Methods and compositions for sequencing library preparation

Abstract

Provided herein are methods of proximity ligation and compositions for use in such methods.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 340,734, filed May 11, 2022, and U.S. Provisional Application No. 63 / 490,192, filed March 14, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Obtaining high-quality, contiguous genome sequences is often difficult, especially when there is limited source material available for sequence analysis. While obtaining raw sequence data has become faster and less expensive, suitable methods for efficiently and accurately analyzing and assembling the data remain a challenge. Summary of the invention

[0004] In one aspect, the present invention provides a method for nucleic acid processing. In some cases, the method includes obtaining a stabilized sample, the stabilized sample includes a nucleic acid molecule compounded with at least one nucleic acid binding protein. In some cases, the method includes cutting the nucleic acid molecule into multiple sections, the multiple sections include at least the first section and the second section, wherein the cutting is achieved by a transposase. In some cases, the method includes connecting the first section to the second section, thereby generating a nucleic acid comprising a first sequence from the first section and a second sequence from the second section. In some cases, the transposase is a Tn5 transposase. In some cases, the method further includes cyclizing the connected nucleic acid by connecting the 5' end of the connected nucleic acid to the 3' end of the connected nucleic acid. In some cases, the method further includes sequencing at least a portion of the connected nucleic acid. In some cases, the sequencing includes sequencing at least a portion of the first sequence and at least a portion of the second sequence. In some cases, the method further includes mapping at least a portion of the first sequence and at least a portion of the second sequence to a genome. In some cases, the method further includes using the information from the sequencing to perform three-dimensional genome analysis. In some cases, the stabilized sample is a cross-linked sample. In some cases, obtaining the stabilized sample includes obtaining a sample and stabilizing the sample. In some cases, obtaining the stabilized sample includes obtaining a previously stabilized sample. In some cases, the nucleic acid binding protein comprises chromatin or a component thereof. In some cases, a linker sequence is connected between the first segment and the second segment. In some cases, the linker sequence comprises a barcode sequence. In some cases, the barcode sequence indicates a source partition. In some cases, the barcode sequence indicates a source cell. In some cases, the barcode sequence indicates a source cell population. In some cases, the barcode sequence indicates a source organism. In some cases, the cutting occurs in open and closed chromatin compartments. In some cases, at least 10% of the cutting occurs in a closed chromatin compartment. In some cases, at least 20% of the cutting occurs in a closed chromatin compartment. In some cases, at least 30% of the cutting occurs in a closed chromatin compartment. In some cases, the stabilized sample comprises no more than 50,000 cells. In some cases, the stabilized sample comprises at least 10,000 cells. In some cases, the stabilized sample comprises stabilized cell nuclei. In some cases, the stabilized sample comprises no more than 50,000 cell nuclei. In some cases, the stabilized sample comprises at least 10,000 cell nuclei. In some cases, the linked nucleic acid does not comprise an affinity tag. In some cases, the linked nucleic acid does not comprise biotin.In some cases, the circularized linked nucleic acid does not include an affinity tag. In some cases, the circularized linked nucleic acid does not include biotin. In some cases, an affinity tag is not used to separate the linked nucleic acid and / or the circularized linked nucleic acid. In some cases, streptavidin is not used to separate the linked nucleic acid and / or the circularized linked nucleic acid.

[0005] Incorporation by reference

[0006] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] An understanding of the features and advantages of the present invention will be gained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments utilizing the principles of the invention, in which:

[0008] Figure 1 Various components of an exemplary computer system according to various embodiments of the present disclosure are illustrated.

[0009] Figure 2 is a block diagram illustrating the architecture of an exemplary computer system that can be used in conjunction with various embodiments of the present disclosure.

[0010] Figure 3 is a schematic diagram illustrating an exemplary computer network that may be used in conjunction with various embodiments of the present disclosure.

[0011] Figure 4 is a block diagram illustrating the architecture of another exemplary computer system that can be used in conjunction with various embodiments of the present disclosure.

[0012] Figure 5 Methods for identifying long noncoding RNA (lncRNA) binding sites are described.

[0013] Figure 6 An example workflow of the labeling and neighbor joining methods is depicted.

[0014] Figure 7 Depicted are example methods for labeling and neighbor joining.

[0015] Figure 8 Two examples of the neighbor joining method are depicted.

[0016] Fig. 9A Depicted is the uniformity of coverage achieved using different chromatin fragmentation methods.

[0017] Fig. 9B Library characterization achieved using different chromatin fragmentation methods is depicted.

[0018] Fig.10 is a table showing the long-range sequence information achieved using different library preparation methods.

[0019] Fig.11 Depicted are chromatin contacts captured using different library preparation methods.

[0020] Fig.12 Depicted are the results of exonuclease treatment in the library preparation method.

[0021] Fig.13 Depicted are the results of exonuclease treatment in the library preparation method.

[0022] Fig.14 Depicted are the results of exonuclease treatment in the library preparation method. DETAILED DESCRIPTION

[0023] In one aspect, compositions, systems and methods are provided herein that relate to determining genomic sequences, including long-range and structural genomic information, determining the physical conformation of nucleic acids in cells, and for generating very long-range read pairs of nucleic acids that outperform some other methods. Techniques that can be utilized by the methods herein include, but are not limited to, transposase fragmentation of cross-linked nucleic acids and ligation-based joining of transposase fragmented nucleic acids.

[0024] Transposase fragments chromatin

[0025] In one aspect, the present invention provides a method for nucleic acid treatment. Such methods may include obtaining a stabilized sample, the stabilized sample comprising a nucleic acid molecule compounded with at least one nucleic acid binding protein, and cutting the nucleic acid molecule into multiple sections, the multiple sections comprising at least a first section and a second section, wherein the cutting is achieved by a transposase. The method herein may also include connecting the first section and the second section, thereby producing a connected nucleic acid. In some cases, the connected nucleic acid is further connected to produce a circularized connected nucleic acid.

[0026] In various aspects of the methods herein, a transposase is used to cut the stabilized nucleic acid. In some cases, the cutting is performed in a permeabilized cell. In some cases, the cutting is performed in a permeabilized nucleus. In some cases, the transposase is Tn5, Tn3, Tn7, Sleeping Beauty transposase, or a combination thereof. In some cases, the transposase is Tn5 transposase.

