molecule into a chromatin complex such that the first segment and the segment are bound independent of their common phosphodiester backbone, for example similarly facilitates such an analysis. Even in the event of cleavage of their common phosphodiester backbone, physical proximity information for the firstAttorney Docket No. 45269-751.601segment and the second segment is preserved such that probing the complex with a first and a second probe will indicate whether the first fragment and the second fragment exist on a common molecule in the original sample.

[0040] Sequencing is another analysis that benefits from preservation of physical linkage information but does not require preservation of physical linkage, or even of physical proximity. Preservation of physical linkage facilitates sequencing, but so do other methods disclosed herein and known to one of skill in the art. Preservation of physical proximity, for example, facilitates sequencing because fragments held in proximity are readily end labeled so as to convey physical linkage information. Exposed internal ends are labeled using oligonucleotide tags that allow adjacent fragment sequence to be mapped to a common molecule. Alternately or in combination, exposed ends are ligated to one another at random, so as to generate read pairs wherein sequence on either side of a marked ligation event is mapped to a common molecule. Even in the absence of physical proximity, sequence analysis is facilitated if a nucleic acid sample is treated so as to add physical proximity markers prior to loss of the physical proximity information. That is, assembly of chromatin on a nucleic acid molecule, exposure of internal double-strand ends and labeling of these exposed ends via cross-ligation or via tagging using common oligonucleotides, if performed prior to subjecting the sample to degradation that may jeopardize or cause loss of physical linkage among segments of a molecule.

[0041] It is for all of these reasons that simple, affordable technologies for extracting physical linkage information encoded by DNA from preserved (e.g., FFPE) samples has become a critical necessity for the field. The methods disclosed herein are useful in many fields including, by way of non-limiting example, forensics, agriculture, environmental studies, renewable energy, epidemiology or disease outbreak response, and species preservation. Techniques of the present disclosure are used for mapping heterogeneity of a tissue sample, such as a tumor sample. For example, a tissue block can be sampled throughout its volume, and techniques of the present disclosure can be used to analyze the samples, allowing for comparison of variation throughout the tissue volume. Infections can also be analyzed throughout a tissue volume, Techniques of the present disclosure can be used for phasing of clinically important regions, analysis of structural variants, analysis of copy number variants, resolution of pseudogenes (e.g., STRC), targeted panels for druggable structural variants in cancer, and other applications.

[0042] In some embodiments of the methods disclosed herein, loss of physical linkage information and / or physical linkage information during sample extraction (e.g., extraction from an FFPE sample) is avoided or reduced by physically preventing or reducing nucleic acid breakage. Loss of phase information and / or physical linkage information is avoided or reduced by holding aAttorney Docket No. 45269-751.601first segment and a second segment in physical proximity independent of their phosphodiester backbone. Alternately or in combination, loss of phase information and / or physical linkage information is avoided or reduced by labeling a first segment and a second segment using a common or reciprocally complementary tag such that, upon loss of physical proximity information and loss of a common phosphodiester backbone tether, sequencing tag information that is affixed to a first segment and a second segment is sufficient to identify the two segments as sharing a common phase or common molecule in the original, un-degraded sample. Additionally, or alternatively, labeling is achieved by ligation of a first segment to a second segment, wherein the second segment is non-adjacent to the first segment, though they are physically linked on the same original DNA molecule.

[0043] Nucleic acid degradation arises from a number of diverse sources. Contemplated herein is protection from DNA degradation of a number of types, in particular DNA degradation that results in the introduction of double-strand breaks such as those that result in loss of physical linkage between a first segment and a second segment on an original common molecule in a nucleic acid sample. Of particular significance is nonenzymatic DNA degradation, such as that which occurs over time to stored nucleic acid samples, or that occurs to samples stored at room temperature. Nonenzymatic nucleic acid degradation includes boiling, proteinase treatment, UV radiation, oxidation, hydrolysis, physical stress such as shearing or tangling, or nucleophilic attach by a free 3’ hydroxyl group onto an internal bond of a nucleic acid molecule such that the molecule is cleaved, or a lariat formed. Also contemplated herein is nucleic acid damage resulting from enzymatic activity, such as nonspecific endonuclease activity, topoisomerase activity involving single strand nicking or double-strand breakage, restriction endonuclease activity, transposase activity, DNA mismatch repair or base excision, or other enzymatic activity that results in nucleic acid damage such as loss of phase information and / or loss of physical linkage information.Enzymatic degradation is exogenous in some cases, such as that which results from incomplete nucleic acid isolation, or initial isolation in a nonsterile environment such as that which may be encountered during collection ‘in the field’ such as a remote location or a location which, due for example to an epidemic or other burden on scientific resources, where sterile conditions are not easily or regularly obtained.

[0044] Some embodiments herein relate to assembling chromatin in vitro onto partially or totally isolated nucleic acids, such as nucleic acids extracted from preserved (e.g., FFPE) samples, such that physical linkage information relating a first segment of a nucleic acid molecule to a second segment of the nucleic acid molecule is not lost in the event that a double strand break occurs between the first nucleic acid molecule and the second nucleic acid molecule. The reassembledAttorney Docket No. 45269-751.601chromatin comprises in some cases nucleic acid binding proteins provided from another source. Alternately, in some cases an incompletely isolated nucleic acid sample, such as a nucleic acid sample treated so as to destroy or disrupt its native chromatin configuration, to inactivate native nuclease activity, or to destroy or disrupt native chromatin and to inactivate native nuclease activity, is contacted to a crosslinking agent so as to stabilize nucleic acids in the sample. In other cases, nucleic acids from preserved samples are analyzed using the native chromatin structures preserved in the sample.

[0045] Double strand breaks often occur during DNA storage over time. As a result, phasing information of DNA molecule is often difficult to obtain since variants cannot be confidently associated with haplotypes over long-distances. Further, nucleic acid segments separated by long repetitive regions cannot be linked or assembled into a common scaffold. These challenges are only amplified by double strand break introduction resulting from FFPE-extraction methods, boiling, proteinase treatments, long term storage, room temperature storage, enzymatic or nonenzymatic degradation, or contamination during or after isolation with a composition having a nuclease activity.

[0046] Sample degradation significantly affects de novo assembly. The disclosure addresses these problems simultaneously in some embodiments by preventing DNA damage through double strand breaks over time and optionally additionally by reducing the impact on phase determination of double-strand breakage. The preserved high DNA integrity enables methods for generating extremely long-range read pair data (XLRPs) that span genomic distances on the order of hundreds of kilobases, and up to megabases, with the appropriate input DNA.

[0047] Such data is invaluable for overcoming the substantial barriers presented by loss of physical linkage information by the loss of physical linkage information due to double strand breaks, DNA fragmentation, and large repetitive regions in genomes, including centromeres; enabling cost-effective de novo assembly; and producing re-sequencing data of sufficient integrity and accuracy for genomic analysis and personalized medicine.

[0048] The disclosure herein addresses these problems by preventing the loss of phase and / or physical linkage information that usually occurs to common extraction (e.g., FFPE extraction) methods, or alternately by preserving phase and / or physical linkage information independent of double strand breakage, such that physical linkage information is preserved even upon downstream processing, such as boiling of proteinase treatment. Physical linkage information can be preserved physically, through binding a first segment and a second segment of a nucleic acid molecule such that they are held together independent of their common phosphodiester backbone. Alternately or in combination, physical linkage information can be preserved through the taggingAttorney Docket No. 45269-751.601or reciprocal labelling of a first segment and a second segment of a common nucleic acid molecule such that, in the event of introduction of a double strand break between the segments, tag or other label information obtained through sequencing the first segment and adjacent sequence and the second segment and adjacent sequence is sufficient to map the first segment and the second segment to a common phase of a common nucleic acid molecule. Tagging can be alternatively achieved through ligating a first segment to a second segment, wherein the second segment is nonadj acent to the first segment, though they are physically linked on the same original DNA molecule. For example, a first segment and a second segment can be non-adj acent along the DNA molecule sequence, but in close physical proximity to each other or at least constituent in a common complex due to folding in a structure such as chromatin. Exposed ends of such segments can be ligated together. In another example, tagging is achieved by ligating barcodes (e.g., oligonucleotide barcodes) or other tags to both the first and second segments such that the first segment and the second segment are recognizably mapped to a common complex or a common molecule. Methods of preserving physical linkage information though chromatin reassembly or nucleic acid labeling or tagging have been previously described (PCT patent application number PCT / US2016 / 024225, incorporated herein in its entirety).Extraction and Recovery of Native Chromatin

[0049] Provided herein are methods for extracting long fragment lengths and / or phase information-containing fragments from preserved samples (e.g., FFPE samples). In some cases, these methods involve treating the nuclei of preserved cells (e.g., FFPE cells) gently in order to preserve the chromatin structures already present in the preserved sample (e.g., FFPE sample).

[0050] Disclosed herein are methods for performing extraction and in situ library preparation for the preservation of long range DNA fragments and / or phase information containing fragments. The released DNA can then further be processed for analysis, such as being used to generate readpair libraries.

[0051] A preserved sample (such as an FFPE sample) can be treated with a dissolving agent to dissolve embedding material (e.g., paraffin). In some cases, the dissolving agent is a solvent, such as xylene. Other examples of suitable solvent agents include but are not limited to organic solvents such as xylene, toluene, and benzene, as well as suitable isomers of each. The composition can be mixed such that the embedding material is dissolved in the dissolving agent. In some cases, mixing involves vortexing or high speed shaking or agitating. Alternately, gentle agitation is used in some cases. The sample is treated to separate the sample from the solvent and dissolved embedding material, such as through centrifugation with sufficient speed as to pellet the sample. Sufficient speeds include, but are not limited to, maximum speed of a tabletop centrifuge, such atAttorney Docket No. 45269-751.60114,000 revolutions per minute. The dissolving agent, comprising the dissolved embedding material, then can be removed, often gently so as not to disturb the pellet. Excess dissolving agent then can be removed with a washing reagent. In some examples, the washing agent is ethanol, for example 100% ethanol. The sample is mixed, vortexed, or agitated to dislodge the sample pellet from the inner wall of the holding vessel. The sample can optionally be re-centrifuged to re-pellet. Any remaining liquid is then removed from the holding vessel and the sample is dried.Representative drying techniques include air drying, vacuum drying, or other drying techniques well known in the art. After drying, a buffer, such as a lysis buffer is added to the sample. Lysis buffer can comprise buffering agents such as tris, salts such as sodium chloride, one or more detergents, such as sodium dodecyl sulfate (SDS), triton, a chelating agent, such as EDTA, and any combination thereof. A representative lysis buffer comprises 50 mM Tris pH 8, 50 mM NaCl, 1% SDS, 0.15% Triton, 1 mM EDTA, though one of skill in the art understands that variants on this composition may be readily generated. Suitable protocols can be employed to remove other embedding agents.

[0052] The sample can be allowed to rehydrate, such as by incubating (e.g., at 37 °C) for a sufficient amount of time, optionally while shaking or gently agitating. The sample then can be agitated, pipetted, or otherwise mixed in order to break up and re-suspend the pellet in the lysis buffer. Remaining non-soluble debris then can be separated from the lysis buffer, such as by centrifugation at a sufficient speed. DNA-protein complexes can be recovered and evaluated using downstream techniques, such as techniques to tag nucleic acid fragments.

[0053] Native DNA-protein complexes (e.g., chromatin) can be isolated from preserved samples (e.g., FFPE samples) such that the complexes rather than the nucleic acids are preserved intact. In these approaches, nucleic acid physical linkage information can be preserved not necessarily by preserving the nucleic acid phosphodiester backbones, but by preserving the linkage information independent of phosphodiester backbone status, such that commonly tagged fragments of a complex can be inferred to have a structural or physical linkage arrangement in the original sample.

[0054] Solubilization of chromatin can be an important step in isolating native DNA-protein complexes and extracting long-range linkage information from preserved samples such as FFPE samples. Chromatin complexes can be solubilized through a variety of methods, including but not limited to proteinase digestion and sonication. Such solubilization methods can disrupt tissue and chromatin to release soluble chromatin.Attorney Docket No. 45269-751.601

[0055] Solubilization via proteinase digestion can employ a variety of proteinase enzymes (also known as peptidase or protease enzymes), including but not limited to one or more of proteinase K, endoproteinase trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C, endoproteinase Lys-C, thermolysin, papain, subtilisin, clostripain, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acylamino-acid-releasing enzyme, and pyroglutamate aminopeptidase. Proteinase enzymes can be serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, or asparagine peptide lyases.

[0056] One protocol for solubilization via proteinase digestion can include removal of embedding material (e.g., paraffin), proteinase digestion, recovery of solubilized chromatin (e.g., with carboxylated beads such as SPRI beads), and sequencing library preparation. For example, first, tissue material can be put into a tube (e.g., 1.5 mL Eppendorf tube). Then, embedding material (e.g., paraffin) can be dissolved using a solvent such as xylene, Hemo-De, or limonene. Ethanol (e.g., 100% EtOH) can be used to remove the solvent, and the sample can be dried to remove the ethanol. The sample can then be digested with a proteinase enzyme (e.g., proteinase K). This can result in most or all of the tissue sample being solubilized. Without being limited by theory, proteinase treatment can be effective because protein-DNA methylene crosslink reversal can be very minor during the conditions of a proteinase treatment (e.g., 1 hour at 37 °C).

[0057] Another protocol for solubilization via sonication can include removal of embedding material (e.g., paraffin), lysis, homogenization, sonication, recovery of solubilized chromatin (e.g., with carboxylated beads such as SPRI beads), and sequencing library preparation. For example, first, embedding material (e.g., paraffin) can be dissolved using a solvent such as xylene, Hemo-De, or limonene. The tissue specimen can then be rehydrated, for example in successive washes of different ethanol concentrations from 100% ethanol to pure water. The tissue material can then be put into a tube and incubated in a lysis buffer (e.g., for one hour). Tissue can then be re-suspended in a buffer, such as a digestion buffer (e.g., MNase digestion buffer). The sample can then be homogenized, by methods including but not limited to Dounce homogenization. The sample can then be sonicated and re-suspended in a sonication buffer. Sonication cycles (e.g., 30 seconds at highest power) can then be repeated for as many cycles as needed to obtain sufficient solubilized chromatin (e.g., 10 cycles, 20 cycles, 30 cycles, 40 cycles). The soluble fraction can then be recovered.

[0058] Another protocol for solubilization includes removal of the embedding material (e.g., paraffin) and rehydration of the sample using xylene and ethanol. This can be followed by anAttorney Docket No. 45269-751.601initial removal of crosslinks in the sample using a solution comprising a Tris buffer, a calcium salt, a guanidinium salt, a Tween 20 detergent, and a Triton X detergent for 15 minutes at 70 °C. This is followed by digestion of the DNA in the protein-DNA complexes with MNase in an amount adjusted for the amount of nucleic acids in the sample. Cells are then lysed with a proteinase K solution and nucleic acid fragments are re-ligated for proximity ligation. The sample is then treated overnight at 78 °C in the crosslink reversal buffer to fully remove the crosslinks in the sample. The resulting DNA is then converted into a sequencing library to sequence the DNA.

[0059] Following solubilization, the sample can then be further processed according to methods discussed herein, such as recovery of solubilized chromatin (e.g., by binding to solid phase reversible immobilization (SPRI) beads), preparation of a sequencing library, such as a Chicago library as described herein (e.g., cleaving, tagging, and ligating of nucleic acids), sequencing (e.g., including long-range information), and sequence assembly.Isolating DNA Segments From DNA-Protein Complexes

[0060] In an embodiment, the method can further comprise isolating ligated DNA segments from the DNA-protein complexes as disclosed herein. In an embodiment, isolating the plurality obligated DNA segments bound in DNA-protein complexes can comprise incubating the DNA-protein complexes in the crosslink reversal buffer as described herein. In an embodiment, the plurality obligated DNA segments bound in DNA-protein complexes are incubated in the buffer at a temperature greater than 60 °C for over an hour. In an embodiment, the plurality obligated DNA segments bound in DNA-protein complexes are incubated in the buffer at a temperature about 70 °C overnight.Preserving DNA connectivity information in preserved extracted nucleic acids

[0061] Preserved samples, such as formalin-fixed, paraffin embedded samples, often comprise nucleic acids having damage, such as damage caused by fixative and / or embedding materials. A relevant component in making use of DNA is preserving the integrity of DNA physical linkage information of isolated DNA subject to a DNA damaging agent. Although DNA is a relatively stable molecule, the integrity of DNA is subject to environmental factors and particularly time. The presence of nuclease contamination, hydrolysis, oxidation, chemical, physical, and mechanical damages represent some of the major threats to DNA preservation. The mechanical, environmental, and physical factors encountered by DNA during transportation frequently leave them in fragments and potentially lose long-range information, which are critical for genomic analysis. Existing methods for preserving DNA information mostly delay the decay of DNA but provide little protection to DNA damage over time, especially when fragmentation occurs. In many cases, such DNA damage can be mitigated by fixing and embedding samples intended forAttorney Docket No. 45269-751.601long term storage. For example, FFPE (formalin-fixation, paraffin embedded) samples can be preserved for a long time. However, the preservation process can result in DNA damage.Additionally, later DNA extraction methods are often harsh and lead to further DNA damage and fragmentation.

[0062] Disclosed herein are methods, compositions, and kits related to recovering long-distance genomic information from preserved and / or stored nucleic acid molecules, such as nucleic acid molecules in DNA complexes or chromatin aggregates, such as crosslinked chromatin stored in preserved (e.g., FFPE) samples (including tissue-based preserved samples and cell culture-based preserved samples). In particular, methods, compositions, systems and kits relate to recovery of nucleic acid samples from these preserved samples such that nucleic acid physical linkage information is preserved. Physical linkage information is preserved either by preservation of the nucleic acids themselves in the FFPE extraction process, or by preserving nucleic acid complexes such that physical linkage information is preserved independent of any damage that may occur to the nucleic acids themselves in the extraction process.

[0063] Often, double strand breaks occur during DNA storage or during extraction of DNA from a preserved sample such as an FFPE sample, causing loss of physical linkage information. Loss of physical linkage information is particularly detrimental, because it precludes a sequence assembler from determining whether, in a diploid organism sample, mutations that map to a common locus are in fact in the same allele or are present on two separate homologous alleles positioned on different strands of the diploid genome. As genome information is used for personalized medicine or for more medicinal or therapeutic purposes, assigning physical linkage information to assembled contig sequence is of increasing importance.

[0064] These challenges to the integrity of DNA are problematic as genomics technologies improve along with expansion of programs for worldwide, prolonged, historical, or large-scale studies of genomes. Such studies are imperative to understand the genomes of current human populations and individuals and their impacts on human health, as well as to preserve present genomes for future studies with ever more powerful techniques. The latter concern also overlaps with forensic interests, which seek to bank DNA samples indefinitely for later analysis and identification.Samples

[0065] Samples herein are preserved, for example as formalin fixed paraffin embedded samples, and in some cases stored for a substantial period of time prior to analysis. Samples may be obtained pursuant to a drug trial and examined years later in an effort to identify genomic structural rearrangements relevant to or predictive of a positive drug treatment outcome. SuchAttorney Docket No. 45269-751.601samples can be used in determining long distance sequence information, such as genomic structural information. Long-range information generated by methods disclosed herein can be used for detecting structural variations, such as inversions, deletions, and duplications. Structural variation detection can also be used for identifying when active enhancers are brought into proximity to oncogenes or when repressive cis-acting elements are brought into proximity to tumor suppressors. Identification of such driver events are applicable to cancer studies, in particular to studies wherein tumor tissue is preserved long after a study is completed, and wherein various cell subpopulations of a tumor harbor differing genomic restructuring events. For example, novel structural variants can be detected and determined to be the causative agent of a cancer type.

[0066] Some formalin fixed paraffin embedded (FFPE) samples provided herein are cut into sections, for example using a microtome, and mounted on a surface, such as a glass slide. The mounted section, in some cases, is also called a FFPE scroll. In some cases, FFPE scrolls are a thickness of about 1 pm, about 2 pm, about 5 pm, about 7 pm, about 10 pm, about 15 pm, or about 20 pm. In some cases, scrolls are a width of about 1 pm, about 2 pm, about 5 pm, about 7 pm, about 10 pm, about 15 pm, or about 20 pm. In some cases, scrolls are a length of about 1 pm, about 2 pm, about 5 pm, about 7 pm, about 10 pm, about 15 pm, or about 20 pm,

[0067] Methods herein are used to obtain genomic structural information from preserved samples, such as samples obtained from a patient, a research animal, or an environmental sample. Some such samples include biopsy samples, surgical samples, tumor samples, whole organs, and other samples. These samples are preserved, often in a fixative such as a formaldehyde, a formalin, UV light, mitomycin C, nitrogen mustard, melphalan, 1,3 -butadiene di epoxide, cis diaminedichloroplatinum (II), or cyclophosphamide. Preserved samples are fixed directly and without homogenization, in some cases, by dropping the sample into a fixative solution. Once preserved, these samples can be stored for months or several years. In addition, the intact nature of the sample preserves positional information of the sample allowing an analysis of genomic structural information spatially throughout the sample. For example, the genomic structural information of the edge of a biopsy sample can be compared to the genomic structural information of the center of a biopsy sample.

[0068] Structural variation detection based on the methods disclosed herein can also be used to determine the DNA structure of gene fusions. Commonly used FISH methods or RNA-seq can determine that a DNA rearrangement has occurred, but the actual sequence of the rearrangement is not provided by these approaches. On the other hand, methods are provided herein for determining the structural variant that created a gene fusion of interest.Attorney Docket No. 45269-751.601

[0069] Provided herein are methods for determining three-dimensional DNA structural information. In some cases, the open or closed state of chromatin is detected by these methods. Structural information gathered by the methods disclosed herein can also be used to determine the presence or absence of insulators or loops, or for detecting novel loops or other new intra or inter chromosomal associations.

[0070] Provided herein are methods for tissue mapping. Tissue mapping is a process by which punch biopsies from different areas of a tissue, such as a tumor, and structural or phasing information is determined from each biopsy in order to determine the genomic heterogeneity in different regions.

[0071] Methods disclosed herein can be used for generating read-pair libraries comprising long range information from preserved (e.g., FFPE) samples. These libraries can be recovered from samples preserved for an indefinite period of time, for example in FFPE tissues.

[0072] Provided herein are methods for determining the structural and phase information of lymphocytes. In some cases, these methods are used to distinguish between different cell or receptor subtypes.

[0073] Methods provided herein are used in some embodiments for the detection of structural variants or genome rearrangements using long range data and phase information containing data. The starting material for these methods is samples which have been fixed in formalin and embedded in paraffin, as is common for most clinical sample preservation. Using the methods provided herein, structural and long range information is obtained from samples; such information is not obtainable using current methods due to high levels of DNA fragmentation. Therefore, use of the methods provided herein provides the opportunity to use this new data in many areas of clinical research and drug discovery.

[0074] Clinical research applications of the methods provided herein include tracking a therapy response or resistance using patient samples. To mitigate library preparation or sequencing variations, it is beneficial to process samples at the same time. This requires early time-point samples to be preserved, such as by FFPE. The methods provided herein provide a way to efficiently extract usable genomic material from these preserved samples, such that samples from multiple time points can be processed and analyzed at the same time.

[0075] In an example, a sample (e.g., a biopsy) is taken from a patient and placed in a fixative (e.g., formalin) during a medical procedure. This fixed sample is subsequently analyzed using the techniques of the present disclosure. For example, genomic features such as rearrangements relevant to cancer can be identified. Tumor / non-tumor phasing can be analyzed to differentiate cancer genomic information from somatic genomic information.Attorney Docket No. 45269-751.601

[0076] Furthermore, using the methods provided herein, useful long range genomic information can also be obtained from older samples that were preserved before the invention of such extraction methods. For example, tumor sample banks can be processed using the methods provided herein and the correlated to the known outcomes of the patients in order to mine this information for clinically relevant information. In this way, methods provided herein allow for prognosis and diagnosis correlations.

[0077] Methods and compositions provided herein can be used to determine structural variation profiles of preserved tissues. These structural variation profiles can be used in conjunction with other data sets, for example gene expression profiles, mutation profiles, methylation profiles, etc., to define distinct subtypes or other clusters.

[0078] Structural variation profiles determined by methods provided herein are also used to determine the structural evolution of mutations over time. For example, one may in some cases monitor the evolution of structural variants in tumor genome structure from inception, through progression or regression. In this way, tumor malignancies and metastasis can be better understood. Monitoring is available to be done both spatially, by examining various subpopulations in a three-dimensional sample, and temporally, by examining a time course of preserved samples, depending upon sample availability.

[0079] Methods provided herein can also be performed on banked, archived, or otherwise long-termed stored genetic samples. For example, archives of preserved tissue samples from now deceased patients who suffered from rare or unknown diseases can be analyzed by the methods provided herein, therefore providing insight not obtainable using standard methods.

[0080] Samples analyzed by the techniques disclosed herein can be degraded or have been subjected to various conditions, including conditions that are detrimental to the preservation of DNA or of long-range DNA information, including structural information. In some cases, samples have been subjected to acid treatment. In some cases, samples have been subjected to crosslinking agents, such as formaldehyde or formalin. In some cases, samples have been subjected to embedding, such as paraffin embedding. In some cases, samples have not been subjected to embedding, such as paraffin embedding. In some cases, samples have been subjected to heat treatment (e.g., to melt an embedding material). In some cases, samples have been subjected to a solvent, such as xylene (e.g., to dissolve an adhesive).

[0081] Fixed samples can have been subjected to various conditions after fixation but prior to subsequent processing or analysis. For example, after fixation, a time can elapse of at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 1 day, 2Attorney Docket No. 45269-751.601days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, 100 years, or more. After fixation, a sample can be subjected to a temperature increase of at least about 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, 100 °C, or more. After fixation, a sample can be subjected to a temperature decrease of at least about 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, 100 °C, or more. After fixation, a sample can be subjected to a pressure (e.g., ambient pressure) decrease of at least about 10 Pascal (Pa), 20 Pa, 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, 100 Pa, 110 Pa, 120 Pa, 130 Pa, 140 Pa, 150 Pa, 160 Pa, 170 Pa, 180 Pa, 190 Pa, 200 Pa, 210 Pa, 220 Pa, 230 Pa, 240 Pa, 250 Pa, 260 Pa, 270 Pa, 280 Pa, 290 Pa, 300 Pa, 310 Pa, 320 Pa, 330 Pa, 340 Pa, 350 Pa, 360 Pa, 370 Pa, 380 Pa, 390 Pa, 400 Pa, 410 Pa, 420 Pa, 430 Pa, 440 Pa, 450 Pa, 460 Pa, 470 Pa, 480 Pa, 490 Pa, 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, 800 Pa, 850 Pa, 900 Pa, 950 Pa, 1000 Pa, 2000 Pa, 3000 Pa, 4000 Pa, 5000 Pa, 6000 Pa, 7000 Pa, 8000 Pa, 9000 Pa, 10000 Pa, 20000 Pa, 30000 Pa, 40000 Pa, 50000 Pa, 60000 Pa, 70000 Pa, 80000 Pa, 90000 Pa, 100000 Pa, 101325 Pa, or more. After fixation, a sample can be subjected to a pressure (e.g., ambient pressure) increase of at least about 10 Pascal (Pa), 20 Pa, 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, 100 Pa, 110 Pa, 120 Pa, 130 Pa, 140 Pa, 150 Pa, 160 Pa, 170 Pa, 180 Pa, 190 Pa, 200 Pa, 210 Pa, 220 Pa, 230 Pa, 240 Pa, 250 Pa, 260 Pa, 270 Pa, 280 Pa, 290 Pa, 300 Pa, 310 Pa, 320 Pa, 330 Pa, 340 Pa, 350 Pa, 360 Pa, 370 Pa, 380 Pa, 390 Pa, 400 Pa, 410 Pa, 420 Pa, 430 Pa, 440 Pa, 450 Pa, 460 Pa, 470 Pa, 480 Pa, 490 Pa, 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, 800 Pa, 850 Pa, 900 Pa, 950 Pa, 1000 Pa, 2000 Pa, 3000 Pa, 4000 Pa, 5000 Pa, 6000 Pa, 7000 Pa, 8000 Pa, 9000 Pa, 10000 Pa, 20000 Pa, 30000 Pa, 40000 Pa, 50000 Pa, 60000 Pa, 70000 Pa, 80000 Pa, 90000 Pa, 100000 Pa, 101325 Pa, or more. After fixation, a sample can be subjected to an altitude change of at least about 0.1 meters (m), 0.2 m, 0.3 m, 0.4 m, 0.5 m, 0.6 m, 0.7 m, 0.8 m, 0.9 m, 1 m, 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, 11 m, 12 m, 13 m, 14 m, 15 m, 16 m, 17 m, 18 m, 19 m, 20 m, or more.

[0082] Fixed samples can be fixed in a fixation reaction that lasts at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, or more. In some cases, fixed samples are fixed in a fixation reaction that lasts at least about 30 minutes. InAttorney Docket No. 45269-751.601some cases, the fixation reaction time can be the time elapsed before the fixation reaction is quenched. In some cases, fixed samples are fixed in a fixation reaction that is not quenched.

[0083] The methods disclosed herein can be used in the analysis of genetic information of selective genomic regions of interest as well as genomic regions which may interact with the selective region of interest. Amplification methods as disclosed herein can be used in the devices, kits, and methods known to the art for genetic analysis, such as, but not limited to those found in U.S. Pat. Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117, 6,225,109, and 6,110,709. In some cases, amplification methods of the present disclosure can be used to amplify target nucleic acid for DNA hybridization studies to determine the presence or absence of polymorphisms. The polymorphisms, or alleles, can be associated with diseases or conditions such as genetic disease. In other cases, the polymorphisms can be associated with susceptibility to diseases or conditions, for example, polymorphisms associated with addiction, degenerative and age related conditions, cancer, and the like. In other cases, the polymorphisms can be associated with beneficial traits such as increased coronary health, or resistance to diseases such as HIV or malaria, or resistance to degenerative diseases such as osteoporosis, Alzheimer's or dementia.

[0084] The compositions and methods of the disclosure can be used for diagnostic, prognostic, therapeutic, patient stratification, drug development, treatment selection, and screening purposes. The present disclosure provides the advantage that many different target molecules can be analyzed at one time from a single biomolecular sample using the methods of the disclosure. This allows, for example, for several diagnostic tests to be performed on one sample.

[0085] The composition and methods of the disclosure can be used in genomics. The methods described herein can provide an answer rapidly which is very acceptable for this application. The methods and composition described herein can be used in the process of finding biomarkers that may be used for diagnostics or prognostics and as indicators of health and disease. The methods and composition described herein can be used to screen for drugs, e.g., drug development, selection of treatment, determination of treatment efficacy and / or identify targets for pharmaceutical development. The ability to test gene expression on screening assays involving drugs is very important because proteins are the final gene product in the body. In some embodiments, the methods and compositions described herein will measure both protein and gene expression simultaneously which will provide the most information regarding the particular screening being performed.

[0086] The composition and methods of the disclosure can be used in gene expression analysis. The methods described herein discriminate between nucleotide sequences. The difference between the target nucleotide sequences can be, for example, a single nucleic acid base difference, aAttorney Docket No. 45269-751.601nucleic acid deletion, a nucleic acid insertion, or rearrangement. Such sequence differences involving more than one base can also be detected. The process of the present disclosure is able to detect infectious diseases, genetic diseases, and cancer.

[0087] The present methods can be applied to the analysis of biomolecular samples obtained or derived from a patient so as to determine whether a diseased cell type is present in the sample, the stage of the disease, the prognosis for the patient, the ability to the patient to respond to a particular treatment, or the best treatment for the patient. The present methods can also be applied to identify biomarkers for a particular disease.

[0088] In some embodiments, the methods described herein are used in the diagnosis of a condition. As used herein the term “diagnose” or “diagnosis” of a condition may include predicting or diagnosing the condition, determining predisposition to the condition, monitoring treatment of the condition, diagnosing a therapeutic response of the disease, or prognosis of the condition, condition progression, or response to particular treatment of the condition. For example, preserved (e.g., FFPE) clinical samples can be assayed according to any of the methods described herein to determine the presence and / or quantity of markers of a disease or malignant cell type in the sample, thereby diagnosing or staging a disease or a cancer.

[0089] In some embodiments, the methods and composition described herein are used for the diagnosis and prognosis of a condition. Numerous immunologic, proliferative and malignant diseases and disorders are especially amenable to the methods described herein. Immunologic diseases and disorders include allergic diseases and disorders, disorders of immune function, and autoimmune diseases and conditions. Allergic diseases and disorders include but are not limited to allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis, and food allergy. Immunodeficiencies include but are not limited to severe combined immunodeficiency (SCID), hypereosinophilic syndrome, chronic granulomatous disease, leukocyte adhesion deficiency I and II, hyper IgE syndrome, Chediak Higashi, neutrophilias, neutropenias, aplasias, Agammaglobulinemia, hyper-IgM syndromes, DiGeorge / Velocardial-facial syndromes and Interferon gamma-THl pathway defects. Autoimmune and immune dysregulation disorders include but are not limited to rheumatoid arthritis, diabetes, systemic lupus erythematosus, Graves' disease, Graves ophthalmopathy, Crohn’s disease, multiple sclerosis, psoriasis, systemic sclerosis, goiter and struma lymphomatosa (Hashimoto's thyroiditis, lymphadenoid goiter), alopecia aerata, autoimmune myocarditis, lichen sclerosis, autoimmune uveitis, Addison's disease, atrophic gastritis, myasthenia gravis, idiopathic thrombocytopenic purpura, hemolytic anemia, primary biliary cirrhosis, Wegener's granulomatosis, polyarteritis nodosa, and inflammatory bowel disease,Attorney Docket No. 45269-751.601allograft rejection and tissue destructive from allergic reactions to infectious microorganisms or to environmental antigens.

[0090] Proliferative diseases and disorders that may be evaluated by the methods of the disclosure include, but are not limited to, hemangiomatosis in newborns; secondary progressive multiple sclerosis; chronic progressive myelodegenerative disease; neurofibromatosis; ganglioneuromatosis; keloid formation; Paget's Disease of the bone; fibrocystic disease (e.g., of the breast or uterus); sarcoidosis; Peronies and Duputren's fibrosis, cirrhosis, atherosclerosis and vascular restenosis.

[0091] Malignant diseases and disorders that may be evaluated by the methods of the disclosure include both hematologic malignancies and solid tumors.

[0092] Hematologic malignancies are especially amenable to the methods of the disclosure when the sample is a blood sample, because such malignancies involve changes in blood-borne cells. Such malignancies include non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, non-B cell lymphomas, and other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma, myelodysplastic disorders, myeloproliferative disorders, myelofibroses, atypical immune lymphoproliferations and plasma cell disorders.

[0093] Plasma cell disorders that may be evaluated by the methods of the disclosure include multiple myeloma, amyloidosis and Waldenstrom’s macroglobulinemia.

[0094] Example of solid tumors include, but are not limited to, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, head and neck tumors, and cervical neoplasms.

[0095] Genetic diseases can also be detected by the process of the present disclosure. This can be carried out by prenatal or post-natal screening for chromosomal and genetic aberrations or for genetic diseases. Examples of detectable genetic diseases include: 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome, Huntington Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors of metabolism, and diabetes.