[0027] In various aspects of the methods herein, cutting occurs in open chromatin and closed chromatin. In some cases, at least about 10% to at least about 50% of the cutting occurs in closed chromatin. For example, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or more of the cutting occurs in closed chromatin. In some embodiments, closed chromatin is transcriptionally inactive and binds to one or more nucleosomes or other chromatin proteins. In some embodiments, open chromatin is transcriptionally active and binds to less or no nucleosomes or other chromatin proteins.

[0028] In various aspects of the method herein, the joint comprising a recombinase site can contact the nucleic acid of cutting in the presence of a recombinase, wherein the recombinase site comprises two recombinase sites that are oriented to repeat in the same direction. In some cases, the existence of the recombinase site that is oriented to repeat in the same direction can prevent the products therefrom from forming a stable hairpin structure. In some cases, the nucleic acid connected does not form a hairpin loop. In some cases, the products therefrom are easier to order-check than the product that the recombinase site is oriented to repeat in reverse.

[0029] In various aspects of the methods herein, the first segment and the second segment of the cleaved nucleic acid are contacted with a linker comprising an integrase site in the presence of a recombinase. In some cases, the recombinase is an integrase. In some cases, the integrase is integrase, Bxb1 integrase, or a combination thereof.

[0030] In various aspects, methods of processing nucleic acids are provided, wherein the joined nucleic acids are circularized. In some cases, the ends of the nucleic acids are removed prior to joining to expose the first segment and the second segment. In some embodiments, the circularized products are amplified using PCR to create a sequencing library. Examples of this method include Figure 6 and Figure 7 shown.

[0031] In an exemplary embodiment, a sample can be prepared and cross-linked, and then the sample is tagged in situ, which fragments the chromatin and leaves mosaic ends at each end of the fragmented chromatin. The tagged chromatin can then be ligated using adapters and ligases. The ends can be removed and the cross-links reversed. The nucleic acid can be captured and then the resulting fragments can be circularized by ligation to form a circular nucleic acid having two genomic DNA fragments connected by mosaic ends on each side of the adapter integrally connected together. The genomic DNA for analysis can be amplified using adapter PCR and purification / size selection suitable for sequence analysis ( Figure 6 and Figure 7 ).

[0032] The method based on cyclization can provide multiple advantages. As mentioned above, cyclization can produce nucleic acid molecules, wherein the adapter site is located around the genomic sequence of interest, so that nucleic acid molecules for sequencing can be directly produced, wherein the proportion of the genome in the sequence is higher (for example, by excluding the adapter sequence). In addition, the method based on cyclization can eliminate the need for affinity tag enrichment methods. Existing proximity connection methods usually use affinity tags to enrich (for example, biotinylated nucleic acids are incorporated into the proximity connection site, which can then be enriched with surface-bound streptavidin) to ensure that the nucleic acid finally sequenced represents the proximity connection event rather than, for example, general genomic DNA; alternatively, as described herein, cyclization can be performed to enrich the nucleic acid that has been adjacent to the connection, because cyclization can require at least a certain length of nucleic acid. For example, nucleic acids less than about 250 base pairs may not be cyclized, such as fragments of the size of mononucleosomes that are not connected to a partner during proximity connection. In some cases, the cyclized molecules can be enriched by, for example, cleaning, bead binding or size selection. In other cases, enrichment for circularized molecules is not required, and primer-based amplification (eg, adapter PCR) is instead used to generate amplification products suitable for sequencing only from the circularized molecules.

[0033] In various aspects of the methods herein, the adapter comprises chimeric ends, sequencing adapters, and attB sequences. Alternatively, the adapter comprises chimeric ends and sequencing adapters, and the attB sequences are added to the transposase products prior to recombination, for example using a ligase.

[0034] In some aspects of the method herein, the method may also include sequencing at least a portion of the nucleic acid connected by any suitable method such as provided herein. In some cases, sequencing may include sequencing at least a portion of the first sequence and at least a portion of the second sequence. In some cases, the method may also include mapping at least a portion of the first sequence and at least a portion of the second sequence to a genome. In various cases, the method may also include using the information from sequencing to perform three-dimensional genome analysis.

[0035] In various aspects of the methods herein, the stabilized sample can be a cross-linked sample. In some cases, the stabilized sample can be a cross-linked cell. In some cases, the stabilized sample can be a cross-linked nucleus. In some cases, the stabilized sample can be a cross-linked chromatin. In some cases, obtaining a stabilized sample can include obtaining a sample and stabilizing the sample. In some cases, obtaining a stabilized sample can include obtaining a previously stabilized sample. In some cases, the nucleic acid binding protein can include chromatin or a component thereof.

[0036] In various aspects of the present method, the recombinase site may include attP and attB integrase sites. In some cases, the first recombinase site may be different from the second recombinase site. In some cases, the first recombinase site may be attP or attB integrase site. In some cases, the second recombinase site may be attP or attB integrase site. In various cases, the first recombinase site is attP integrase site, and the second recombinase site is attB integrase site. In various cases, the first recombinase site is attB integrase site, and the second recombinase site is attP integrase site. In some cases, the first recombinase site and the second recombinase site may include transposase chimeric ends.

[0037] In various aspects of the methods herein, the joint may comprise an additional sequence. In some cases, the joint sequence may comprise a barcode sequence. In some cases, the barcode sequence may indicate a source partition. In some cases, the barcode sequence may indicate a source cell. In some cases, the barcode sequence may indicate a source cell population. In some cases, the barcode sequence may indicate a source organism. In some cases, the barcode sequence may indicate a source species. In some cases, the joint may comprise an adapter. In some cases, the adapter may comprise a P5 sequence. In some cases, the adapter may comprise a P7 sequence.

[0038] In various aspects of the methods herein, the method can be completed in less than one day. In some cases, the method can be completed in less than 8 hours. In some cases, the method can be completed in less than 6 hours. In some cases, the method can be completed in no more than 4 hours. In some cases, the method can be completed in 4-6 hours. In some cases, the method can be completed in 4-8 hours. In some cases, the method can be completed in 3-4 hours.