[0096] The methods described herein can be used to diagnose pathogen infections, for example infections by intracellular bacteria and viruses, by determining the presence and / or quantity of markers of bacterium or virus, respectively, in the sample.Attorney Docket No. 45269-751.601

[0097] A wide variety of infectious diseases can be detected by the process of the present disclosure. The infectious diseases can be caused by bacterial, viral, parasite, and fungal infectious agents. The resistance of various infectious agents to drugs can also be determined using the present disclosure.

[0098] Bacterial infectious agents which can be detected by the present disclosure include Escherichia co , Salmonella, Shigella, Klesbiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium aviumintracellulare , Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes.

[0099] Fungal infectious agents which can be detected by the present disclosure include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis.

[0100] Viral infectious agents which can be detected by the present disclosure include human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus), Epstein - Barr virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.

[0101] Parasitic agents which can be detected by the present disclosure include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp. , Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.

[0102] The present disclosure is also useful for detection of drug resistance by infectious agents. For example, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multi-drug resistant Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus can all be identified with the present disclosure.Attorney Docket No. 45269-751.601

[0103] Thus, the target molecules detected using the compositions and methods of the disclosure can be either patient markers (such as a cancer marker) or markers of infection with a foreign agent, such as bacterial or viral markers.

[0104] The compositions and methods of the disclosure can be used to identify and / or quantify a target molecule whose abundance is indicative of a biological state or disease condition, for example, blood markers that are upregulated or downregulated as a result of a disease state.

[0105] In some embodiments, the methods and compositions of the present disclosure can be used for cytokine expression. The low sensitivity of the methods described herein would be helpful for early detection of cytokines, e.g., as biomarkers of a condition, diagnosis or prognosis of a disease such as cancer, and the identification of subclinical conditions.

[0106] The different samples from which the target polynucleotides are derived can comprise multiple samples from the same individual, samples from different individuals, or combinations thereof. In some embodiments, a sample comprises a plurality of polynucleotides from a single individual. In some embodiments, a sample comprises a plurality of polynucleotides from two or more individuals. An individual is any organism or portion thereof from which target polynucleotides can be derived, non-limiting examples of which include plants, animals, fungi, protists, monerans, viruses, mitochondria, and chloroplasts. Sample polynucleotides can be isolated from a subject, such as a preserved (e.g., FFPE) cell sample, preserved (e.g., FFPE) tissue sample, or organ sample derived therefrom, including, for example, tissue or tumor biopsy. The subject may be an animal, including but not limited to, an animal such as a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, etc., and is in some cases a mammal, such as a human. Samples can also be artificially derived, such as by chemical synthesis. In some embodiments, the samples comprise DNA. In some embodiments, the samples comprise genomic DNA. In some embodiments, the samples comprise mitochondrial DNA, chloroplast DNA, plasmid DNA, bacterial artificial chromosomes, yeast artificial chromosomes, oligonucleotide tags, or combinations thereof. In some embodiments, the samples comprise DNA generated by primer extension reactions using any suitable combination of primers and a DNA polymerase, including but not limited to polymerase chain reaction (PCR), reverse transcription, and combinations thereof. Where the template for the primer extension reaction is RNA, the product of reverse transcription is referred to as complementary DNA (cDNA). Primers useful in primer extension reactions can comprise sequences specific to one or more targets, random sequences, partially random sequences, and combinations thereof. Reaction conditions suitable for primer extension reactions are known in the art. In general, sample polynucleotides comprise any polynucleotide present in a sample, which may or may not include target polynucleotides.Attorney Docket No. 45269-751.601Size Selection

[0107] Nucleic acid obtained from preserved (e.g., FFPE) biological samples can be fragmented to produce suitable fragments for analysis. Template nucleic acids may be fragmented or sheared to desired length, using a variety of mechanical, chemical and / or enzymatic methods. DNA may be randomly sheared via sonication, e.g., Covaris method, brief exposure to a DNase, or using a mixture of one or more restriction enzymes, or a transposase or nicking enzyme. RNA may be fragmented by brief exposure to an RNase, heat plus magnesium, or by shearing. The RNA may be converted to cDNA. If fragmentation is employed, the RNA may be converted to cDNA before or after fragmentation. In some embodiments, nucleic acid from a biological sample is fragmented by sonication. In other embodiments, nucleic acid is fragmented by a hydroshear instrument. Generally, individual nucleic acid template molecules can be from about 2 kb bases to about 40 kb. In various embodiments, nucleic acids can be about 6kb-10 kb fragments. Nucleic acid molecules may be single-stranded, double-stranded, or double-stranded with single-stranded regions (for example, stem- and loop-structures).

[0108] In some embodiments, crosslinked DNA molecules may be subjected to a size selection step. Size selection of the nucleic acids may be performed to crosslinked DNA molecules below or above a certain size. Size selection may further be affected by the frequency of crosslinks and / or by the fragmentation method, for example by choosing a frequent or rare cutter restriction enzyme. In some embodiments, a composition may be prepared comprising crosslinking a DNA molecule in the range of about 1 kb to 5 Mb, about 5kb to 5 Mb, about 5 kB to 2Mb, about 10 kb to 2Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb about 20 kb to 500 kb, about 50 kb to 500 kb, about 50 kb to 200 kb, about 60 kb to 200 kb, about 60 kb to 150 kb, about 80 kb to 150 kb, about 80 kb to 120 kb, or about 100 kb to 120 kb, or any range bounded by any of these values (e.g. about 150 kb to 1 Mb).

[0109] In some embodiments, sample polynucleotides are fragmented into a population of fragmented DNA molecules of one or more specific size range(s). In some embodiments, fragments can be generated from at least about 1, about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, about 10,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, about 1,000,000, about 2,000,000, about 5,000,000, about 10,000,000, or more genome-equivalents of starting DNA. Fragmentation may be accomplished by methods known in the art, including chemical, enzymatic, and mechanical fragmentation. In some embodiments, the fragments have an average length from about 10 to about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 150,000, about 200,000, aboutAttorney Docket No. 45269-751.601300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 2,000,000, about 5,000,000, about 10,000,000, or more nucleotides. In some embodiments, the fragments have an average length from about 1 kb to about 10 Mb. In some embodiments, the fragments have an average length from about 1 kb to 5 Mb, about 5 kb to 5 Mb, about 5 kB to 2 Mb, about 10 kb to 2 Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb about 20 kb to 500 kb, about 50 kb to 500 kb, about 50 kb to 200 kb, about 60 kb to 200 kb, about 60 kb to 150 kb, about 80 kb to 150 kb, about 80 kb to 120 kb, or about 100 kb to 120 kb, or any range bounded by any of these values (e.g. about 60 to 120 kb). In some embodiments, the fragments have an average length less than about 10 Mb, less than about 5 Mb, less than about 1 Mb, less than about 500 kb, less than about 200 kb, less than about 100 kb, or less than about 50 kb. In other embodiments, the fragments have an average length more than about 5 kb, more than about 10 kb, more than about 50 kb, more than about 100 kb, more than about 200 kb, more than about 500 kb, more than about 1 Mb, more than about 5 Mb, or more than about 10 Mb. In some embodiments, the fragmentation is accomplished mechanically comprising subjection sample DNA molecules to acoustic sonication. In some embodiments, the fragmentation comprises treating the sample DNA molecules with one or more enzymes under conditions suitable for the one or more enzymes to generate double-stranded nucleic acid breaks. Examples of enzymes useful in the generation of DNA fragments include sequence specific and non-sequence specific nucleases. Non-limiting examples of nucleases include DNase I, Fragmentase, restriction endonucleases, variants thereof, and combinations thereof. For example, digestion with DNase I can induce random double-stranded breaks in DNA in the absence of Mg++and in the presence of Mn++. In some embodiments, fragmentation comprises treating the sample DNA molecules with one or more restriction endonucleases. Fragmentation can produce fragments having 5' overhangs, 3' overhangs, blunt ends, or a combination thereof. In some embodiments, such as when fragmentation comprises the use of one or more restriction endonucleases, cleavage of sample DNA molecules leaves overhangs having a predictable sequence. In some embodiments, the method includes the step of size selecting the fragments via standard methods such as column purification or isolation from an agarose gel.Sequencing Library Preparation

[0110] In an embodiment, the method as disclosed herein can further comprise obtaining a sequence of at least a portion of the first DNA segment and a portion of the second DNA segment of the plurality obligated DNA segments. In an embodiment, obtaining the sequence further comprises assigning contigs having a sequence common to the sequence of the first DNA segment and the second DNA segment to a common scaffold in a nucleicAttorney Docket No. 45269-751.601acid assembly. In an embodiment, the method can further comprise analysis of the obtained sequences. In an embodiment, analysis can comprise sequencing, immunoprecipitation, nucleic acid hybridization, polymerase chain reaction (PCR), quantitative PCR, mass spectrometry, or a combination thereof. In an embodiment, the analysis can comprise deriving information indicative of a phase status for a first segment and a second segment of the nucleic acids.

[0111] FIG. IB shows an exemplary schematic of chromatin-based next generation sequencing (NGS) library preparation (e.g., “Chicago”) for obtaining the sequence of the portion of the first DNA segment and the portion of the second DNA segment of the plurality obligated DNA segments. In a first step 111, chromatin nucleases (circles) are crosslinked (gray lines) forming chromatin aggregates. In a second step 112, chromatin aggregates are cut with restriction endonuclease. In a third step 113, cut ends are blunt ended, ligated, and marked (e.g., with biotin) (small gray circles). In a fourth step 114, blunt ends are randomly ligated forming short, medium, and long-range associations. In a fifth step (115), crosslinks are reversed, DNA is purified, and informative ligation-containing fragments are selected for with marker pulldown. Then, a conventional sequencing library preparation can be performed. Resulting read pairs can span genomic distances up to the maximum size of the input DNA. Such libraries can be used to construct highly-contiguous genome assemblies with chromosome-scale super-scaffolds.

[0112] FIG. 1C shows an exemplary schematic of a workflow for chromatin extraction and library preparation (e.g., Chicago library preparation) from a preserved sample (e.g., an FFPE sample). Preserved samples can be processed to extract fixed chromatin that can then be put through methods for generating and sequencing long range genomic linkage information. For example, a preserved sample 121 can have chromatin extracted 122 and fragmented (e.g., with a restriction enzyme, such as DpnII). The chromatin can comprise cross-links 123. Overhangs (e.g., 4 bp 5’ overhangs) can be filled in with a nucleotide mix including biotinylated nucleotides 124.Blunt ends can then be ligated 125, and markers (e.g., biotin) can be pulled down (e.g., using streptavidin) 126. Non-marked (e.g., non-biotinylated) blunt ends can then be removed, and sequencing adapters (e.g., Illumina sequencing adapters, Pacific Biosciences sequencing adapters, nanopore sequencing adapters) can be attached and a sequencing library 127 can be prepared. The library can be enriched for molecules containing biotinylated ligated junctions, amplified (e.g., by PCR), and sequenced (e.g., using an Illumina sequencer such as a MiSeq or HiSeq, using a Pacific Biosciences long-read sequencer, or using a nanopore sequencer such as Oxford Nanopore or Genia). In some cases, such as when using a long-read sequencer like Pacific Biosciences orAttorney Docket No. 45269-751.601nanopore sequencers, multiple molecules can be joined (e.g., ligated) into a longer molecule prior to sequencing.

[0113] Enrichment can be performed, alternatively or in addition to enrichment for labeled nucleotides (e.g., biotinylated nucleotides, epigentically modified nucleotides), for genetic regions of interest. For example, a sample or a library can be enriched for a fusion gene, such as by targeting a known relevant half of a fusion gene. Other genetic and genomic features as discussed herein can also be targeted for enrichment.

[0114] In many cases, no fixative agent is added to the previously obtained sample (such as an FFPE sample) as part of the purification process. Rather, crosslinks previously generated pursuant to an original sample preservation process can be relied upon to stabilize the DNA-protein (e.g., chromatin) complexes isolated herein, and the extraction process preserves linked complexes rather than generating substantial amounts of new ones. The fraction of the sample solubilized in the lysis buffer is then processed by any of the methods disclosed herein.

[0115] Alternatively, in some embodiments, in vitro proximity ligation (e.g., Chicago in vitro proximity ligation) or other protein-DNA complex tagging methods are used to generate read-pair libraries from native chromatin generated from high quality nucleic acids extracted from preserved samples (such as FFPE preserved samples) comprising DNA. For example, a preserved sample (e.g., an FFPE sample) can be processed to extract nucleic acids such as DNA so as to minimize DNA damage in the extraction process. In some cases, one or more of vortexing, shearing, boiling, high-temperature incubation or DNase-related enzymatic treatment are excluded from the nucleic acid extraction protocol, so as to decrease the damage to isolated DNA. The isolated DNA recovered can be of quality sufficient to preserve physical linkage, phase, or genome structural information. Native chromatin can be crosslinked, such as with formaldehyde, in order to preserve proximal information of DNA sequences within the same DNA molecule, independent of their common phosphodiester backbone. Importantly, the crosslinking can be performed on the DNA extracted from the preserved sample (such as an FFPE sample) after isolation from the preserved sample. As discussed above in the context of isolation of DNA-protein complexes, in many cases no crosslinking agent is added during the isolation process. These crosslinked complexes can be labeled, such as with biotin, methylation, sulfylation, acetylation, or other base modification, and then isolated, such as with streptavidin beads in the case of biotin labelling. The isolated complexes then can be digested with restriction enzymes in order to generate free sticky ends which are then filled in with labelled nucleotides, such as with biotinylated nucleotides or other nucleotides as mentioned.Attorney Docket No. 45269-751.601

[0116] Exposed DNA ends in DNA-protein complexes can be ligated to generate paired ends between DNA sequences within the same DNA molecule. These ligated paired ends can often be originally not adjacent to one another on the DNA molecule. Paired ends can be blunt, in some cases as a result of filling in sticky ends.

[0117] Alternately or additionally, exposed nucleic acid complex ends can be ligated to one another through a punctuation oligonucleotide as discussed herein, or can be tagged using a population of oligonucleotide tags such that nucleic acid fragments are identifiably mapped to a common DNA protein complex. In some cases, paired end reads are generated not from cleaved ends of a DNA-complex that are directly ligated, but from cleaved ends that are joined to a common punctuation oligonucleotide. A punctuation oligonucleotide includes any oligonucleotide that can be joined to a target polynucleotide, so as to bridge two cleaved internal ends of a sample molecule undergoing phase-preserving rearrangement. Punctuation oligonucleotides can comprise DNA, RNA, nucleotide analogues, non-canonical nucleotides, labeled nucleotides, modified nucleotides, or combinations thereof. In many examples, double-stranded punctuation oligonucleotides comprise two separate oligonucleotides hybridized to one another (also referred to as an “oligonucleotide duplex”), and hybridization may leave one or more blunt ends, one or more 3’ overhangs, one or more 5’ overhangs, one or more bulges resulting from mismatched and / or unpaired nucleotides, or any combination of these. In some instances, different punctuation oligonucleotides are joined to target polynucleotides in sequential reactions or simultaneously. For example, the first and second punctuation oligonucleotides can be added to the same reaction. Alternately, punctuation oligo populations are uniform in some cases. Punctuation molecule and methods of use in preserving and determining genomic structural and proximity information has been described previously (US provisional application numbers 62 / 298906, 62 / 298966, and 62 / 305957, all three of which are incorporated herein in their entirety). Some punctuation oligonucleotides comprise a tag or label to facilitate isolation, such as a biotin tag, such that fragments of a library comprising punctuation oligonucleotides are easily isolated. Alternative tags include but are not limited to methylation, acetylation, or other base modification. Generally, punctuation oligonucleotides are ligated to exposed nucleic acid ends, but alternate approaches of incorporating punctuation oligonucleotides into a library are also contemplated.

[0118] Nucleotides, such as those used to fill in sticky ends, can be labeled. Labelled nucleotides can biotinylated, sulphated, attached to a fluorophore, dephosphorylated, or any other number of nucleotide modifications. Nucleotide modifications can also include epigenetic modifications, such as methylation (e.g., 5-mC, 5-hmC, 5-fC, 5-caC, 4-mC, 6-mA, 8-oxoG, 8-oxoA). Labels or modifications can be selected from those detectable during sequencing, such asAttorney Docket No. 45269-751.601epigenetic modifications detectable by nanopore sequencing; in this way, the locations of ligation junctions can be detected during sequencing. These labels or modifications can also be targeted for binding or enrichment; for example, antibodies targeting methyl -cytosine can be used to capture, target, bind, or label blunt ends filled in with methyl -cytosine. Non-natural nucleotides, non-canonical or modified nucleotides, and nucleic acid analogs can also be used to label the locations of blunt-end fill-in. Non-canonical or modified nucleotides can include pseudouridine (T), dihydrouridine (D), inosine (I), 7-methylguanosine (m7G), xanthine, hypoxanthine, purine, 2,6-diaminopurine, and 6,8-diaminopurine. Nucleic acid analogs can include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). In some cases, overhangs are filled in with un-labeled dNTPs, such as dNTPs without biotin. In some cases, such as cleavage with a transposon, blunt ends are generated that do not require filling in. These free blunt ends are generated when the transposase inserts two unlinked punctuation oligonucleotides. The punctuation oligonucleotides, however, can be synthesized to have sticky or blunt ends as desired. Proteins associated with sample nucleic acids, such as histones, can also be modified. For example, histones can be acetylated (e.g., at lysine residues) and / or methylated (e.g., at lysine and arginine residues).