[0039] In various aspects of the methods herein, the method may require very low sample material input. In some cases, the stabilized sample may contain no more than 50,000 cells. In some cases, the sample may contain no more than 40,000 cells. In some cases, the sample may contain no more than 30,000 cells. In some cases, the sample may contain no more than 20,000 cells. In some cases, the sample may contain at least 10,000 cells. In some cases, the sample may contain at least 20,000 cells. In some cases, the sample may contain at least 30,000 cells. In some cases, the sample may contain at least 40,000 cells. In some cases, the sample may contain about 10,000 cells to about 50,000 cells. In some cases, the sample may contain about 20,000 cells to about 50,000 cells. In some cases, the sample may contain about 30,000 cells to about 50,000 cells. In some cases, the sample may include about 40,000 cells to about 50,000 cells. In some cases, the sample may include about 10,000 cells to about 40,000 cells. In some cases, the sample may include about 10,000 cells to about 30,000 cells. In some cases, the sample may include about 10,000 cells to about 20,000 cells. In some cases, the sample may include about 20,000 cells to about 50,000 cells. In some cases, the sample may include about 20,000 cells to about 40,000 cells. In some cases, the sample may include about 20,000 cells to about 30,000 cells. In some cases, the sample may include about 30,000 cells to about 50,000 cells. In some cases, the sample may include about 30,000 cells to about 40,000 cells.

[0040] In various aspects of the methods herein, the stabilized sample may include cell nuclei. In some cases, the stabilized sample may include no more than 50,000 cell nuclei. In some cases, the sample may include no more than 40,000 cell nuclei. In some cases, the sample may include no more than 30,000 cell nuclei. In some cases, the sample may include no more than 20,000 cell nuclei. In some cases, the sample may include at least 10,000 cell nuclei. In some cases, the sample may include at least 20,000 cell nuclei. In some cases, the sample may include at least 30,000 cell nuclei. In some cases, the sample may include at least 40,000 cell nuclei. In some cases, the sample may include about 10,000 cell nuclei to about 50,000 cell nuclei. In some cases, the sample may include about 20,000 cell nuclei to about 50,000 cell nuclei. In some cases, the sample may include about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 40,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 10,000 nuclei to about 40,000 nuclei. In some cases, the sample may include about 10,000 nuclei to about 30,000 nuclei. In some cases, the sample may include about 10,000 nuclei to about 20,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 40,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 30,000 nuclei. In some cases, the sample can contain about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample can contain about 30,000 nuclei to about 40,000 nuclei.

[0041] Recombinase site orientation is direct repeats

[0042] On the other hand, a method for treating nucleic acids is provided, comprising obtaining a stabilized sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein. Next, the method may include cutting the nucleic acid molecule into a plurality of sections comprising at least a first section and a second section, and attaching a first recombinase site to the first section and the second section. The method may then include contacting the first section and the second section with a joint comprising a second recombinase site in the presence of a recombinase, thereby producing a nucleic acid adjacent to a connection, the nucleic acid adjacent to the connection comprising a first sequence from the first section, a joint sequence from the joint, and a second sequence from the second section, wherein the second recombinase site comprises two recombinase sites oriented as unidirectional repeats. In some cases, the stabilized sample may not be ultrasonically treated.

[0043] In various aspects of the methods herein, a transposase can be used to cut the stabilized nucleic acid. In some cases, the cutting can be performed in a permeabilized cell. In some cases, the cutting can be performed in a permeabilized cell nucleus. In some cases, the transposase can be Tn5, Tn3, Tn7, Sleeping Beauty transposase, or a combination thereof. In some cases, the transposase can be a Tn5 transposase.

[0044] In various aspects of the method herein, the joint comprising a recombinase site can contact the nucleic acid of cutting in the presence of a recombinase, wherein the recombinase site comprises two recombinase sites that are oriented to repeat in the same direction. In some cases, the existence of the recombinase site that is oriented to repeat in the same direction can prevent the products therefrom from forming a stable hairpin structure. In some cases, the nucleic acid adjacent to the connection does not form a hairpin loop. In some cases, the products therefrom can be easier to sequence than the product that the recombinase site is oriented to repeat in reverse.

[0045] In various aspects of the methods herein, the first segment and the second segment of the cleaved nucleic acid can be contacted with a linker comprising an integrase site in the presence of a recombinase. In some cases, the recombinase can be an integrase. In some cases, the integrase can be integrase, Bxb1 integrase, or a combination thereof.

[0046] In various aspects of the method herein, the method may also include sequencing at least a portion of the nucleic acid adjacent to the connection by any suitable method such as provided herein. In some cases, sequencing may include sequencing at least a portion of the first sequence and at least a portion of the second sequence. In some cases, the method further includes mapping at least a portion of the first sequence and at least a portion of the second sequence to a genome. In some cases, the method further includes using the information from sequencing to perform three-dimensional genome analysis.

[0047] In various aspects of the methods herein, the stabilized sample can be a cross-linked sample. In some cases, the stabilized sample can be a cross-linked cell. In some cases, the stabilized sample can be a cross-linked nucleus. In some cases, the stabilized sample can be a cross-linked chromatin. In some cases, obtaining a stabilized sample includes obtaining a sample and stabilizing the sample. In some cases, obtaining a stabilized sample includes obtaining a previously stabilized sample. In some cases, the nucleic acid binding protein comprises chromatin or a component thereof.

[0048] In various aspects of the present invention, the recombinase site may include attP and attB integrase sites. In some cases, the first recombinase site may be different from the second recombinase site. In some cases, the first recombinase site may be attP or attB integrase site. In some cases, the second recombinase site may be attP or attB integrase site. In various cases, the first recombinase site is attP integrase site and the second recombinase site is attB integrase site. In various cases, the first recombinase site is attB integrase site and the second recombinase site is attP integrase site. In some cases, the first recombinase site and the second recombinase site may include transposase chimeric ends.

[0049] In various aspects of the methods herein, the joint may comprise an additional sequence. In some cases, the joint sequence may comprise a barcode sequence. In some cases, the barcode sequence may indicate a source partition. In some cases, the barcode sequence may indicate a source cell. In some cases, the barcode sequence may indicate a source cell population. In some cases, the barcode sequence may indicate a source organism. In some cases, the barcode sequence may indicate a source species. In some cases, the joint may comprise an adapter. In some cases, the adapter may comprise a P5 sequence. In some cases, the adapter may comprise a P7 sequence.

[0050] In various aspects of the methods herein, the method can be completed in less than one day. In some cases, the method can be completed in less than 8 hours. In some cases, the method can be completed in less than 6 hours. In some cases, the method can be completed in no more than 4 hours. In some cases, the method can be completed in 4-6 hours. In some cases, the method can be completed in 4-8 hours. In some cases, the method can be completed in 3-4 hours.

[0051] In various aspects of the methods herein, the method may require very low sample material input. In some cases, the stabilized sample may include no more than 50,000 cells. In some cases, the sample may include no more than 40,000 cells. In some cases, the sample may include no more than 30,000 cells. In some cases, the sample may include no more than 20,000 cells. In some cases, the sample may include at least 10,000 cells. In some cases, the sample may include at least 20,000 cells. In some cases, the sample may include at least 30,000 cells. In some cases, the sample may include at least about 40,000 cells. In some cases, the sample may include about 10,000 cells to about 50,000 cells. In some cases, the sample may include about 20,000 cells to about 50,000 cells. In some cases, the sample may include about 30,000 cells to about 50,000 cells. In some cases, the sample may include about 40,000 cells to about 50,000 cells. In some cases, the sample may include about 10,000 cells to about 40,000 cells. In some cases, the sample may include about 10,000 cells to about 30,000 cells. In some cases, the sample may include about 10,000 cells to about 20,000 cells. In some cases, the sample may include about 20,000 cells to about 50,000 cells. In some cases, the sample may include about 20,000 cells to about 40,000 cells. In some cases, the sample may include about 20,000 cells to about 30,000 cells. In some cases, the sample may include about 30,000 cells to about 50,000 cells. In some cases, the sample may include about 30,000 cells to about 40,000 cells.

[0052] In various aspects of the methods herein, the stabilized sample may include cell nuclei. In some cases, the stabilized sample may include no more than 50,000 cell nuclei. In some cases, the sample may include no more than 40,000 cell nuclei. In some cases, the sample may include no more than 30,000 cell nuclei. In some cases, the sample may include no more than 20,000 cell nuclei. In some cases, the sample may include at least 10,000 cell nuclei. In some cases, the sample may include at least 20,000 cell nuclei. In some cases, the sample may include at least 30,000 cell nuclei. In some cases, the sample may include at least 40,000 cell nuclei. In some cases, the sample may include about 10,000 cell nuclei to about 50,000 cell nuclei. In some cases, the sample may include about 20,000 cell nuclei to about 50,000 cell nuclei. In some cases, the sample may include about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 40,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 10,000 nuclei to about 40,000 nuclei. In some cases, the sample may include about 10,000 nuclei to about 30,000 nuclei. In some cases, the sample includes about 10,000 nuclei to about 20,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 50,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 40,000 nuclei. In some cases, the sample may include about 20,000 nuclei to about 30,000 nuclei. In some cases, the sample can contain about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample can contain about 30,000 nuclei to about 40,000 nuclei.

[0053] Connect adjacently to create concatemers

[0054] Provided herein are compositions, systems and methods that allow for the use of proximity ligation to form concatemers. For example, a biological sample, such as a stabilized biological sample having a nucleic acid molecule complexed with a nucleic acid binding protein, can be contacted with a dendrimer to form a complex. In another example, the biological sample is stabilized by contacting with a dendrimer to form a complex. Next, the nucleic acid molecule can be cut into a plurality of segments, such as at least a first segment and a second segment. Then, the plurality of segments can be attached at a plurality of splicing sequences, such as a first segment and a second segment can be attached at a splicing sequence.

[0055] In certain aspects of the methods herein, a biological sample, such as a stabilized biological sample having a nucleic acid molecule complexed with a nucleic acid binding protein and a dendrimer. In some cases, the dendrimer is conjugated to psoralen. In some cases, the dendrimer is conjugated to azido-Peg4-N-hydroxysuccinimide (NHS) ester. In some cases, the NHS ester of the azido-Peg4-NHS ester reacts with a primary amine on the dendrimer to produce a dendrimer with a reactive azide group. In some cases, carboxylated beads (e.g., magnetic beads) are prepared by conjugating with dibenzocyclooctyne-amine (DBCO)-Peg4-amine building blocks using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) / sulfoNHC chemistry. These prepared beads can be used to separate dendrimers, such as by magnetic separation methods before proximity ligation.

[0056] In some cases, the dendrimer is modified with a compound or contacted with a compound. For example, in some cases, the dendrimer is modified with psoralen. In some cases, the psoralen comprises N-hydroxysuccinimide (NHS) ester-conjugated psoralen. In some cases, the dendrimer comprises a polyamidoamine (PAMAM) dendrimer. In some cases, the dendrimer is modified with a cross-linking agent such as nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis(2-chloroethyl)ethylamine, bis(2-chloroethyl)methylamine, tris(2-chloroethyl)amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, cicycloplatin, epplatin, lobaplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin, or idarubicin. In some cases, the dendrimer is modified with an intercalator, antibiotic, or minor groove binder.

[0057] The methods herein may include uncoupling a compound from a dendrimer. For example, heat may be used to uncouple a compound such as psoralen from a dendrimer. In some cases, alkaline conditions or high pH are used to uncouple a compound such as psoralen from a dendrimer. Alternatively, heat and alkaline conditions are used to uncouple a compound such as psoralen from a dendrimer. The compound such as psoralen may also be uncoupled from a dendrimer using UV radiation.

[0058] Any suitable dendrimer can be used in the methods herein. The dendrimer can have a molecular weight of about 5 kilodaltons (kDa) to about 125 kDa. In some cases, the dendrimer has a molecular weight of 6 kDa to 8 kDa. In some cases, the dendrimer has a molecular weight of 25 kDa to 35 kDa. In some cases, the dendrimer has a molecular weight of 110 kDa to 125 kDa. In some cases, the dendrimer comprises 32 to 512 reactive groups. In some cases, the dendrimer comprises about 32 reactive groups. In some cases, the dendrimer comprises about 128 reactive groups. In some cases, the dendrimer comprises about 512 reactive groups. In some cases, the dendrimer is a Gen3 dendrimer. In some cases, the dendrimer is a Gen5 dendrimer. In some cases, the dendrimer is a Gen7 dendrimer.