[0119] In some embodiments, Hi-C or other ligation or tagging-mediated methods can be used to generate read-pair libraries from naturally occurring chromatin that is crosslinked, for example chromatin that is crosslinked pursuant to sample preservation. The DNA can be crosslinked, such as with formaldehyde, to preserve native chromatin structures during the preservation process. Extraction can be performed as above to separate these DNA-protein structures from any sample preservative or fixative such as paraffin, without disrupting the crosslinked DNA-protein structures, thereby preserving proximal information between DNA molecules independent of a phosphodiester backbone. These crosslinked structures can be digested with a restriction enzyme to generate free sticky ends which are subsequently filled in with tagged nucleotides, such as biotin labelled nucleotides. The resulting blunt ends can be ligated together to generate paired ends of DNA fragments. These paired ends represent DNA molecules that are in proximity to each other in the chromatin structure. Hi-C methods and variations are known in the art (Liberman-Aiden et al., 2009, Science 326, 289, incorporated herein in its entirety;US20130096009, incorporated herein in its entirety).

[0120] The paired ends can be released from the chromatin protein, such as by enzymatic digestion (e.g., with a proteinase such as proteinase K). Released paired ends can be treated with an exonuclease to remove labelled nucleotides from remaining free ends, such that the only labelled nucleotides reside between the ligated paired ends. These paired ends then can beAttorney Docket No. 45269-751.601purified, such as with streptavidin beads in the case of biotin labels. Purification can also be conducted by other means, such as with SPRI beads (e.g., carboxylated beads) or via electrophoresis (e.g., gel electrophoresis, capillary electrophoresis). Paired ends then can be prepared for sequencing. For example, the paired ends can be attached to sequencing adapters and then sequenced to generate read pair libraries. Chicago in vitro proximity ligation methods have been described previously (see, e.g., U.S. Pat. Pub. No. 20140220587, incorporated herein by reference in its entirety; U.S. Pat. Pub. No. 20150363550, incorporated herein by reference in its entirety).

[0121] In an embodiment, a library is created from cells previously embedded in FFPE, in sections 15-20 microns thick having about 3 x 105cells per section. Alternatively, cells embedded in FFPE are provided in sections 1-5, 5-10, 10-15, 15-20, 25-30, 35-40, or 45-50 thick having about 103, 104, 105, 106, or 107cells per section. In some cases, the samples are AJ GIAB (‘Genome in a Bottle’) samples GM24149 (father) and GM24385 (son). The sections are washed with a solvent to remove the embedding material, for example xylene, toluene, or benzene. The solvent is removed by washing the sections with an ethanol solution, in some cases 100% ethanol is used to wash the sections. The paraffin-free tissue samples are then solubilized in a buffer, for example in a detergent buffer. Nucleic acids in the samples are then digested with an endonuclease, for example a restriction enzyme such as Mbol . Blunt ends are created in the digested nucleic acids by filling in the overhangs resulting from the restriction enzyme digest using a DNA polymerase and nucleotides, such as biotinylated dNTPs. The blunt ends are ligated together using a DNA ligase, for example T4 DNA ligase in a reaction favoring blunt end ligation, resulting in biotinylated fragments of DNA. These fragments are prepared for use in a sequencing reaction.Sequencing

[0122] Also disclosed herein are methods and compositions for generating nucleic acid sequencing libraries that harbor genomic structural information such as physical linkage information. DNA complexes are generated from preserved samples such as FFPE derived nucleic acid samples. Paired ends, ligation junctions, punctuation ends, or commonly tagged ends are generated through the isolation of nucleic acid complexes bound such that a first segment and a second segment are held together independently of any phosphodiester backbone bond, exposed ends are tagged, and tag junctions are isolated. Tagging variously comprises tagging one exposed end using a second exposed end directly, such that the junction is identifiable from the fact that sequences on either side of the junction map to contigs that correspond to distal positions on a genome scaffold, are unscaffolded, or map to different chromosomes in an unrearranged genome.Attorney Docket No. 45269-751.601Alternately, tagging involves joining exposed ends using a punctuation oligo, or adding a common oligo tag to exposed ends of a complex such that sequence adjacent to tagged ends is confidently mapped to a common DNA complex and therefore a common phase of a source nucleic acid from which the DNA complex was generated.

[0123] Paired ends, concatemerized paired ends, or punctuated molecules are sequenced using an appropriate short read or long read sequencing technology platform, and the sequence reads are then analyzed.

[0124] In some cases, a plurality of paired end molecules is generated as described herein, and subsequently sequenced using short read sequencing technology. In these cases, either short sequence reads across the paired end ligation junction are generated, or short reads from each end of the paired end fragment are generated to make a read pair. If sequences from the first and second nucleic acid segments are detected in a single sequence read or read pair, it is determined that the first and second nucleic acid segments are in phase on the same DNA molecule in the input DNA sample. In such cases, the generated sequence libraries yield phase and structural information for DNA segments.

[0125] For a given punctuated molecule sequence read or read pair, sequence segments are observed that are locally uninterrupted by punctuation elements. Sequence in these segments is presumed to be in phase, and locally correctly ordered and oriented. Segments are observed to be separated by punctuation oligos. Segments on either side of a punctuation oligo are inferred to be in phase with one another on a common sample nucleic acid molecule but not to be correctly ordered and oriented relative to one another on the punctuation molecule. A benefit of the rearrangement is that segments positioned far removed from one another are sometimes brought into proximity, such that they are read in a common read and confidently assigned to a common phase even if in the sample molecule they are separated by large distances of identical, difficult to phase sequence. Another benefit is that the segment sequences themselves comprise most, substantially all or all of the original sample sequence, such that in addition to phase information, in some cases contig information is determined sufficient to perform de novo sequence assembly in some cases. This de novo sequence is optionally used to generate a novel scaffold or contig set, or to augment a previously or independently generated contig or scaffold sequence set.

[0126] In some cases, a plurality of punctuated DNA molecules is generated as disclosed herein, concatemerized into a single long nucleic acid molecule or preserved without shearing or cleavage as a single, rearranged long molecule, and subsequently sequenced using long-read sequencing technology. Each punctuated molecule is sequenced, and the sequence reads are analyzed. In preferred examples, sequence reads average 10 kb for the sequence reaction. In otherAttorney Docket No. 45269-751.601examples, sequence reads average about 5kb, 6kb, 7kb, 8kb, 9kb, lOkb, llkb, 12kb, 13kb, 14kb, 15kb, 16kb, 17kb, 18kb, 19kb, 20kb, 21 kb, 22kb, 25kb, 3Okb, 35kb, 40kb, or greater. In favored examples, sequence reads are identified that comprise at least 500 bases of a first segment and 500 bases of a second segment, joined by a punctuation oligo sequence. In other examples, the sequence reads comprise at least about 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, or greater of a first DNA segment and at least about 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, or greater of a second DNA segment. In some examples, the first and second segment sequences are mapped to a scaffold genome and are found to map to contigs that are separated by at least lOOkb. In other examples, the separation distance is 8kb, 9kb, lOkb, 12.5kb, 15kb, 17.5kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 60kb, 70kb, 80kb, 90kb, lOOkb, 125kb, 150kb, 200kb, 300kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1Mb, or greater. In most cases, the first contig and the second contig each comprise a single heterozygous position, the phase of which is not determined in a scaffold. In preferred examples, the heterozygous position of the first contig is spanned by the first segment of the long read, and the heterozygous position of the second contig is spanned by the second segment of the long read. In such cases, the reads each span their contigs’ respective heterozygous regions and sequence of the read segments indicates that a first allele of the first contig and a first allele of the second contig are in phase. If sequences from the first and second nucleic acid segments are detected in a single long sequence read, it is determined that the first and second nucleic acid segments are comprised on the same DNA molecule in the input DNA sample. In these embodiments, nucleic acid sequence libraries generated by the methods and compositions disclosed herein provide phase information for contigs that are positioned far apart from one another on a genome scaffold.

[0127] Alternatively, a plurality of paired end molecules is generated as described herein, and subsequently sequenced using long read sequencing technology. In some cases, the average read length for the library is determined to be about 1 kb. In other cases, the average read length for the library is about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, or greater. In most examples, paired end molecules comprise a first DNA segment and a second DNA segment that, within the input DNA sample, are in phase and separated by a distance greater than 10 kb. In some examples, the separation distance between two such DNA segments is greater than about 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 20 kb, 23 kb, 25 kb, 30 kb, 32 kb, 35 kb, 40 kb, 50 kb, 60 kb, 75 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 750 kb, 1 Mb, or greater. In most cases, sequence reads are generated from paired end molecules, some of which comprise at least 300 bases of sequenceAttorney Docket No. 45269-751.601from a first nucleic acid segment and at least 300 bases of sequence from a second nucleic acid segment. In other examples, the sequence reads comprise at least about 50 bases, 100 bases, 150 bases, 200 bases, 250 bases, 300 bases, 350 bases, 400 bases, 450 bases, 500 bases, 550 bases, 600 bases, 650 bases, 700 bases, 750 bases, 800 bases, or greater of a first DNA segment and at least about 50 bases, 100 bases, 150 bases, 200 bases, 250 bases, 300 bases, 350 bases, 400 bases, 450 bases, 500 bases, 550 bases, 600 bases, 650 bases, 700 bases, 750 bases, 800 bases, or greater of a second DNA segment. If sequences from the first and second nucleic acid segments are detected in a single sequence read or read pair, it is determined that the first and second nucleic acid segments are in phase on the same DNA molecule in the input DNA sample. In such cases, the generated sequence libraries yield phase information for DNA segments that are separated in the nucleic acid sample by greater than the read length of the sequencing technology used to sequence them.

[0128] In various embodiments, suitable sequencing methods described herein or otherwise known in the art are used to obtain sequence information from nucleic acid molecules within a sample. Sequencing can be accomplished through classic Sanger sequencing methods which are well known in the art. Sequence can also be accomplished using high-throughput systems some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, such as detection of sequence in real time or substantially real time. In some cases, high throughput sequencing generates at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000 or at least 500,000 sequence reads per hour; where the sequencing reads can be at least about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 150, about 180, about 210, about 240, about 270, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, or about 1000 bases per read.

[0129] In some embodiments, high-throughput sequencing involves the use of technology available by Illumina’s Genome Analyzer IIX, MiSeq personal sequencer, or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000 machines. These machines use reversible terminator-based sequencing by synthesis chemistry. These machines can do 200 billion DNA reads or more in eight days. Smaller systems may be utilized for runs within 3, 2, 1 days or less time.

[0130] In some embodiments, high-throughput sequencing involves the use of technology available by ABI Solid System. This genetic analysis platform that enables massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology is based on sequential ligation with dye-labeled oligonucleotides.Attorney Docket No. 45269-751.601

[0131] The next generation sequencing can comprise ion semiconductor sequencing (e.g., using technology from Life Technologies (Ion Torrent)). Ion semiconductor sequencing can take advantage of the fact that when a nucleotide is incorporated into a strand of DNA, an ion can be released. To perform ion semiconductor sequencing, a high density array of micromachined wells can be formed. Each well can hold a single DNA template. Beneath the well can be an ion sensitive layer, and beneath the ion sensitive layer can be an ion sensor. When a nucleotide is added to a DNA, H+ can be released, which can be measured as a change in pH. The H+ ion can be converted to voltage and recorded by the semiconductor sensor. An array chip can be sequentially flooded with one nucleotide after another. No scanning, light, or cameras can be required. In some cases, an IONPROTON™ Sequencer is used to sequence nucleic acid. In some cases, an IONPGM™ Sequencer is used. The Ion Torrent Personal Genome Machine (PGM). The PGM can do 10 million reads in two hours.

[0132] In some embodiments, high-throughput sequencing involves the use of technology available by Helicos BioSciences Corporation (Cambridge, Massachusetts) such as the Single Molecule Sequencing by Synthesis (SMSS) method. SMSS is unique because it allows for sequencing the entire human genome in up to 24 hours. Finally, SMSS is described in part in US Publication Application Nos. 20060024711; 20060024678; 20060012793; 20060012784; and 20050100932.

[0133] In some embodiments, high-throughput sequencing involves the use of technology available by 454 Lifesciences, Inc. (Branford, Connecticut) such as the PicoTiterPlate device which includes a fiber optic plate that transmits chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. This use of fiber optics allows for the detection of a minimum of 20 million base pairs in 4.5 hours.

[0134] Methods for using bead amplification followed by fiber optics detection are described in Marguiles, M., etal. “Genome sequencing in microfabricated high-density picolitre reactors”, Nature, doi: 10.1038 / nature03959; and well as in US Publication Application Nos. 20020012930; 20030068629; 20030100102; 20030148344; 20040248161; 20050079510, 20050124022; and 20060078909.

[0135] In some embodiments, high-throughput sequencing is performed using Clonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis (SBS) utilizing reversible terminator chemistry. These technologies are described in part in US Patent Nos. 6,969,488; 6,897,023; 6,833,246; 6,787,308; and US Publication Application Nos. 20040106110;20030064398; 20030022207; and Constans, A., The Scientist 2003, 17(I3):36.Attorney Docket No. 45269-751.601

[0136] The next generation sequencing technique can comprise real-time (SMRT™) technology by Pacific Biosciences. In SMRT, each of four DNA bases can be attached to one of four different fluorescent dyes. These dyes can be phospho linked. A single DNA polymerase can be immobilized with a single molecule of template single stranded DNA at the bottom of a zeromode waveguide (ZMW). A ZMW can be a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that can rapidly diffuse in an out of the ZMW (in microseconds). It can take several milliseconds to incorporate a nucleotide into a growing strand. During this time, the fluorescent label can be excited and produce a fluorescent signal, and the fluorescent tag can be cleaved off. The ZMW can be illuminated from below. Attenuated light from an excitation beam can penetrate the lower 20-30 nm of each ZMW. A microscope with a detection limit of 20 zepto liters (10" liters) can be created. The tiny detection volume can provide 1000-fold improvement in the reduction of background noise. Detection of the corresponding fluorescence of the dye can indicate which base was incorporated. The process can be repeated.

[0137] In some cases, the next generation sequencing is nanopore sequencing (See, e.g., Soni GV and Meller A. (2007) Clin Chem 53: 1996-2001). A nanopore can be a small hole, of the order of about one nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential across it can result in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows can be sensitive to the size of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule can obstruct the nanopore to a different degree. Thus, the change in the current passing through the nanopore as the DNA molecule passes through the nanopore can represent a reading of the DNA sequence. The nanopore sequencing technology can be from Oxford Nanopore Technologies; e.g., a GridlON system. A single nanopore can be inserted in a polymer membrane across the top of a microwell. Each microwell can have an electrode for individual sensing. The microwells can be fabricated into an array chip, with 100,000 or more microwells (e.g., more than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000) per chip. An instrument (or node) can be used to analyze the chip. Data can be analyzed in real-time. One or more instruments can be operated at a time. The nanopore can be a protein nanopore, e.g., the protein alphahemolysin, a heptameric protein pore. The nanopore can be a solid-state nanopore made, e.g., a nanometer sized hole formed in a synthetic membrane (e.g., SiNx, or SiO2). The nanopore can be a hybrid pore (e.g., an integration of a protein pore into a solid-state membrane). The nanopore can be a nanopore with integrated sensors (e.g., tunneling electrode detectors, capacitive detectors, or graphene based nano-gap or edge state detectors (see e.g., Garaj et al. (2010) Nature vol. 67, doi:Attorney Docket No. 45269-751.60110.1038 / nature09379)). A nanopore can be functionalized for analyzing a specific type of molecule (e.g., DNA, RNA, or protein). Nanopore sequencing can comprise "strand sequencing" in which intact DNA polymers can be passed through a protein nanopore with sequencing in real time as the DNA translocates the pore. An enzyme can separate strands of a double stranded DNA and feed a strand through a nanopore. The DNA can have a hairpin at one end, and the system can read both strands. In some cases, nanopore sequencing is "exonuclease sequencing" in which individual nucleotides can be cleaved from a DNA strand by a processive exonuclease, and the nucleotides can be passed through a protein nanopore. The nucleotides can transiently bind to a molecule in the pore (e.g., cyclodextran). A characteristic disruption in current can be used to identify bases.

[0138] Nanopore sequencing technology from GENIA can be used. An engineered protein pore can be embedded in a lipid bilayer membrane. "Active Control" technology can be used to enable efficient nanopore-membrane assembly and control of DNA movement through the channel. In some cases, the nanopore sequencing technology is from NABsys. Genomic DNA can be fragmented into strands of average length of about 100 kb. The 100 kb fragments can be made single stranded and subsequently hybridized with a 6-mer probe. The genomic fragments with probes can be driven through a nanopore, which can create a current -versus- time tracing. The current tracing can provide the positions of the probes on each genomic fragment. The genomic fragments can be lined up to create a probe map for the genome. The process can be done in parallel for a library of probes. A genome-length probe map for each probe can be generated. Errors can be fixed with a process termed "moving window Sequencing by Hybridization (mwSBH)." In some cases, the nanopore sequencing technology is from IBM / Roche. An electron beam can be used to make a nanopore sized opening in a microchip. An electrical field can be used to pull or thread DNA through the nanopore. A DNA transistor device in the nanopore can comprise alternating nanometer sized layers of metal and dielectric. Discrete charges in the DNA backbone can get trapped by electrical fields inside the DNA nanopore. Turning off and on gate voltages can allow the DNA sequence to be read.