[0059] The method herein can cause at least a portion of the sections to be connected into concatemers. For example, at least two sections, at least three sections, at least four sections, at least five sections, at least six sections, at least seven sections, at least eight sections, at least nine sections, at least ten sections or more can be attached to form concatemers. In some cases, the oligonucleotide is attached between each section. In some cases, the oligonucleotide is a bridging oligonucleotide. In some cases, the oligonucleotide is an adapter oligonucleotide. In some cases, the oligonucleotide is a punctuation oligonucleotide. In some cases, the bridging oligonucleotide, the adapter oligonucleotide and / or the punctuation oligonucleotide include a barcode sequence. In some cases, the bridging oligonucleotide, the adapter oligonucleotide and / or the punctuation oligonucleotide are partially modified with dibenzocyclooctyne (DBCO). In some cases, the DBCO part promotes copper-free click chemistry. In some cases, multiple oligonucleotides are attached in series between each section. Attachment can cause a sample, cell, nucleus, chromosome or nucleic acid molecule of a stabilizing biological sample to receive a unique sequence of an oligonucleotide (bridging oligonucleotide).

[0060] In some cases, after the dendrimer is contacted with the stabilized biological sample to form a complex, the complex is photoactivated, for example by exposing the complex to UV radiation having a wavelength of about 360 nm, thereby generating a cross-linked complex. In some cases, the cross-linking is reversible and does not leave adducts on the nucleic acid.

[0061] The method herein may further comprise size-selecting the plurality of segments to obtain a plurality of selected segments. The size selection herein may comprise any suitable range of segment sizes.

[0062] The cleavage in the methods provided herein can be performed using any suitable method, such as by using a nuclease or deoxyribonuclease (DNase). In some cases, the DNase includes DNase I, DNase II, micrococcal nuclease, restriction endonuclease or a combination thereof.

[0063] The stabilized biological sample in the method herein can be stabilized by treatment with a stabilizer or a cross-linking agent. In some cases, the cross-linking agent is a chemical fixative, such as formaldehyde, psoralen, disuccinimidyl glutarate (DSG), ethylene glycol bis (succinimidyl succinate) (EGS), ultraviolet light or a combination thereof. In some cases, the cross-linking agent comprises nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolomide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. The stabilization biological sample can be a paraffin-embedded tissue sample cross-linked. In some cases, the stabilization biological sample comprises a stabilization intact cell or a stabilization intact cell nucleus. In some cases, the method comprises lysing cells and / or cell nuclei in the stabilized biological sample.The cleavage step of the methods herein can be performed prior to lysis of intact cells or intact cell nuclei.

[0064] The methods herein can be performed on a stabilized biological sample comprising a small number of cells. For example, in some cases, the stabilized biological sample comprises less than about 3,000,000 cells. The stabilized biological sample may comprise less than about 1,000,000 cells, less than about 500,000 cells, less than about 400,000 cells, less than about 300,000 cells, less than about 200,000 cells, less than about 100,000 cells or less.

[0065] In aspects of the methods herein, the method may further include obtaining at least some sequences on each side of the spliced ​​sequence to generate a first read pair. In addition, the method may further include mapping the first read pair to a set of overlapping groups; and determining a path through the set of overlapping groups, the path representing the order and / or orientation to the genome. Alternatively or in combination, the method may include mapping the first read pair to a set of overlapping groups; and determining the presence of structural variants or loss of heterozygosity in the stabilized biological sample from the set of overlapping groups. Alternatively or in combination, the method may further include mapping the first read pair to a set of overlapping groups; and assigning variants in the set of overlapping groups to phases. Alternatively or in combination, the method may further include mapping the first read pair to a set of overlapping groups; determining the presence of variants in the set of overlapping groups from the set of overlapping groups; and performing one or more steps selected from the following: identifying the disease stage, prognosis or course of treatment of the stabilized biological sample; selecting drugs based on the presence of variants; or identifying the efficacy of drugs for the stabilized biological sample.

[0066] In aspects of the methods herein, proximity ligation can be performed with click chemistry, including copper-free click chemistry, such as with a DBCO-modified bridging oligonucleotide connected between each segment of the concatemer. The concatemers can then be connected, for example, by dendrimers. In order to enrich the connected molecules, the characteristics of the bridging oligonucleotide can be targeted. In one example, the DBCO containing oligonucleotide can be reacted with an azide-biotin moiety, which can be separated with a streptavidin substrate such as beads. In another example, the DBCO containing oligonucleotide can be reacted with an azide-modified NHS-SS-dPEG4-biotin containing a disulfide bond; azide can be added to NHS-SS-dPEG4-biotin using azido-PEG3-amine, and the disulfide bond can be reduced, for example, by using DTT and heating to separate nucleic acids for library preparation, such as 70°C for about 10 minutes.

[0067] In aspects of the methods herein, prior to proximity ligation of nucleic acid fragments, the dendrimers with nucleic acid fragments in contact therewith can be separated or isolated from the rest of the nucleic acids in the sample. This step can ensure that the concatemers formed by proximity ligation contain fragments in contact with the same dendrimer. This can mean that all fragments of a given concatemer are close to each other in the original stabilized sample. Thus, this method is not just pairwise information about which nucleic acid regions are adjacent to which other regions, but can also generate more complex proximity information - for example, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid regions are all adjacent to each other.

[0068] In some cases, dendrimers with nucleic acid fragments in contact therewith can be separated or isolated from the rest of the nucleic acid to enable barcoding or tagging of those fragments rather than proximity ligation. Fragments associated with a given dendrimer can be barcoded or tagged—for example, in a droplet or well. After sequencing, the sequences can be associated based on their barcodes, and proximity information can be derived based on the barcodes rather than based on presence in the same concatemer as described above. This proximity information can then be used as discussed herein. In one example, dendrimers are complexed with nucleic acids in a sample, thereby stabilizing them; the nucleic acids are then fragmented; the dendrimers and their complexed nucleic acid fragments are then separated and encapsulated in droplets; the nucleic acids in the droplets are labeled with droplet-specific barcodes or tags; the nucleic acids are then sequenced, using the barcode or tag information to associate fragments that are adjacent to each other in the sample.

[0069] Long noncoding RNA analysis

[0070] Provided herein is a method for analyzing long non-coding RNA binding sites. In some cases, such methods include obtaining a stabilized biological sample, the stabilized biological sample comprising a DNA molecule compounded with at least one nucleic acid binding protein and at least one non-coding RNA. Next, the method may include contacting the DNA molecule with a Tn5 transposase and an oligonucleotide comprising a chimeric end and a detectable label, thereby fragmenting the DNA molecule and attaching the oligonucleotide to the end of the fragmented DNA molecule. The fragment may then be contacted with a T4 RNA ligase, thereby connecting the non-coding RNA to the oligonucleotide, and reversing cross-linking. The connected RNA may then be extended with a reverse transcriptase to produce a double-stranded DNA fragment. The double-stranded DNA fragment may then be contacted with an endonuclease connected to a reagent to digest the DNA near the detectable label, and the reagent may bind the detectable label. Sequencing adapters may then be attached to create a sequencing library. In some cases, one end of the oligonucleotide is adenylated to facilitate connection to the non-coding RNA. In some cases, the oligonucleotide further comprises a barcode. In some cases, the sample stabilization biological sample is contacted with RNase H before the transposase is treated.

[0071] In aspects of the methods herein, in some cases, the non-coding RNA is a long non-coding RNA. In some cases, the non-coding RNA is an enhancer RNA. In some cases, the non-coding RNA is a miRNA. In some cases, the non-coding RNA is a gamma RNA. In some cases, the non-coding RNA is RNase P. In some cases, the non-coding RNA is a piRNA. In some cases, the non-coding RNA is Xist.

[0072] In aspects of the methods herein, in some cases, the detectable label comprises a modified nucleotide capable of a click chemistry reaction. In some cases, the detectable label comprises biotin. In some cases, the reagent comprises an antibody, protein A, protein G, or streptavidin. In some cases, the DNA attached to the non-coding RNA is enriched prior to further analysis.

[0073] In aspects of the methods herein, an endonuclease is used to cleave the foreign sample DNA prior to analysis. In some cases, the endonuclease comprises DNase I, DNase II, micrococcal nuclease, restriction endonuclease, or a combination thereof.

[0074] In aspects of the methods herein, the sequence of a double-stranded DNA fragment containing a non-coding RNA is obtained. Any suitable sequencing method may be used, including the methods further described herein.

[0075] Various suitable stabilized biological samples are contemplated for use in the methods herein. Stabilized biological samples (described in more detail elsewhere herein) have been crosslinked with a crosslinking agent (e.g., a fixative) or with UV light. For example, in some cases, the stabilized biological sample is a crosslinked paraffin-embedded tissue sample. In some cases, the stabilized biological sample comprises a stabilized cell lysate. In some cases, the stabilized biological sample comprises a stabilized intact cell. In some cases, the stabilized biological sample comprises a stabilized intact cell nucleus.

[0076] Nucleic acid conformation assessment

[0077] Disclosed herein are compositions, systems and methods for determining the physical conformation of nucleic acids in cells (such as single cells or cell groups) that are distinguished from the physical conformation of a second cell or cell group. Through the practice of the disclosure herein, nucleic acid molecules indicating the relative position of three-dimensional nucleic acids and optionally having a label (e.g., a nucleic acid barcode) can be generated to distinguish the common cells or source populations of multiple molecules.

[0078] By the practice of the disclosed method herein, nucleic acids can be obtained so as to retain all or at least some of their three-dimensional configurations in cells. The exposed nucleic acid loops of such nucleic acids can be cut to expose the internal segment ends, which are randomly reattached to each other, making it more likely that the exposed ends physically adjacent will be attached to each other (adjacent attachment). Therefore, by determining which exposed ends will be attached to each other, data information about the physical proximity of the adjacent nucleic acids at the ends in the native cell conformation can be obtained.

[0079] For example, related methods are disclosed in US9434985B2 published by Dekker et al. on September 6, 2016, which is incorporated herein by reference in its entirety.

[0080] By the practice of the method disclosed herein, the paired end library components can be further labeled or otherwise provided with sequence information of the cell indicating the source, so that the conformational differences between the individual cells in the cell population can be easily distinguished for the cell population, or even when the analysis is performed simultaneously, the conformational differences between the first cell population and the second cell population can be easily distinguished. The label can include, for example, a nucleic acid barcode. In some cases, the label can include a splicing sequence between two discontinuous nucleic acid segments in the genome. Nucleic acid molecules can be generated so that when all or part of the sequencing is performed, at least some genome sequences sufficient to map each genome end to its genome site are usually obtained, and further obtained to be sufficient to identify the labeling or connection sequence of the accurate or possible source cell or cell population. Therefore, sequence information about two genome regions physically close to each other is obtained, and information about the cell or cell population in which this physical conformation occurs is also obtained, so that it can be evaluated in the context of other physical conformation information co-occurring in the cell or cell population.

[0081] The genome or other nucleic acid in the cell can be stabilized and, for eukaryotic cells, the nucleus is optionally isolated according to known methods, such as those incorporated herein or otherwise known.

[0082] Nucleic acids consistent with the disclosure herein include any number of cellular nucleic acids, such as prokaryotic primary genome or plasmid nucleic acids, eukaryotic nuclear, mitochondrial or plastid nucleic acids, or in some cases cytoplasmic nucleic acids, such as rRNA, mRNA, or exogenous nucleic acids in a sample, such as viruses or other pathogens or other exogenous nucleic acids to the sample.

[0083] In some cases, the stabilized nucleic acids can be distributed so that at least some of the nucleic acids are distributed in separate partitions. Exemplary partitions include droplets in wells, emulsions, or surface locations (e.g., array spots, beads, etc.) that contain different patches of differential sequencing adapter molecules as described elsewhere herein. Additional partitions are also contemplated and are consistent with the methods, compositions, and systems disclosed herein.

[0084] Stabilized nucleic acid can be fragmented to expose internal fractures for later reconnection, thereby obtaining nucleic acid configuration information of specific cells. Many fragmentation methods are known and consistent with the disclosure herein. Restriction endonucleases, programmable endonucleases (e.g., CRISPR / Cas molecules coupled to guide RNA), non-specific endonucleases (e.g., DNase), labeling, shearing, ultrasonic treatment, heating or other mechanisms can be used. One or more groups are used to fragment nucleic acids. In some cases, DNase is non-sequence specific. In some cases, DNase is active to both single-stranded DNA and double-stranded DNA. In some cases, DNase is specific to double-stranded DNA. In some cases, DNase is preferred over double-stranded DNA. In some cases, DNase is specific to single-stranded DNA. In some cases, DNase is preferred over single-stranded DNA. In some cases, DNase is DNase I. In some cases, DNase is DNase II. In some cases, DNase is selected from one or more of DNase I and DNase II. In some cases, DNase is micrococcal nuclease. In some cases, the DNase is selected from one or more of DNase I, DNase II, and micrococcal nuclease. Other suitable nucleases are also within the scope of the present disclosure.