[0139] The next generation sequencing can in some cases comprise DNA nanoball sequencing (as performed, e.g., by Complete Genomics; see e.g., Drmanac etal. (2010) Science 327: 78-81). DNA can be isolated, fragmented, and size selected. For example, DNA can be fragmented (e.g., by sonication) to a mean length of about 500 bp. Adaptors (Adi) can be attached to the ends of the fragments. The adaptors can be used to hybridize to anchors for sequencing reactions. DNA with adaptors bound to each end can be PCR amplified. The adaptor sequences can be modified so that complementary single strand ends bind to each other forming circularAttorney Docket No. 45269-751.601DNA. The DNA can be methylated to protect it from cleavage by a type IIS restriction enzyme used in a subsequent step. An adaptor (e.g., the right adaptor) can have a restriction recognition site, and the restriction recognition site can remain non-methylated. The non-methylated restriction recognition site in the adaptor can be recognized by a restriction enzyme (e.g., Acul), and the DNA can be cleaved by Acul 13 bp to the right of the right adaptor to form linear double stranded DNA. A second round of right and left adaptors (Ad2) can be ligated onto either end of the linear DNA, and all DNA with both adapters bound can be PCR amplified (e.g., by PCR). Ad2 sequences can be modified to allow them to bind each other and form circular DNA. The DNA can be methylated, but a restriction enzyme recognition site can remain non-methylated on the left Adi adapter. A restriction enzyme (e.g., Acul) can be applied, and the DNA can be cleaved 13 bp to the left of the Adi to form a linear DNA fragment. A third round of right and left adaptor (Ad3) can be ligated to the right and left flank of the linear DNA, and the resulting fragment can be PCR amplified. The adaptors can be modified so that they can bind to each other and form circular DNA. A type III restriction enzyme (e.g., EcoP15) can be added; EcoP15 can cleave the DNA 26 bp to the left of Ad3 and 26 bp to the right of Ad2. This cleavage can remove a large segment of DNA and linearize the DNA once again. A fourth round of right and left adaptors (Ad4) can be ligated to the DNA, the DNA can be amplified (e.g., by PCR), and modified so that they bind each other and form the completed circular DNA template.

[0140] Rolling circle replication (e.g., using Phi 29 DNA polymerase) can be used to amplify small fragments of DNA. The four adaptor sequences can contain palindromic sequences that can hybridize and a single strand can fold onto itself to form a DNA nanoball (DNB™) which can be approximately 200-300 nanometers in diameter on average. A DNA nanoball can be attached (e.g., by adsorption) to a microarray (sequencing flow cell). The flow cell can be a silicon wafer coated with silicon dioxide, titanium and hexamehtyl di silazane (HMDS) and a photoresist material. Sequencing can be performed by unchained sequencing by ligating fluorescent probes to the DNA. The color of the fluorescence of an interrogated position can be visualized by a high resolution camera. The identity of nucleotide sequences between adaptor sequences can be determined.

[0141] In some embodiments, high-throughput sequencing can take place using AnyDot.chips (Genovoxx, Germany). In particular, the AnyDot.chips allow for lOx - 50x enhancement of nucleotide fluorescence signal detection. AnyDot.chips and methods for using them are described in part in International Publication Application Nos. WO 02088382, WO 03020968, WO 03031947, WO 2005044836, PCT / EP 05 / 05657, PCT / EP 05 / 05655; and German Patent Application Nos. DE 101 49786, DE 102 14395, DE 103 56837, DE 102004009704,Attorney Docket No. 45269-751.601DE 102004025 696, DE 102004025 746, DE 102004025 694, DE 102004025 695, DE 10 2004025 744, DE 102004025 745, and DE 102005 012301.

[0142] Other high-throughput sequencing systems include those disclosed in Venter, J., et al. Science 16 February 2001; Adams, M. etal. Science 24 March 2000; and M. J. Levene, et al. Science 299:682-686, January 2003; as well as US Publication No. 20030044781 and 2006 / 0078937. Overall, such systems involve sequencing a target nucleic acid molecule having a plurality of bases by the temporal addition of bases via a polymerization reaction that is measured on a molecule of nucleic acid, such as the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. Sequence can then be deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labeled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labeled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended, and the sequence of the target nucleic acid is determined.

[0143] Prior to sequencing, nucleic acid molecules can be barcoded or otherwise labeled. Barcoding can allow for easier grouping of sequence reads. For example, barcodes can be used to identify sequences originating from the same nucleic acid molecule or DNA protein complex. Barcodes can also be used to uniquely identify individual junctions. For example, each junction can be marked with a unique (e.g., randomly generated) barcode which can uniquely identify the junction. Multiple barcodes can be used together, such as a first barcode to identify sequences originating from the same nucleic acid molecule or DNA protein complex and a second barcode that uniquely identifies individual junctions.

[0144] Barcoding can be achieved through a number of techniques. In some cases, barcodes can be included as a sequence within a punctuation oligonucleotide. In other cases, a nucleic acid molecule can be contacted to oligonucleotides comprising at least two segments: oneAttorney Docket No. 45269-751.601segment contains a barcode, and a second segment contains a sequence complementary to a punctuation sequence. After annealing to the punctuation sequences, the barcoded oligonucleotides can be extended with polymerase to yield barcoded molecules from the same punctuated nucleic acid molecule. Since the punctuated nucleic acid molecule is a rearranged version of the input nucleic acid molecule, in which phase information is preserved, the generated barcoded molecules are also from the same input nucleic acid molecule. These barcoded molecules comprise a barcode sequence, the punctuation complementary sequence, and genomic sequence.

[0145] For nucleic acid molecules (e.g., nucleic acids part of or recovered from a DNA protein complex) with or without punctuation, molecules can be barcoded by other means. For example, nucleic acid molecules can be contacted with barcoded oligonucleotides which can be extended to incorporate sequence from the nucleic acid molecule. Barcodes can hybridize to punctuation sequences, to restriction enzyme recognition sites, to sites of interest (e.g., genomic regions of interest), or to random sites (e.g., through a random n-mer sequence on the barcode oligonucleotide). Nucleic acid molecules can be contacted to the barcodes using appropriate concentrations and / or separations (e.g., spatial or temporal separation) from other nucleic acid molecules in the sample such that multiple nucleic acid molecules are not given then same barcode sequence. For example, a solution comprising nucleic acid molecules can be diluted to such a concentration that only one nucleic acid molecule or only one DNA protein complex will be contacted to a barcode or group of barcodes with a given barcode sequence. Barcodes can be contacted to nucleic acid molecules in free solution, in fluidic partitions (e.g., droplets or wells), or on an array (e.g., at particular array spots).

[0146] Barcoded nucleic acid molecules (e.g., extension products) can be sequenced, for example, on a short-read sequencing machine and sequence information is determined by grouping sequence reads having the same barcode into a common alignment, scaffold, phase, or other group. In this way, synthetic long reads can be achieved via short-read sequencing.Alternatively, prior to sequencing, the barcoded products can be linked together, for example though bulk ligation, to generate long molecules which are sequenced, for example, using long-read sequencing technology. In these cases, the embedded read pairs can be identifiable via the amplification adapters and punctuation sequences. Further information is obtained from the barcode sequence of the read pair.

[0147] Alternately, in some cases library molecules generated as described herein are concatenated without punctuation oligo insertion. These molecules are nonetheless suitable for sequencing using long read chemistries commercially available for generating reads of as long asAttorney Docket No. 45269-751.6015kb, lOkb, 20kb or longer. In these cases, concatenation junctions are readily identified through sequence analysis.

[0148] Long reads (e.g., synthetic or actual long reads) can be used to obtain information, such as phasing information, that may be otherwise difficult or impossible to determine from short reads. Phasing information includes matemal / patemal phasing as well as tumor / non-tumor phasing information. Tumor / non-tumor phasing can be used to differentiate cancer genomic information from somatic genomic information.

[0149] In an example, fragments from a library, such as a library created from an FFPE sample, as described above, are end sequenced. Read pairs are observed which indicate that the contigs where each end mapped are physically linked on a common nucleic acid molecule in the sample. The resulting library is further analyzed by sequencing in order to determine the distance between paired ends of the recovered fragments by comparing the location of the isolated sequences to a genome assembly. The long distance read pair frequencies in the FFPE samples are compared to the long distance read pair frequencies of a non-FFPE sample. In an exemplary library, such as the above library, sequencing reveals that the FFPE-Chicago method results in long distance read pair frequencies comparable to (>200 kbp insert) or greater than (100 kbp - 200 kbp inserts) Chicago methods performed on non-FFPE samples. The complexity and raw sequencing coverage of the FFPE-Chicago library are also determined. Complexity of a library refers to the variety of different molecules within the library.Genetic Information

[0150] Phasing information, chromosome conformation, sequence assembly, and genetic features including but not limited to structural variations (SVs), copy number variants (CNVs), loss of heterozygosity (LOH), single nucleotide variants (SNVs), single nucleotide polymorphisms (SNPs), chromosomal translocations, gene fusions, and insertions and deletions (INDELs) can be determined by analysis of sequence read data produced by methods disclosed herein. Other inputs for analysis of genetic features can include a reference genome (e.g., with annotations), genome masking information, and a list of candidate genes, gene pairs, and / or coordinates of interest. Configuration parameters and genome masking information can be customized, or default parameters and genome masking can be used. In an example, read pairs are mapped to a genome, then each pair is represented as a point in the plane with x and y coordinates equal to the mapped position on concatenated reference chromosomes of read 1 and read 2 of the read pair, respectively. The x-y plane can be divided into non-overlapping square bins and the number of read pairs mapping to each bin can be tabulated. The bin counts can be visualized as an image (e.g., a heat map) with bins made to correspond to pixels. A variety of analysis techniques, such asAttorney Docket No. 45269-751.601image processing techniques, can be used to identify the signatures of genetic features such as different rearrangements.

[0151] Inputs, such as sequence read data, can be formatted in appropriate file formats. For example, sequence read data can be contained in FASTA files, FASTQ files, BAM files, SAM files, or other file formats. Input sequence read data can be unaligned. Input sequence read data can be aligned.

[0152] Sequence read data can be prepared for analysis. For example, reads can be trimmed for quality. Reads can also be trimmed to remove sequencing adapters, if necessary.

[0153] Sequence read data can be aligned. For example, read pairs can be aligned to a specified reference genome. In some cases, the reference genome is CRCh38. Alignment can be performed with a variety of algorithms or tools, including but not limited to SNAP, Burrows-Wheeler aligners (e.g., bwa-sw, bwa-mem, bwa-aln), Bowtie2, Novoalign, and modifications or variations thereof.

[0154] Quality control (QC) reports of the analysis can also be generated. QC reports can be used to identify failed libraries before conducting deeper sequencing. Such quality control reports can include a variety of metrics. QC metrics can include but are not limited to total read pairs, percent of duplicates (e.g., PCR duplicates), percent of unmapped reads, percent of reads with low map quality (e.g., Q < 20), percent of read pairs mapped to different chromosomes, percent of read pair inserts (such as distance between mapping positions) between 0 and 1 kbp, percent of read pair inserts between 1 kbp and 100 kbp, percent of read pair inserts between 100 kbp and 1 Mbp, percent of read pair inserts above 1 Mbp, percent of read pairs containing a ligation junction, proximity to restriction fragment ends, a read pair separation plot, and an estimate of library complexity. QC metrics can be used to optimize the analysis, and to identify quality problems in reagents, samples, and users. Sequence alignments can be filtered based on one or more of the QC metrics. Duplicate reads can also be filtered, for example based on comparison of reads at closely corresponding positions.

[0155] Sequence read analysis results can include link density results. Link density results can include whole genome, one locus, and two locus views of link density results. Link density results can be output as a data set. Link density results can be presented as a linkage density plot (LDP), such as a heat map of interactions (e.g., contacts) between regions of a chromosome or a genome. Link density results can be associated with a score, such as a quality score. In some cases, link density visualizations are output for results that exceed a score threshold. In an example, visualizations are included for the whole genome, for de novo calls that exceed a score threshold, for single-sided candidate calls that exceed a score threshold and for all double-sidedAttorney Docket No. 45269-751.601candidates, including those classified as negative. Link density visualization can include a scale (e.g., a color scale), a length scale bar, gene name labels, exon / intron structure glyphs for genes, and highlighting of detected rearrangements.

[0156] Linkage information can be normalized to control for effects and biases such as coverage, fragment mappability, fragment GC content, and fragment length. Normalization can be conducted by matrix balancing or other factor-agnostic methods. Matrix balancing can employ algorithms such as the Sinkhorn-Knopp algorithm or Knight-Ruiz normalization. Normalization can also be conducted to correct for background signal that may lead to false positives.

[0157] Aligned sequence data can be analyzed for rearrangements, including rearrangements through the whole genome and rearrangements at specific two-locus (or two-sided) candidate genes. Analysis can also include identification of contacts, fusions, and joins. Alignments of sequence read data (e.g., in a BAM file or other suitable format) can be input into the analysis. Genome masking information can be input as well, or default genome masking information can be used in the analysis. Analysis can be conducted across the entire genome. Additionally or alternatively, analysis can be conducted for a list of two-sided candidate fusions. In some cases, the analysis conducted on a list of candidate fusions is more sensitive than the analysis conducted on a whole genome. Analysis of two-sided candidate fusions can detect fusions involving translocations of relatively short segments of DNA that may be missed by a genomewide scan.

[0158] Analysis to identify features such as contacts and rearrangements (including but not limited to deletions, duplications, insertions, inversions or reversals, translocations, joins, fusions, and fissions), and other interactions can be conducted with a variety of techniques. Analysis techniques can include statistical and probability analysis, signal processing including Fourier analysis, computer vision and other image processing, language processing (e.g., natural language processing), and machine learning. For example, interaction plots such as contact matrixes can be analyzed for features indicative of features. In some cases, filters can be applied to plots or other data. Filters can be convolution filters including but not limited to smoothing filters (e.g., kernel smoothing or Savitzky-Golay filter, Gaussian blur).

[0159] Some embodiments involve machine learning as a component of genome structure determination, and accordingly some computer systems are configured to comprise a module having a machine learning capacity. Machine learning modules comprise at least one of the following listed modalities, so as to constitute a machine learning functionality.

[0160] Modalities that constitute machine learning variously demonstrate a data filtering capacity, so as to be able to perform automated mass spectrometric data spot detection and calling.Attorney Docket No. 45269-751.601This modality is in some cases facilitated by the presence of predicted patterns indicative of various genomic structural changes, such as inversions, insertions, deletions, or translocations.

[0161] Modalities that constitute machine learning variously demonstrate a data treatment or data processing capacity, so as to render read pair frequencies in a form conducive to downstream analysis. Examples of data treatment include but are not necessarily limited to log transformation, assigning of scaling ratios, or mapping data to crafted features so as to render the data in a form that is conducive to downstream analysis.

[0162] Machine learning data analysis components as disclosed herein regularly process a wide range of features in a read pair data set, such as 1 to 10,000 features, or 2 to 300,000 features, or a number of features within either of these ranges or higher than either of these ranges. In some cases, data analysis involves at least Ik, 2k, 3k, 4k, 5k, 6k, 7k, 8k, 9k, 10k, 20k, 30k, 40k, 50k, 60k, 70k, 80k, 90k, 100k, 120k, 140k, 160k, 180k, 200k, 220k, 2240k, 260k, 280k, 300k, or more than 300k features.

[0163] Read pair distribution patterns are identified using any number of approaches consistent with the disclosure herein. In some cases, read pair distribution patterns selection comprises elastic net, information gain, random forest imputing or other feature selection approaches consistent with the disclosure herein and familiar to one of skill in the art.

[0164] Selected read pair distribution patterns are matched against predicted patterns indicative of a genomic structural change, again using any number of approaches consistent with the disclosure herein. In some cases, read pair pattern detection comprises logistic regression, SVM, random forest, KNN, or other classifier approaches consistent with the disclosure herein and familiar to one of skill in the art.

[0165] Applying machine learning, or providing a machine learning module on a computer configured for the analyses disclosed herein, allows for the detection of relevant genomic structural changes for asymptomatic disease detection or early detection as part of an ongoing monitoring procedure, so as to identify a disease or disorder either ahead of symptom development or while intervention is either more easily accomplished or more likely to bring about a successful outcome.

[0166] Applying machine learning, or providing a machine learning module on a computer configured for the analyses disclosed herein also allows identification of structural rearrangements in individuals subjected to a drug treatment, for example as part of a drug trial, so that outcome of the trial for the individual or for the population may be concurrently or retrospectively correlated so as to identify particular genomic structural events that correspond positively or negatively with drug efficacy.Attorney Docket No. 45269-751.601

[0167] Applying machine learning or providing a machine learning module on a computer configured for the analyses disclosed herein also allows identification of structural rearrangements that correspond with particular regions of genetically heterogeneous samples, such as tumor tissue samples collected without homogenization so as to preserve positional information in the sample. As some tumor regions are known to correspond to cell populations particularly adept at metastasis or tumor spread, identifying genomic rearrangements or other phase information that correlates with such cell populations assists in selecting a treatment regimen to target these particularly dangerous cell populations.

[0168] Monitoring is often but not necessarily performed in combination with or in support of a genetic assessment indicating a genetic predisposition for a disorder for which a signature of onset or progression is monitored. Similarly, in some cases machine learning is used to facilitate monitoring of or assessment of treatment efficacy for a treatment regimen, such that the treatment regimen can be modified over time, continued or resolved as indicated by the ongoing proteomics mediated monitoring.

[0169] Machine learning approaches and computer systems having modules configured to execute machine learning algorithms facilitate identification of phase information or genomic rearrangement in datasets of varying complexity. In some cases the phase information or genomic rearrangements are identified from an untargeted database comprising a large amount of mass spectrometric data, such as data obtained from a single individual at multiple time points, samples taken from multiple individuals such as multiple individuals of a known status for a condition of interest or known eventual treatment outcome or response, or from multiple time points and multiple individuals.