[0085] In particular, the disclosure of WO2014121091A1 published by Green et al. on August 7, 2014 (later published as US20150363550A1 on December 17, 2015 and as US10089437B2 on October 2, 2018) is incorporated herein in its entirety. Similarly, the disclosure of WO2016019360A1 published by Fields et al. on February 4, 2016 (later published as US20170335369A1 on November 23, 2017) is incorporated herein in its entirety. Similarly, the disclosure of WO2017147279A1 published by Green et al. on August 31, 2017 is incorporated herein in its entirety.

[0086] The nucleic acid can be bound to the surface before or after attachment. Exemplary surfaces include, but are not limited to, beads, arrays, and wells. In some cases, the surface is a solid phase reversible immobilization (SPRI) surface, such as SPRI beads. Binding the nucleic acid to the surface before attachment can improve the performance of downstream steps, such as reducing interchromosomal connections or attachments and increasing intrachromosomal connections or attachments.

[0087] The nucleic acid can be immunoprecipitated before or after attachment. Such a method may include fragmenting the chromatin and then contacting the fragments with antibodies that specifically identify and bind to acetylated histones (particularly H3). Examples of such antibodies include, but are not limited to, anti-acetylated histone H3, available from Upstate Biotechnology, Lake Placid, NY. Polynucleotides from the immunoprecipitate can then be collected from the immunoprecipitate. Similar targeted enrichment methods can also be used for target-specific compounds, including but not limited to aptamers, oligonucleotides or other nucleic acid probes and nucleic acid-guided nucleases (e.g., Cas family enzymes, such as Cas9, including catalytically inactive or "dead" nucleases).

[0088] A linked nucleic acid (such as a linked nucleic acid having a barcode, partition-specific sequence, or partition identification sequence) can be attached to the exposed internal end to produce a nucleic acid segment having a left genomic segment, a linked region typically having a partition-specific or partition identification sequence (e.g., a nucleic acid barcode), and a right genomic segment, wherein the left genomic segment and the right genomic segment map to physically adjacent genomic segments in the source cell.

[0089] Before attaching the exposed nucleic acid ends, the ends can be processed. Such processing can include end polishing or blunt end. For example, the exposed nucleic acid ends of the blunt end can be directly connected to other exposed nucleic acid ends of the blunt end or connected to an adapter or a joint. Such processing can include, for example, generating an overhang by tailing (for example, adding an A tail or adenylation). In one example, the size of the overhang is one nucleotide. In one example, the overhang is a single A nucleotide. For example, the exposed nucleic acid ends of the tailing can be directly connected to other exposed nucleic acid ends of the tailing or connected to an adapter or a joint. In some cases, the nucleic acid of the blunt end or tailing can be incorporated into an affinity tag, such as a biotinylated nucleic acid. Affinity tags can be used for example for downstream capture or enrichment steps. In other cases, blunt end or tailing can be performed in the case of a nucleic acid without incorporating an affinity tag (for example, without biotinylated nucleic acid). If desired, an affinity tag can be subsequently added to, for example, an adapter or a joint (for example, bridging). In one example, the exposed nucleic acids are end-finished to generate overhangs, and the exposed ends are attached via bridging oligos.

[0090] The attachment may be direct, such as via a connection.

[0091] Attachment can be via linkers or bridges, such as by linking one or more linkers or bridge nucleic acids connecting one exposed nucleic acid end to another linker or bridge nucleic acid.

[0092] Attachment can be performed by using, for example, capped nucleic acid adaptor segments coincident with recombinase incorporation, such as integrase or transposase incorporation. Adaptors with recombinase sites can be added to exposed nucleic acid ends, and those ends can then be joined, for example, by recombination.

[0093] by Taking integrase barcode delivery as an example, linkers, such as cell-identifying or cell-specific linkers (eg, nucleic acid barcodes), can be enzymatically added as follows.

[0094] After exposing the internal nucleic acid termini, an integrase site can be attached to the exposed nucleic acid termini, such as the internal termini or exposed linear chromosome termini (such as chromosome termini from which telomeres have been removed). An exemplary integration site is attP Integrase integration site or nucleic acid comprising attP integration site, although other integration sites are consistent with the disclosure herein. Ligation produces a population of nucleic acid fragments, at least some of which individually comprise segments of cellular nucleic acid bounded at each end by integration sites, such as segments comprising attP segments. In various embodiments, one or both of fragmentation and integration site attachment occurs before partitioning, or one or both of fragmentation and integration site attachment occurs after partitioning.

[0095] Alternatively, transposases (such as Tn3, Tn5, Tn7 or Sleeping Beauty transposases) can be used for barcode delivery. After exposing the internal nucleic acid ends, the chimeric ends can be connected to the exposed nucleic acid ends, such as the internal ends or the exposed linear chromosome ends (such as the chromosome ends from which the telomeres have been removed). Exemplary chimeric ends are Tn5 chimeric ends or nucleic acids comprising Tn5 chimeric ends, although other chimeric ends are consistent with the disclosure herein. The connection produces a population of nucleic acid fragments, at least some of which are individually contained in cellular nucleic acid segments bounded by chimeric ends (such as Tn5 chimeric ends) at each end.

[0096] In various embodiments, one or both of fragmentation and chimeric end attachment occurs before partitioning, or one or both of fragmentation and chimeric end attachment occurs after partitioning. In an exemplary system for single-cell HiC (or other proximity ligation techniques), integrase-mediated intra-aggregate ligation is used. A single cell nucleus is encapsulated in a first set of partitions together with an integrase. In this case, the partition is a droplet in an emulsion. The cell nucleus is subjected to chain breakage to produce an internally exposed end and retain local three-dimensional information. An adapter is connected to the exposed internal end. The adapter optionally includes an exonuclease-resistant end. In this embodiment, the adapter does not convey partition-distinguishing information. In the second set of partitions, a connector having a partition-distinguishing sequence such as a unique molecular identifier (UMI) is encapsulated and optionally amplified and cut-oriented linearization is performed. The first and second sets of partitions are merged at a ratio of approximately 1: 1, or merged under conditions where nucleic acids from two cells are unlikely to merge into a single resulting partition.