[0170] Alternately, in some cases machine learning facilitates the refinement of a genomic rearrangement or phase information through the analysis of a database targeted to that a genomic rearrangement or phase information, by for example collecting a genomic rearrangement or phase information from a single individual over multiple time points, when a health condition for the individual is known for the time points, or collecting sequence information from multiple individuals of known status for a condition of interest, or collecting sequence information from multiple individuals at multiple time points. As is readily apparent, in some cases collection of sequence information is facilitated through the use of preserved sample such as crosslinked samples collected pursuant to surgery or FFPE samples collected pursuant to a drug trial.

[0171] Thus, sequence information is collected either alone or in combination with drug trial outcome or surgical intervention outcome information. Sequence data is subjected to machine learning, for example on a computer system configured as disclosed herein, so as to identify aAttorney Docket No. 45269-751.601subset of read pairs indicative of a pattern corresponding to a genomic rearrangement that either alone or in combination with one or more additional markers, account for a health status signal . Thus, machine learning in some cases facilitates identification of sequence, either DNA or RNA sequence, or of a genomic rearrangement that is individually informative of a health status in an individual.

[0172] The minimum distance between breakpoints for detectable rearrangements can be less than, about, or a number in a range defined by two numbers selected from the list of nucleic acid lengths comprising 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1 Mb, 2 Mb, 3 Mb, 4 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 60 Mb, 70 Mb, 80 Mb, 90 Mb, 100 Mb, 200 Mb, 300 Mb, 400 Mb, 500 Mb, 600 Mb, 700 Mb, 800 Mb, 900 Mb, or 1 Gb.

[0173] Rearrangement analysis can produce a list of pairs of breakpoints that are deemed joined in the subject genome. The list of pairs of breakpoint coordinates can also include statistical significance or confidence metrics (e.g., p-value) for the breakpoint coordinate pairs. These pairs of breakpoints can be output in an appropriate format, such as browser extensible data (BED) or BED-PE.

[0174] Analysis of chromosome conformation can also be conducted using the techniques disclosed herein. For example, topologically associating domains (TADs) and TAD boundaries can be determined. Other topological domains and boundaries can also be determined, including but not limited to lamina-associated domains (LADs), replication time zones, and large organized chromatin K9-modification (LOCK) domains.

[0175] In an exemplary embodiment, sequencing data is used to determine phasing information for polymorphisms known to be in the starting FFPE sample. For example, the sequencing data is used to determine whether certain polymorphisms such as SNPs were present on the same or different DNA molecules. Accuracy of the phasing determined using this method is measured by comparing to a known sequence, such as the sequence of the GIAB sample. For example, in some cases it is found that between 0-10,000, there were 132,796 SNPS found and 99.059 % were in the correct phase. A high concordance (>95%) is seen up until about 1.5 MB (with the exception of the 70-80 kb bin, which missed 1 of 13 and the 1.1 - 1.3 MB bin which missed 2 of 15). In the 1.7 - 1.9 MB range, 7 of 7 SNP pair phases were properly called. From these data, it is concluded that, despite low levels of spurious linkage, proper long-rangeAttorney Docket No. 45269-751.601information is determined using the FFPE-Chicago method, even up to the megabase range.Importantly, these ‘concordance’ prediction rates are 95% or greater, significantly higher than the 50% success rate one would expect from random chance).Structural phasing information

[0176] Currently, structural and phasing analyses (e.g., for medical purposes) remain challenging. For example, there is astounding heterogeneity among cancers, individuals with the same type of cancer, or even within the same tumor. Teasing out the causative from consequential effects can require very high precision and throughput at a low per-sample cost. In the domain of personalized medicine, one of the gold standards of genomic care is a sequenced genome with all variants thoroughly characterized and phased, including large and small structural rearrangements and novel mutations. To achieve this with previous technologies demands effort akin to that required for a de novo assembly, which is currently too expensive and laborious to be a routine medical procedure.

[0177] Phasing information includes maternal / paternal phasing as well as tumor / non-tumor phasing information. Tumor / non-tumor phasing can be used to differentiate cancer genomic information from somatic genomic information.

[0178] In some embodiments of the disclosure, a preserved tissue (e.g., an FFPE tissue) from a subject can be provided and the method can return an assembled genome, alignments with called variants (including large structural variants and copy number variants), phased variant calls, or any additional analyses. In other embodiments, the methods disclosed herein can provide long distance read pair libraries directly for the individual.

[0179] In various embodiments of the disclosure, the methods disclosed herein can generate long-range read pairs separated by large distances. The upper limit of this distance may be improved by the ability to collect DNA samples of large size. In some cases, the read pairs can span up to 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000 kbp or more in genomic distance. In some examples, the read pairs can span up to 500 kbp in genomic distance. In other examples, the read pairs can span up to 2000 kbp in genomic distance. The methods disclosed herein can integrate and build upon standard techniques in molecular biology, and are further well -suited for increases in efficiency, specificity, and genomic coverage.

[0180] In other embodiments, the methods disclosed herein can be used with currently employed sequencing technology. For example, the methods can be used in combination with well-tested and / or widely deployed sequencing instruments. In further embodiments, the methodsAttorney Docket No. 45269-751.601disclosed herein can be used with technologies and approaches derived from currently employed sequencing technology.

[0181] In various embodiments, the disclosure provides for one or more methods disclosed herein that comprise the step of probing the physical layout of chromosomes within preserved (e.g., FFPE) samples or cells. Examples of techniques to probe the physical layout of chromosomes through sequencing include the “C” family of techniques, such as chromosome conformation capture ("3C"), circularized chromosome conformation capture ("4C"), carbon-copy chromosome capture ("5C"), and Hi-C based methods; and ChIP based methods, such as ChlP-loop, ChIP -PET. These techniques utilize the fixation of chromatin in live cells to cement spatial relationships in the nucleus. Subsequent processing and sequencing of the products allows a researcher to recover a matrix of proximate associations among genomic regions. With further analysis these associations can be used to produce a three-dimensional geometric map of the chromosomes as they are physically arranged in the preserved (e.g., FFPE) sample. Such techniques describe the discrete spatial organization of chromosomes and provide an accurate view of the functional interactions among chromosomal loci.

[0182] In some embodiments, the intrachromosomal interactions correlate with chromosomal connectivity. In some cases, the intrachromosomal data can aid genomic assembly. In some cases, the chromatin is reconstructed in vitro. This can be advantageous because chromatin - particularly histones, the major protein component of chromatin - is important for fixation under the most common “C” family of techniques for detecting chromatin conformation and structure through sequencing: 3C, 4C, 5C, and Hi-C. Chromatin is highly non-specific in terms of sequence and will generally assemble uniformly across the genome. In some cases, the genomes of species that do not use chromatin can be assembled on a reconstructed chromatin and thereby extend the horizon for the disclosure to all domains of life.

[0183] Read pair data can be obtained from a chromatin conformation capture technique. In some examples, ligation or other tagging is accomplished so as to mark genome regions that are in close physical proximity. Crosslinking of the complex such that proteins (such as histones) are stably bound in a complex with the DNA molecule, e.g. genomic DNA, within chromatin can be accomplished according to a suitable method described in further detail elsewhere herein or otherwise known in the art. In some cases, crosslinks arising from sample preservation (e.g., from fixation) are utilized by extracting DNA-protein complexes under conditions such that such complexes are not degraded, such as through the exclusion of proteinase K treatment. For example, nucleotide segments that are not in close proximity along a genome sequence can be in close physical proximity when part of a structure such as chromatin. Such nucleotide segments canAttorney Docket No. 45269-751.601be ligated together and subsequently analyzed according to methods of the present disclosure. For example, ligated nucleotide segments can be sequenced and the distance between the sequenced ends of two ligated segments (insert distance) can be analyzed.

[0184] In some cases, two or more nucleotide sequences can be crosslinked via proteins bound to one or more nucleotide sequences. One approach is to expose the chromatin to ultraviolet irradiation (Gilmour etal., Proc. Nat’l. Acad. Sci. USA 81:4275-4279, 1984). Crosslinking of polynucleotide segments may also be performed utilizing other approaches, such as chemical or physical (e.g. optical) crosslinking. Suitable chemical crosslinking agents include, but are not limited to, formaldehyde and psoralen (Solomon etal., Proc. Natl. Acad. Sci. USA 82:6470-6474, 1985; Solomon etal., Cell 53:937-947, 1988). For example, crosslinking can be performed by adding 2% formaldehyde to a mixture comprising the DNA molecule and chromatin proteins. Other examples of agents that can be used to crosslink DNA include, but are not limited to, UV light, mitomycin C, nitrogen mustard, melphalan, 1,3 -butadiene di epoxide, cis diaminedichloroplatinum (II) and cyclophosphamide. Suitably, the crosslinking agent will form crosslinks that bridge relatively short distances — such as about 2 A — thereby selecting intimate interactions that can be reversed.

[0185] Universally, procedures for probing the physical layout of chromosomes, such as Hi-C based techniques, utilize chromatin that is formed within a cell / organism, such as chromatin isolated from cultured cells or primary tissue. These techniques can be achieved through Chicago based methods. In some cases, the methods disclosed herein can reduce inter-chromosomal crosslinking. In some cases, reverse crosslinking can reduce inter-chromosomal crosslinking. In some cases, a sample may have less than about 30, 25, or 20% or less inter-chromosomal or intermolecular crosslinking according to the methods and compositions of the disclosure. The frequency intramolecular crosslinks within the polynucleotide can be adjusted by crosslink reversal. For example, crosslink reversal with the buffer disclosed herein can reduce inter-chromosomal crosslink or intramolecular crosslinks within the polynucleotide. Accordingly, the distribution of crosslinks can be altered to favor longer-range interactions. In some embodiments, sub-samples with varying crosslinking density may be prepared to cover both short- and long-range associations. For example, the crosslinking conditions can be adjusted such that at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the crosslinks occur between DNA segments that are at least about 50 kb, about 60 kb, about 70 kb, about 80 kb, aboutAttorney Docket No. 45269-751.60190 kb, about 100 kb, about 110 kb, about 120 kb, about 130 kb, about 140 kb, about 150 kb, about 160 kb, about 180 kb, about 200 kb, about 250 kb, about 300 kb, about 350 kb, about 400 kb, about 450 kb, or about 500 kb apart on the sample DNA molecule.

[0186] High degrees of accuracy required by cancer genome sequencing can be achieved using the methods and systems described herein. Inaccurate reference genomes can make basecalling challenging when sequencing cancer genomes. Heterogeneous samples and small starting materials, for example a sample obtained by biopsy introduce additional challenges. Further, detection of large scale structural variants and / or losses of heterozygosity is often crucial for cancer genome sequencing, as well as the ability to differentiate between somatic variants and errors in base-calling.

[0187] Systems and methods described herein may generate accurate long sequences from complex samples containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more varying genomes. Mixed samples of normal, benign, and / or tumor origin may be analyzed, optionally without the need for a normal control. In some embodiments, starting samples as little as lOOng or even as little as hundreds of genome equivalents are utilized to generate accurate long sequences. Systems and methods described herein may allow for detection of copy number variants, large scale structural variants and rearrangements, phased variant calls may be obtained over long sequences spanning about 1 kbp, about 2 kbp, about 5 kbp, about 10 kbp, 20 kbp, about 50 kbp, about 100 kbp, about 200 kbp, about 500 kbp, about 1 Mbp, about 2 Mbp, about 5 Mbp, about 10 Mbp, about 20 Mbp, about 50 Mbp, or about 100 Mbp or more nucleotides. For example, phase variant calls may be obtained over long sequences spanning about 1 Mbp or about 2 Mbp.

[0188] Samples can comprise tissue sections of various volumes and surface areas. In some cases, a sample comprises a tissue section between about 5 pm and 10 pm in thickness. In some cases, a sample comprises a tissue section about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 850 pm, 900 pm, 950 pm, 1000 pm, or more in thickness. In some cases, a sample comprises a tissue section at least about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 850 pm, 900 pm, 950 pm, 1000 pm, or more in thickness. In some cases, a sample comprises a tissue section at mostAttorney Docket No. 45269-751.601about 1 pm, 2 m, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 850 pm, 900 pm, 950 pm, 1000 pm, or more in thickness. In some cases, a sample comprises a tissue section with a surface area between about 100 and 300 mm2. In some cases, a sample comprises a tissue section about 10 mm2, 20 mm2, 30 mm2, 40 mm2, 50 mm2, 60 mm2, 70 mm2, 80 mm2, 90 mm2, 100 mm2, 200 mm2, 300 mm2, 400 mm2, 500 mm2, 600 mm2, 700 mm2, 800 mm2, 900 mm2, 1000 mm2, or more in surface area. In some cases, a sample comprises a tissue section at least about 10 mm2, 20 mm2, 30 mm2, 40 mm2, 50 mm2, 60 mm2, 70 mm2, 80 mm2, 90 mm2, 100 mm2, 200 mm2, 300 mm2, 400 mm2, 500 mm2, 600 mm2, 700 mm2, 800 mm2, 900 mm2, 1000 mm2, or more in surface area. In some cases, a sample comprises a tissue section at most about 10 mm2, 20 mm2, 30 mm2, 40 mm2, 50 mm2, 60 mm2, 70 mm2, 80 mm2, 90 mm2, 100 mm2, 200 mm2, 300 mm2, 400 mm2, 500 mm2, 600 mm2, 700 mm2, 800 mm2, 900 mm2, 1000 mm2, or more in surface area.

[0189] Haplotypes determined using the methods and systems described herein may be assigned to computational resources, for example computational resources over a network, such as a cloud system. Short variant calls can be corrected, if necessary, using relevant information that is stored in the computational resources. Structural variants can be detected based on the combined information from short variant calls and the information stored in the computational resources. Problematic parts of the genome, such as segmental duplications, regions prone to structural variation, the highly variable and medically relevant MHC region, centromeric and telomeric regions, and other heterochromatic regions including but limited to those with repeat regions, low sequence accuracy, high variant rates, ALU repeats, segmental duplications, or any other relevant problematic parts known in the art, can be reassembled for increased accuracy.

[0190] A sample type can be assigned to the sequence information either locally or in a networked computational resource, such as a cloud. In cases where the source of the information is known, for example when the source of the information is from a cancer or normal tissue, the source can be assigned to the sample as part of a sample type. Other sample type examples generally include, but are not limited to, tissue type, sample collection method, presence of infection, type of infection, processing method, size of the sample, etc. In cases where a complete or partial comparison genome sequence is available, such as a normal genome in comparison to a cancer genome, the differences between the sample data and the comparison genome sequence can be determined and optionally output.Attorney Docket No. 45269-751.601Methods for Haplotype Phasing

[0191] Because the read pairs generated by the methods disclosed herein are generally derived from intra-chromosomal contacts, any read pairs that contain sites of heterozygosity will also carry information about their phasing. Using this information, reliable phasing over short, intermediate and even long (megabase) distances can be performed rapidly and accurately.Experiments designed to phase data from one of the 1000 genomes trios (a set of mother / father / offspring genomes) have reliably inferred phasing. Additionally, haplotype reconstruction using proximity -ligation similar to Selvaraj el al. (Nature Biotechnology 31:1111- 1118 (2013)) can also be used with haplotype phasing methods disclosed herein.

[0192] For example, a haplotype reconstruction using proximity -ligation based method can also be used in the methods disclosed herein in phasing a genome. A haplotype reconstruction using proximity -ligation based method combines a proximity -ligation and DNA sequencing with a probabilistic algorithm for haplotype assembly. First, proximity-ligation sequencing is performed using a chromosome capture protocol, such as the Hi-C protocol. These methods can capture DNA fragments from two distant genomic loci that looped together in three-dimensional space. After shotgun DNA-sequencing of the resulting DNA library, paired-end sequencing reads have ‘insert sizes’ that range from several hundred base pairs to tens of millions of base pairs. Thus, short DNA fragments generated in a Hi-C experiment can yield small haplotype blocks, long fragments ultimately can link these small blocks together. With enough sequencing coverage, this approach has the potential to link variants in discontinuous blocks and assemble every such block into a single haplotype. This data is then combined with a probabilistic algorithm for haplotype assembly. The probabilistic algorithm utilizes a graph in which nodes correspond to heterozygous variants and edges correspond to overlapping sequence fragments that may link the variants. This graph might contain spurious edges resulting from sequencing errors or trans interactions. A maxcut algorithm is then used to predict parsimonious solutions that are maximally consistent with the haplotype information provided by the set of input sequencing reads. Because proximity ligation generates larger graphs than conventional genome sequencing or mate-pair sequencing, computing time and number of iterations are modified so that the haplotypes can be predicted with reasonable speed and high accuracy. The resulting data can then be used to guide local phasing using Beagle software and sequencing data from the genome project to generate chromosome-spanning haplotypes with high resolution and accuracy.Determining phase information with paired ends

[0193] Further provided herein are methods and compositions for determining phase information from paired ends derived from FFPE-samples. Paired ends can be generated by any ofAttorney Docket No. 45269-751.601the methods disclosed or those further illustrated in the provided Examples. For example, in the case of a DNA molecule bound to a solid surface which was subsequently cleaved, following religation of free ends, re-ligated DNA segments are released from the solid-phase attached DNA molecule, for example, by restriction digestion. This release results in a plurality of paired end fragments. In some cases, the paired ends are ligated to amplification adapters, amplified, and sequenced with short read technology. In these cases, paired ends from multiple different solid phase-bound DNA molecules are within the sequenced sample. However, it is confidently concluded that for either side of a paired end junction, the junction adjacent sequence is derived from a common phase of a common molecule. In cases where paired ends are linked with a punctuation oligonucleotide, the paired end junction in the sequencing read is identified by the punctuation oligonucleotide sequence. In other cases, the pair ends were linked by modified nucleotides, which can be identified based on the sequence of the modified nucleotides used.

[0194] Alternatively, following release of paired ends, the free paired ends are ligated to amplification adapters and amplified. In these cases, the plurality of paired ends is then bulk ligated together to generate long molecules which are read using long-read sequencing technology. In other examples, released paired ends are bulk ligated to each other without the intervening amplification step. In either case, the embedded read pairs are identifiable via the native DNA sequence adjacent to the linking sequence, such as a punctuation sequence or modified nucleotides. The concatenated paired ends are read on a long-sequence device, and sequence information for multiple junctions is obtained. Since the paired ends derived from multiple different solid phase-bound DNA molecules, sequences spanning two individual paired ends, such as those flanking amplification adapter sequences, are found to map to multiple different DNA molecules. However, it is confidently concluded that for either side of a paired end junction, the junction-adjacent sequence is derived from a common phase of a common molecule. For example, in the case of paired ends derived from a punctuated molecule, sequences flanking the punctuation sequence are confidently assigned to a common DNA molecule. In preferred cases, because the individual paired ends are concatenated using the methods and compositions disclosed herein, one is able to sequence multiple paired ends in a single read.

[0195] Sequencing data generated using the methods and compositions described herein are used, in preferred embodiments, to generate phased de novo sequence assemblies, determine phase information, and / or identify structural variations.Determining structural variations and other genetic features

[0196] An example is provided of mapped locations on a reference sequence, e.g., GRCh38, of read pairs generated from proximity ligation of DNA from re-assembled chromatinAttorney Docket No. 45269-751.601are plotted in the vicinity of structural differences between GM12878 and the reference. Each read pair generated is represented both above and below the diagonal. Above the diagonal, shades indicate map quality score on scale shown; below the diagonal shades indicate the inferred haplotype phase of generated read pairs based on overlap with a phased SNPs. In some embodiments, plots generated depict inversions with flanking repetitive regions. In some embodiments, plots generated depict data for a phased heterozygous deletion.

[0197] Mapping paired sequence reads from one individual against a reference is the most commonly used sequence-based method for identifying differences in contiguous nucleic acid or genome structure like inversions, deletions and duplications (Tuzun et al., 2005). To estimate the sensitivity and specificity of the read pair data for identifying structural differences, a maximum likelihood discriminator on simulated data sets constructed to simulate the effect of heterozygous inversions was tested. The test data was constructed by randomly selecting intervals of a defined length L from the mapping of the NA12878 reads generated to the GRCh38 reference sequence and assigning each generated read pair independently at random to the inverted or reference haplotype and editing the mapped coordinates accordingly. Non-allelic homologous recombination is responsible for much of the structural variation observed in human genomes, resulting in many variation breakpoints that occur in long blocks of repeated sequence (Kidd et al., 2008). The effect of varying lengths of repetitive sequence surrounding the inversion breakpoints was simulated by removing all reads mapped to within a distance W of them. In the absence of repetitive sequences at the inversion breakpoints, for 1 Kbp, 2 Kbp and 5 Kbp inversions respectively, the sensitivities (specificities) were 0.76 (0.88), 0.89 (0.89) and 0.97 (0.94) respectively. When 1 Kbp regions of repetitive (unmappable) sequence at the inversion breakpoints was used in a simulation, the sensitivity (specificity) for 5 Kbp inversions was 0.81 (0.76).Performance

[0198] Analysis conducted with the techniques disclosed herein can be performed at high accuracy. Analysis can be conducted with an accuracy of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or more. Analysis can be conducted with an accuracy of at least 70%. Analysis can be conducted with an accuracy of at least 80%. Analysis can be conducted with an accuracy of at least 90%.

[0199] Analysis conducted with the techniques disclosed herein can be performed at high specificity. Analysis can be conducted with a specificity of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or more. Analysis can beAttorney Docket No. 45269-751.601conducted with a specificity of at least 70%. Analysis can be conducted with a specificity of at least 80%. Analysis can be conducted with a specificity of at least 90%.

[0200] Analysis conducted with the techniques disclosed herein can be performed at high sensitivity. Analysis can be conducted with a sensitivity of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or more. Analysis can be conducted with a sensitivity of at least 70%. Analysis can be conducted with a sensitivity of at least 80%. Analysis can be conducted with a sensitivity of at least 90%.

[0201] Use of the techniques of the present disclosure can improve the functioning of the computer systems on which they are implemented. For example, the techniques can reduce the processing time for a given analysis by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The techniques can reduce the memory requirements for a given analysis by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0202] Use of the techniques of the present disclosure can enable conducting analyses that were previously not possible. For example, certain genetic features can be detected from sequence information that would not be detectable from such information without the methods of the present disclosure.Computer Systems

[0203] FIG. 3 shows a computer system 1001 that is programmed or otherwise configured to implement the methods provided herein. The computer system 1001 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0204] The computer system 1001 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 1005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1001 also includes memory or memory location 1010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1015 (e.g., hard disk), communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage and / or electronic display adapters. The memory 1010, storage unit 1015, interface 1020 and peripheral devices 1025 are in communication with the CPU 1005 through a communication bus (solid lines), such as a motherboard. The storage unit 1015 can be a data storage unit (or data repository) for storing data. The computer system 1001 can be operatively coupled to a computer network ("network") 1030 with the aid of the communication interface 1020. The network 1030 can be the Internet, an internet and / or extranet, or an intranetAttorney Docket No. 45269-751.601and / or extranet that is in communication with the Internet. The network 1030 in some cases is a telecommunication and / or data network. The network 1030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1030, in some cases with the aid of the computer system 1001, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1001 to behave as a client or a server.

[0205] The CPU 1005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions can be directed to the CPU 1005, which can subsequently program or otherwise configure the CPU 1005 to implement methods of the present disclosure. Examples of operations performed by the CPU 1005 can include fetch, decode, execute, and writeback.

[0206] The CPU 1005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0207] The storage unit 1015 can store files, such as drivers, libraries and saved programs. The storage unit 1015 can store user data, e.g., user preferences and user programs. The computer system 1001 in some cases can include one or more additional data storage units that are external to the computer system 1001, such as located on a remote server that is in communication with the computer system 1001 through an intranet or the Internet.

[0208] The computer system 1001 can communicate with one or more remote computer systems through the network 1030. For instance, the computer system 1001 can communicate with a remote computer system of a user (e.g., service provider). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1001 via the network 1030.

[0209] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine readable code can be provided in the form of software.

[0210] During use, the code can be executed by the processor 1005. In some cases, the code can be retrieved from the storage unit 1015 and stored on the memory 1010 for ready access by the processor 1005. In some situations, the electronic storage unit 1015 can be precluded, and machine-executable instructions are stored on memory 1010.Attorney Docket No. 45269-751.601

[0211] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[0212] Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., readonly memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0213] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable mediaAttorney Docket No. 45269-751.601therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD- ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0214] The computer system 1001 can include or be in communication with an electronic display 1035 that comprises a user interface (UI) 1040 for providing, for example, an output or readout of the trained algorithm. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0215] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1005.

[0216] Computer systems herein are in some cases configured to execute machine learning operations such as those disclosed in the specification herein or otherwise known to one of skill in the art.Non-Sequencing Based Assays

[0217] Non-sequencing based assays, such as hybridization (e.g., labeling, array hybridization, fluorescent probe hybridization such as FISH, antibody hybridization) or amplification (e.g., PCR) can be employed to detect genetic features (e.g., genetic rearrangements) on DNA-protein complexes (e.g., chromatin) or other bound DNA complexes (e.g., DNA complexed with beads or other substrates).

[0218] DNA complexes (e.g., DNA-protein complexes such as chromatin or other bound DNA complexes) can be collected using techniques discussed herein. For example, DNA complexes can be recovered from preserved samples (e.g., FFPE samples). In an example, chromatin can be liberated from a preserved sample (e.g., an FFPE sample) by heat treatment and proteolysis.

[0219] DNA complexes can be captured or purified. For example, DNA complexes (e.g., chromatin) can be captured on a solid phase. In some cases, the solid phase comprises a carboxylated substrate, such as carboxylated paramagnetic beads.Attorney Docket No. 45269-751.601

[0220] DNA complexes can be fragmented and ligated by methods disclosed herein, including but not limited to enzymatic (e.g., restriction enzymes, fragmentase, transposase), thermal, and physical fragmentation. Ligation can be preceded by blunt ending.

[0221] DNA complexes can be partitioned for further analysis. For example, DNA complexes (e.g., chromatin) can be partitioned into droplets (e.g., microfluidic droplets), wells, array spots, or other partitions.

[0222] DNA complexes can be analyzed by a variety of means. Amplification (e.g., PCR) can be conducted (e.g., in a partition such as droplet PCR) targeting variant breakpoints (e.g., targeting with primer pairs). Hybridization assays, such as with fluorescent oligonucleotide probes, can be used to target variant breakpoints. Rearrangements can be detected by a change in signal due to changed probability of proximity ligation of nearby loci. In some cases, Taq-Man probes can be used. In some cases, SYBR probes can be used. Such an analysis can be multiplexed, for example in droplets, wells, array spots, or other partitions.

[0223] In an example, chromatin is liberated from a preserved sample (e.g., FFPE) by mild heat treatment and proteolysis. The liberated chromatin is captured on a solid phase comprising paramagnetic carboxylated polystyrene beads. DNA bound to the captured chromatin is fragmented (e.g., enzymatically) and fragmented ends are blunted. Blunt ended DNA associated with chromatin is ligated to other nearby DNA. The presence of inter-chromosomal variants is quantified, such as by droplet-based PCR or fluorescent oligonucleotide probe hybridization. Deletions and inversions change (e.g., increase) the signal due to a change (e.g., increase) in probability of proximity ligation of nearby loci.

[0224] Rearrangement assays can be combined with sequencing-based assays such as those described herein, including sequencing-based assays of rearrangement. For example, after a PCR or hybridization assay, chromatin can be sequenced and analyzed as disclosed herein.Kits

[0225] Disclosed herein are kits for conducting the techniques disclosed herein. Kits can be contained in packaging such as boxes, with materials for a certain number of reactions in each unit of packaging. In some cases, a kit contains reagents for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more reactions.

[0226] Kits as disclosed herein comprise some or all reagents necessary to practice the methods and generate or analyze the compositions disclosed herein. In some cases, the kits comprise a subset of reagents necessary to practice the methods and generate or analyze theAttorney Docket No. 45269-751.601compositions disclosed herein, and optionally include instructions relevant to reagents not included in a kit but often readily available from a reagent vendor.

[0227] Some kits disclosed herein comprise a buffer, a DNA binding agent, an affinity tag binding agent, deoxynucleotides, tagged deoxynucleotides, a DNA fragmenting agent, an end repair enzyme, a ligase, a protein removal agent, and instructions for use in obtaining genomic structural information from the preserved sample. Kits optionally comprise reagents for PCR, such as a buffer, nucleotides, a forward primer, a reverse primer, and a thermostable DNA polymerase.

[0228] Buffers in some kits comprise at least one of a restriction digest buffer, an end repair buffer, a ligation buffer, a TE buffer, a wash buffer, a TWB solution a NTB solution, a LWB solution, a NWB solution, and a crosslink reversal buffer. A representative digest buffer is a DpnII buffer, or a commercial buffer such as or functionally analogous to NEB buffer 2.Exemplary ligation buffers include T4 DNA ligase buffer, BSA, and Triton X-100.

[0229] Other suitable reagents, either included in a kit or referred to in instructions for use in combination with kit reagents, include a TE buffer comprising tris and EDTA, a wash buffer comprising tris and sodium chloride, a TWB solution comprising one or more of tris, EDTA, and Tween 20, an NTB solution comprising one or more of tris, EDTA, and sodium chloride, an LWB solution comprising one or more of tris, lithium chloride, EDTA, and Tween 20, an NWB solution comprising at least one of tris, sodium chloride, EDTA, and Tween 20, and a crosslink reversal buffer comprising one or more of tris, SDS, and calcium chloride.

[0230] Some kits are configured to include or to be compatible with an affinity tag binding agent such as streptavidin beads, for example dynabeads.

[0231] Kits include or are compatible with nucleotides, such as dATP, dCTP, dGTP and dTTP, and in some cases biotinylated versions of the nucleotides.

[0232] DNA fragmenting agents included in kits herein or compatible therewith include at least one of a restriction enzyme such as Dpnl, a transposase, a nuclease, a sonication device, a hydrodynamic shearing device, and a divalent metal cation.

[0233] End repair enzymes included in or compatible with kits herein comprise at least one of T4 DNA polymerase, klenow DNA polymerase, and T4 polynucleotide kinase.

[0234] One suitable ligase in or compatible with kits herein includes T4 ligase.

[0235] Protein removal reagents included in or to be used in combination with kits herein include phenol and proteinases, such as proteinase K, Streptomyces griseus protease, a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamic protease, a metalloprotease, and an asparagine peptide lyase.Attorney Docket No. 45269-751.601

[0236] Kits optionally include or are compatible with solvents, such as solvents to be used to remove an embedding material such as paraffin.Definitions

[0237] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "contig" includes a plurality of such contigs and reference to "probing the physical layout of chromosomes" includes reference to one or more methods for probing the physical layout of chromosomes and equivalents thereof known to those skilled in the art, and so forth.

[0238] Also, the use of "and" means "and / or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0239] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of' or "consisting of."

[0240] The term "sequencing read" as used herein, refers to a fragment of DNA in which the sequence has been determined.

[0241] The term "contigs" as used herein, refers to contiguous regions of DNA sequence. "Contigs" can be determined by any number methods known in the art, such as, by comparing sequencing reads for overlapping sequences, and / or by comparing sequencing reads against databases of known sequences in order to identify which sequencing reads have a high probability of being contiguous.

[0242] The term "subject" as used herein can refer to any eukaryotic or prokaryotic organism.

[0243] The term "naked DNA" as used herein can refer to DNA that is substantially free of complexed proteins. For example, it can refer to DNA complexed with less than about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1% of the endogenous proteins found in the cell nucleus.

[0244] The term "read pair" or "read-pair" as used herein can refer to two or more elements that are linked to provide sequence information. In some cases, the number of read-pairs can refer to the number of mappable read-pairs. In other cases, the number of read-pairs can refer to the total number of generated read-pairs.

[0245] A “tissue sample” as used herein, refers to a biological sample from an individual or an environment potentially comprising nucleic acids. Tumors, for example, are consideredAttorney Docket No. 45269-751.601tissues, and a sample taken from a tumor constitutes a tissue sample, but in some cases the term refers to samples taken from a heterogeneous environment such as a stomach or intestine section, or an environmental sample comprising nucleic acids from a plurality of sources spatially distributed relative to one another.

[0246] About,” as used herein in reference to a number refers to that number + / - 10% of that number. As used in reference to a range, ‘about’ refers to a range having a lower limit 10% less than the indicated lower limit of the range and an upper limit that is 10% greater than the indicated upper limit of the range.

[0247] A “probe” as used herein refers to a molecule that conveys information through binding to a target. Suitable probes include but are not limited to oligonucleotide molecules and antibodies. Oligonucleotide molecules may act as probes by annealing to a target and conveying information either by changing a fluorescence characteristic, or alternately by annealing to a target and facilitating synthesis of a product such as an amplicon indicative of presence of the target. That is, the term probe as used herein variously contemplates antibody probes and other small molecule probes, as well as oligonucleic acid molecules, either acting by generating a signal directly through hybridization to a target leading to, for example, a change in fluorescence status, or acting by facilitating synthesis of an amplicon indicative of target presence.

[0248] As used herein, a DNA protein complex is destroyed or disrupted when proteins and nucleic acids are no longer assembled so as to form a complex. In some cases, the complexes are completely denatured or disassembled, so that no protein DNA binding remains. Alternately, in some cases a DNA protein complex is substantially destroyed when a first nucleic acid segment and a second nucleic acid segment are no longer held together independent of any phosphodiester bond.

[0249] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the example methods and materials are now described.

[0250] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.Attorney Docket No. 45269-751.601EXAMPLESExample 1. DNA Library Preparation on Formalin Fixed Paraffin Embedded Samples

[0251] Provided herein is a method of DNA Library preparation from DNA extracted from formalin fixed paraffin embedded (FFPE) tissue.

[0252] Conditions were tested for preparing DNA Libraries from formalin fixed paraffin embedded (FFPE) tissue. The FFPE tissue blocks were received from AMSBIO (Cambridge, MA) and tissue block information, DNA extraction information, and an indication of whether Library preparation was successful are summarized in Table 1. FFPE tissue was cut into scrolls and were processed to prepare DNA for proximity ligation. For example, scrolls were cut at various dimensions, including 1 x 5 pM, 1 x 10 pM, 2 x 10 pM, or 1 x 15 pM. First, the scrolls were treated to dissolve the paraffin, for example using xylene, and to rehydrate the tissue in the scrolls, for example using ethanol and water (FEO). Next, crosslinks were reversed in a first crosslink reversal step using a crosslink reversal solution. The crosslink reversal solution had a buffer (Tris at a pH of 8), a detergent (sodium dodecyl sulfate (SDS)), Proteinase K, and a guanidium salt (guanidium chloride). Samples were treated at a temperature of 55 °C, or 70 °C for 15 minutes to perform crosslink reversal. Following crosslink reversal, DNA were digested with 0.01 pL Micrococcal nuclease (MNase) for 15 minutes. Following DNA digestion, cell lysis with or without filtration was performed for 5 minutes. Proximity ligation was then performed on the lysis product for 3 hours, followed by a second round of crosslink reversal overnight. Finally, library conversion was performed on the resulting DNA product for 3.5 hours.

[0253] An example of the optimized method for preparing DNA Libraries as disclosed herein is provided in FIG. 3. FFPE tissue blocks are assessed for quality 301, for example by assessing a genomic DNA integrity number (DIN). Samples with sufficient genomic DNA were then sectioned into various tissue scroll sizes 302, as disclosed herein. Tissue scrolls were deparaffinized with xylene for 10 minutes 303. Tissue was rehydrated with 100% ethanol (EtOH) for 10 minutes, 70% EtOH for 10 minutes, 50% EtOH for 10 minutes, 20% EtOH for 10 minutes, and water for 10 minutes 303. Rehydrated tissue underwent crosslink reversal at 70 °C for 15 minutes with a solution comprising a buffer (Tris at a pH of 8), a detergent (sodium dodecyl sulfate (SDS)), Proteinase K, and a guanidium salt (guanidium chloride) 304. Tissue was then provided 0.01 pL MNase for 15 minutes 305. Tissue was then lysed with Proteinase K and lysate was filtered 306. Filtered lysate underwent proximity ligation as disclosed herein 307. Ligated DNA underwent crosslink reversal at 78 °C overnight 308. DNA underwent library conversion to generate DNA Libraries 309.Attorney Docket No. 45269-751.601

[0254] Steps of the method disclosed herein were optimized through experimentation to increase the quantity of DNA extracted from FFPE tissue, increase the yield of DNA prepared into DNA Libraries, increase the number of paired reads from the DNA Libraries, increase the length of paired reads from the DNA Libraries, increase the number of unique paired reads out of total paired reads, and to decrease the number of inter-chromosomal reads.First Crosslink Reversal Optimization

[0255] To optimize the first crosslink reversal conditions, crosslink reversal was performed on FFPE tissue scrolls with a solution (Solution 1) of a buffer (Tris at a pH of 8), a detergent (sodium dodecyl sulfate (SDS)), Proteinase K, and a guanidium salt (guanidium chloride) at either 55 °C, or 70 °C, for 15 minutes. In addition, the crosslink reversal procedure using Solution 1 was tested followed by Liberase™ treatment at 37 °C overnight. In another condition, instead of treating the FFPE tissue with Solution 1, the samples were only treated with Liberase™ at 37 °C overnight. As an alternative to crosslink reversal, nuclei preparation was tested on FFPE tissue using a solution of 20 mM Tris with 0.3% SDS at 62 °C overnight. Each of these experimental condition parameters were followed by MNase digestion, proximity ligation, a second round of crosslink reversal at about 78 °C overnight, and library conversion. The amount of pre-library conversion DNA (PL) and library input DNA (Lib input) were quantified. Following library conversion, the amount of library DNA was quantified (Lib), the total number of read pairs were quantified (Total Read Pairs), the proportion of inter-chromosomal reads were quantified (inter.chr), the proportion of reads less than 1 kb were quantified (< Ikb), the proportion of reads greater than 1 kb were quantified (> Ikb), and the proportion of unique reads out of 400 millionAttorney Docket No. 45269-751.601reads were quantified (Complexity). Of these parameters, conditions were considered acceptable if proximity ligation resulted in greater quantities of library prepared DNA, and if the DNA Libraries contained lower proportions of inter-chromosomal DNA, contained longer DNA fragments (i.e., > 1 kb), and a greater proportion of unique reads (complexity). The results of the crosslink reversal conditions, and the nuclei preparation condition are summarized in Table 2.< >< >Attorney Docket No. 45269-751.601< >< >

[0256] The results of the crosslink reversal optimization experiments (Table 2) revealed that crosslink reversal with Solution 1 at 70 °C for 15 minutes with or without Liberase™ treatment at 37 °C for 1 hour resulted in the highest proportion of library prepared DNA (Lib), total read pairs, reads greater than 1 kb, and unique reads. Similarly, the resultsAttorney Docket No. 45269-751.601revealed that these conditions resulted in the lowest proportion of inter-chromosomal DNA (inter.chr) and reads less than 1 kb. To determine if either of these crosslink reversal conditions were superior, the library preparation protocol disclosed herein was compared with either a first crosslink reversal with Solution 1 at 70 °C for 15 minutes, or a first crosslink reversal with Solution 1 at 70 °C for 15 minutes with Liberase™ treatment at 37 °C for 1 hour. The results of this comparison are summarized in Table 3.< >< >< >< >< >Attorney Docket No. 45269-751.601< >< >< >

[0257] Head-to-head comparison of the two crosslink reversal conditions revealed similar outcomes across the parameters disclosed herein. Thus, the less reagent-intensive condition using Solution 1 at 70 °C for 15 minutes without Liberase™ treatment was chosen as the optimized condition for the method.Tissue Rehydration Optimization

[0258] Following cutting FFPE tissue blocks into scrolls, the paraffin must be dissolved, and the tissue must be rehydrated before crosslink reversal. Paraffin was dissolved using xylene for 10 minutes. Following paraffin dissolution, the tissue was rehydrated with 100% ethanol (EtOH) followed by increasing dilutions of ethanol in water (H2O). The number and lengths of hydration steps were optimized through experimentation to determineAttorney Docket No. 45269-751.601acceptable conditions for DNA Library preparation. In each test condition, paraffin was dissolved through a 10-minute xylene exposure, followed subsequent rehydration steps. In some conditions, the tissue was briefly vortexed during hydration. The conditions that were tested are summarized in Table 4.

[0259] Following paraffin dissolution and tissue rehydration according to the experimental conditions of Table 4, the tissue scrolls underwent a first crosslink reversal as described herein. Following crosslink reversal, the tissue was digested with MNase as described herein. Next, the digested DNA was proximity ligated, followed by a second crosslink reversal overnight, and DNA library conversion as described herein. The amount of pre-library conversion DNA (PL) and library input DNA (Lib input) were quantified.Following library conversion, the amount of library DNA was quantified (Lib), the total number of read pairs were quantified (Total Read Pairs), the total number of aligned read pairs were quantified (Total Ain. Pairs), the total number of reads that correspond to a transcript were quantified (f. aligned), the proportion of inter-chromosomal reads were quantified (inter.chr), the proportion of reads less than 1 kb were quantified (< Ikb), the proportion of reads greater than 1 kb were quantified (> Ikb), and the total number of unique reads out of 400 million reads were quantified (Complexity). Of these parameters, conditions were considered acceptable if proximity ligation resulted in greater quantities of library prepared DNA, and if the DNA Libraries contained lower proportions of inter-chromosomal DNA, longer DNA fragments (i.e., > 1 kb), and a greater proportion of unique reads (complexity). The results of the tissue rehydration conditions are summarized in Table 5.Attorney Docket No. 45269-751.601>>>>>

[0260] The rehydration conditions that resulted in acceptable levels of crosslink reversal and DNA Library conversion was determined to be Condition 1 or Condition 2, as specified in Table 5.Attorney Docket No. 45269-751.601Micrococcal Nuclease Digest Optimization

[0261] Following the first crosslink reversal, FFPE samples underwent Micrococcal nuclease (MNase) digestion. The MNase conditions were also tested with varying amounts of MNase added to an input of 5x10 pm scrolls with crosslink reversal with Solution 1 further comprising calcium chloride and a detergent (Tween 20 and Triton X-100) at 55 °C for 15 minutes. Results from libraries obtained with these conditions are summarized in Table 6.Table 6: Library Results from MNase testing< >

[0262] The input was also tested with various amounts of input scrolls added with crosslink reversal with Solution 1 further comprising calcium chloride and a detergent (Tween 20 and Triton X-100) at 55 °C for 15 minutes and MNase treatment of 0.5 pl.Results from libraries obtained with these conditions are summarized in Table 7.< >

[0263] MNase digestion conditions were also tested with various low input amounts. Results from libraries obtained with these conditions are summarized in Table 8.Attorney Docket No. 45269-751.601Table 8: Optimizing Low Input Conditions< >

[0264] The MNase optimization experiments revealed that digestion with 0.01 pL of MNase provided acceptable Library DNA yields.Second Crosslink Reversal Optimization

[0265] Following proximity ligation, proximity ligated DNA underwent a second crosslink reversal as disclosed herein. To optimize the conditions for the second crosslink reversal, proximity ligated DNA was provided crosslink reversal buffer (Solution 1, as disclosed herein) for 1 hour at 78 °C, for 1 hour at 78 °C 7.5 pL Proteinase K (ProK), for 2 hours at 78 °C, or overnight at 78 °C. The results of these optimization conditions are presented in Table 9.>Attorney Docket No. 45269-751.601>>

[0266] Experimentation with these conditions revealed that the second round of crosslink reversal overnight yielded greater quantities of Library input DNA, Library DNA, and in cases where tissue had poor DNA quality (Block 557), reduced inter-chromosomal DNA, reduced DNA fragments >lkb, and increased DNA fragments >lkb.Attorney Docket No. 45269-751.601Cell Lysate Filtration

[0267] Following MNase digestion and cell lysis as disclosed herein, it was contemplated whether filtration would improve library conversion. To test this, cell lysate was either filtered, or not filtered, prior to proximity ligation. Results of the filtration optimization experiment are summarized in Table 10.>>>>Attorney Docket No. 45269-751.601>>

[0268] In some cases, filtration resulted in increased levels of DNA Library quantity and complexity (Table 10, Block 661 and 189). Therefore, the filtration step was included in the optimized method as described herein.DNA Repair Optimization

[0269] Some FFPE tissues provide low quality DNA; therefore, a DNA repair was considered for improving Library input yield, and DNA Library yield. FFPE tissue was rehydrated as disclosed herein. DNA digestion and proximity ligation were not performed.DNA was repaired using NEBNext® FFPE DNA Repair mix. DNA Integrity Number (DIN) and Library yields with and without DNA repair were compared. The comparison is summarized in Table 11.Attorney Docket No. 45269-751.601

[0270] FFPE DNA Repair resulted in creased Library yields. Therefore, FFPE DNA repair was tested in the DNA Library preparation method as disclosed herein. Before proximity ligation or after the second crosslink reversal step, FFPE DNA repair was performed using NEBNext® FFPE DNA Repair mix. For optimization of the method, the method was performed as disclosed herein with a DNA repair step before proximity ligation and after the second crosslink reversal. The second crosslink reversal step included incubation with Solution 1 at 70 °C for 15 minutes, or incubation with Solution 1 at 70 °C for 15 minutes, followed by Liberase™ incubation at 70 °C for 1 hour. The results of this optimization experiment are summarized in Table 12.>>>Attorney Docket No. 45269-751.601

[0271] Comparing pre-Library input DNA and Library DNA yields with or without FFPE DNA repair revealed no difference. Therefore, the DNA repair step was omitted.

[0272] The optimization experimentation disclosed herein resulted in an optimized library conversion protocol comprising cutting FFPE tissue samples into tissue scrolls, deparaffinization with xylene for 10 minutes, tissue rehydration with about 100% EtOH for about 10 minutes, about 70% EtOH for about 10 mins, about 50% EtOH for about 10 minutes, about 20% EtOH for about 10 minutes, and water for about 10 minutes, a first crosslink reversal with Solution 1 at about 70 °C for about 15 minutes, cell lysis with lysate filtration for about 5 minutes, proximity ligation for about 3 hours, a second round of crosslink reversal with Solution 1 at about 78 °C overnight, and library conversion.Example 2, DNA Library Preparation on Formalin Fixed Paraffin Embedded Samples

[0273] FFPE samples were prepared for sequencing according to methods of the present disclosure. FFPE tissue was cut into scrolls and were processed to prepare DNA for proximity ligation.

[0274] First, quality control (QC) was performed on 5 um or 10 um FFPE sample scrolls using an FFPE prep kit (e.g., Zymo FFPE Quick DNA / RNA Prep Kit). The amount of DNA recovered was assessed using Qubit values, and DNA quality was assessed using a Tapestation. If the Qubit reading was > 5ng / uL, then the sample was run on Genomic tape to determine the DNA Integrity Number (DIN), or if Qubit reading is < 5ng / uL then run on HS D5000 tape. Samples with DNA yield > 20 ng, DIN > 2, HS D5000 > 1000 bp were good candidates for successful library preparation.

[0275] Second, sample preparation and lysate preparation were conducted. The number of scrolls or slide sections input into the assay was based on the Qubit reading (ng) from the QC stage. Scrolls or slides equal to 200-1000 ng of DNA (up to 10 scrolls) were input, with an optimal input of 200-500 ng of DNA. Tissue scrolls were deparaffinized with 1 mL xylene for 10 minutes. Tissue was then rehydrated with 1 mL 100% ethanol for 10 minutes followed by 200 uL water for 10 minutes. Rehydrated tissue underwent crosslink reversal at 70 °C for 15 minutes with a solution comprising a buffer (Tris at a pH of 8), a detergent (sodium dodecyl sulfate (SDS)), Proteinase K, and a guanidium salt (guanidium chloride). Tissue was then provided 0.01 pL MNase for 15 minutes. Tissue was then lysed with Proteinase K and lysate was filtered.

[0276] Third, proximity ligation was conducted as disclosed herein. Following proximity ligation, the percentage of recovered DNA was calculated as (ng of crosslinked DNA) / (ng of input DNA). If recovered DNA was > 20%, then the sample proceeded toAttorney Docket No. 45269-751.601library prep with up to 1000 ng of crosslink reversed DNA. If the recovery was < 20%, then the sample preparation and lysate preparation stage was repeated, but with the crosslink reversal step omitted.

[0277] Fourth, library preparation was conducted as disclosed herein and samples were sequenced.

Claims

Attorney Docket No. 45269-751.601CLAIMS WHAT IS CLAIMED IS:

1. A method of analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) sample comprising:(a) providing an FFPE sample comprising cells comprising cross-linked DNA-protein complexes;(b) reversing at least a portion of crosslinks in the FFPE sample using a buffer comprising a guanidinium salt, wherein crosslinks in DNA-protein complexes are not disrupted;(c) isolating the DNA-protein complexes; and(d) performing an analysis of nucleic acids of the DNA-protein complexes.

2. The method of claim 1, wherein (b) comprises incubating the FFPE sample in the buffer at a temperature about 70 °C for less than an hour.

3. The method of claim 1, wherein (b) comprises incubating the FFPE sample in the buffer at a temperature about 70 °C for about 15 minutes.

4. The method of claim 1, wherein (b) comprises incubating the FFPE sample in the buffer at a temperature about 55 °C for less than an hour.

5. The method of claim 1, wherein (b) comprises incubating the FFPE sample in the buffer at a temperature about 55 °C for about 15 minutes.

6. The method of any one of claims 1 to 3, further comprising cleaving DNA of the DNA-protein complexes to obtain a plurality of DNA segments bound in DNA-protein complexes.

7. The method of claim 6, wherein cleaving is effected by a nuclease.

8. The method of claim 7, wherein the nuclease is selected from micrococcal nuclease, a transposase, an integrase, a restriction endonuclease, or a combination thereof.

9. The method of claim 8, wherein the nuclease is micrococcal nuclease.

10. The method of any one of claims 1 to 9, further comprising lysing cells in the FFPE sample to generate a cell lysate.

11. The method of claim 10, further comprising filtering the cell lysate.

12. The method of any one of claims 1 to 11, further comprising ligating at least a first DNA segment of the plurality of DNA segments to a second DNA segment of the plurality of DNA segments to create a plurality of ligated DNA segments bound in DNA-protein complexes.Attorney Docket No. 45269-751.60113. The method of claim 12, wherein ligating the first DNA segment and the second DNA segment further comprises ligating a tag oligonucleotide between the first DNA segment and the second DNA segment.

14. The method of claim 13, wherein the tag comprises a barcode sequence.

15. The method of any one of claims 1 to 14, further comprising isolating the plurality of ligated DNA segments from the DNA-protein complexes.

16. The method of claim 15, wherein isolating the plurality obligated DNA segments comprises incubating the plurality obligated DNA segments bound in DNA- protein complexes in the buffer at a temperature greater than 60 °C for over an hour.

17. The method of claim 16, wherein isolating the plurality obligated DNA segments comprises incubating the plurality obligated DNA segments bound in DNA-protein complexes in the buffer at a temperature about 78 °C overnight.

18. The method of any one of claims 1 to 17, further comprising obtaining a sequence of at least a portion of the first DNA segment and a portion of the second DNA segment of the plurality obligated DNA segments.

19. The method of claim 18, further comprising assigning contigs having a sequence common to the sequence of the first DNA segment and the second DNA segment to a common scaffold in a nucleic acid assembly.

20. The method of any one of claims 1 to 19, wherein the analysis comprises sequencing, immunoprecipitation, nucleic acid hybridization, polymerase chain reaction (PCR), quantitative PCR, mass spectrometry, or a combination thereof.

21. The method of any one of claims 1 to 20, wherein the analysis comprises deriving genomic structural information indicative of an inversion, a deletion, or a translocation relative to a reference genome.

22. The method of any one of claims 1 to 21, wherein the analysis comprises deriving information indicative of a phase status for a first segment and a second segment of the nucleic acids.

23. The method of any one of claims 1 to 22, further comprising treating the FFPE sample with ethanol and / or xylene.

24. The method of claim 23, wherein treating the FFPE samples with ethanol comprises treating the FFPE samples with 100% ethanol, 70% ethanol, 50% ethanol, and 20% ethanol each for about 10 minutes.

25. The method of claim 23, wherein treating the FFPE samples with ethanol comprises treating the FFPE samples with 100% ethanol for about 10 minutes.Attorney Docket No. 45269-751.60126. The method of any one of claims 1 to 25, wherein the FFPE sample comprises 200 ng to 1000 ng of DNA.

27. The method of claim 26, wherein the FFPE sample comprises 200 ng to 500 ng of DNA.

28. The method of any one of claims 1 to 27, wherein the FFPE sample comprises a 1x10 pm scroll, a 5x10 pm scroll, a 10x10 pm scroll, or a 20x10 pm scroll.

29. The method of any one of claims 1 to 28, wherein the buffer further comprises at least one of a buffering agent and a detergent.

30. The method of claim 29, wherein the detergent is Tween 20, Triton X-100, or a combination thereof.

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