[0097] In some cases, recombinase sites such as integrase sites or chimeric ends can be carried on unmodified single-stranded or double-stranded fragments to be connected to internal nucleic acid ends. Alternatively, to facilitate subsequent sequencing library purification (clean-up), some single-stranded or double-stranded fragments containing integration sites (such as attP sequences) or chimeric ends (such as Tn3, Tn5, Tn7 or Sleeping Beauty transposase chimeric ends) can include at least one modification (such as a modification that interferes with nuclease exonuclease or other nucleic acid degradation activity). Examples include thiosulfate modifications for preventing exonuclease degradation of fragments, in which double-stranded fragments containing integration sites are added to each end.

[0098] Typically, recombinase sites (such as integration sites or chimeric ends) are non-specific because the sequences in such integration sites or chimeric ends (such as attP sequences or Tn3, Tn5, Tn7 or Sleeping Beauty transposase chimeric ends) are not used to specify the cellular origin of the adjacent nucleic acid. Alternatively, typically after partitioning the nucleic acid, the partitions can be provided with adapters having different, specific or cell-distinguishing sequences (e.g., nucleic acid barcodes) adjacent to the integration site or chimeric end, or different integration sites or chimeric ends can be provided, such that the nucleic acid of the first partition receives an integration segment or chimeric end having a first identification segment, while the nucleic acid segments of the second partition receive an integration segment having a second identification segment.

[0099] Fragments with recombinase borders (such as borders containing an integrase attP segment) can then be combined with a recombinase site (such as attB) in a common solution. In one example, the integrase may include Integrase, the integration borders may comprise attP segments, and the integration site may comprise an attB integration site. Alternatively, the fragments have chimeric end borders, such as Tn3, Tn5, Tn7 or Sleeping Beauty transposase chimeric end borders.

[0100] When a recombinase site (such as an attB integration site or a Tn3, Tn5, Tn7 or Sleeping Beauty transposase chimeric end) is flanked by a joining segment having a sequence that identifies a partition or cell, such as a sequence that is specific to the segment or cell source (e.g., a nucleic acid barcode), the sequence identifies the adjacent cellular nucleic acid as originating from a specific or shared cellular source or partition, so that multiple exposed ends from a common cell joined by shared cell-distinguishing or partition-distinguishing segments can be easily identified as originating from a shared cell, even if they are populated with fragments of a second partition before or simultaneously with sequencing.

[0101] When the cell-distinguishing sequence is delivered via the recombinase site boundary fragment, integration or transposition is preferably performed after partitioning. The nucleic acid content of at least some partitions can thus be distinguished by the cell-distinguishing sequence of its adapter, so that even after nucleic acids from multiple cell sources are formed and used in large quantities for sequencing, internal end pairs (and neighboring information assigned to the vicinity to which they are mapped in the set of overlapping groups, until and including the genome that is mostly or completely sequenced) can be assigned to a common cell that is different from at least one other cell of the sample, so that the difference in the predicted three-dimensional conformation of the nucleic acid can be determined.

[0102] The fragments bordered by the recombination site variously include left and right border fragments (e.g., attB sites or Tn3, Tn5, Tn7, or Sleeping Beauty transposase chimeric ends), which are connected by a linker region that optionally includes a cell or partition specifying sequence (e.g., a nucleic acid barcode). The linker region optionally further includes a portion that facilitates subsequent separation. Many affinity tags or modified bases are consistent with the disclosure herein. After integrase or transposase treatment, the exemplary portion promotes physical or chemical separation of the linker. Any number of affinity tags are consistent with the disclosure herein, such as one or more biotin tags that can promote avidin or streptavidin-based separation. Alternatively, any antigen, receptor, or ligand that promotes separation without interfering with integrase or transposase activity is suitable for some embodiments herein.

[0103] As mentioned above, some library generation methods include purification steps, such as the step of selectively removing unbound reagents.For example, exonuclease treatment is generally used to selectively remove unattached linker molecules, unattached genomic fragments of integration sites or unattached linker molecules and unattached genomic fragments of integration sites.The genomic fragment connected with the integration site fragment with anti-exonuclease modification (such as thiosulfate backbone) can resist the exonuclease degradation from the end, and the nucleic acid molecules with bounds at both ends by the integration site fragment with exonuclease resistance modification (such as thiosulfate backbone) can resist degradation at both ends and can survive after exonuclease treatment.

[0104] Alternatively or in combination, some linker molecules include an anti-affinity tag on the opposite side of the recombination site (such as an attP integration site or a Tn3, Tn5, Tn7 or Sleeping Beauty transposase chimeric end), so that the anti-affinity tag is removed upon successful recombination reaction. In such a case, the unwanted reagent can be removed by contact with the binding partner of the anti-affinity tag.

[0105] As part of the integration event, integrase activity partially destroys two integration sites, such as attB and attP sites. Therefore, by designing primers to anneal to ligated adapter sites (such as attP integration sites) alone or in combination with linker-based separation, clonal amplicons can be generated that span at least one linker, thereby amplifying cell or aliquot distinguishing information and internal end-adjacent information, facilitating sequencing or other downstream analysis in some cases.

[0106] After library generation and optionally library purification, nucleic acid can be sequenced completely or partially to obtain enough information for cell differentiation or cell-specific three-dimensional nucleic acid position assessment. As mentioned above, preferably sequenced to obtain at least some genomic sequences that are enough to map each genome end of library components to its genomic site, and further obtain the connection sequence that is enough to identify accurate or possible source cells. Therefore, sequence information about two genomic regions physically close to each other is obtained, and information about the cell in which this physical conformation occurs is also obtained, so that it can be evaluated under the background of other physical conformation information co-occurring in the cell. Usually, this information is obtained by paired end sequencing rather than full-length sequencing, although these two methods and other methods are consistent with the disclosure of this article.

[0107] Compositions and methods related to the physical conformation of nucleic acids in a determined cell (such as a single cell that can be distinguished from a second cell in physical conformation) can be implemented on many systems consistent with the disclosure herein. Some systems include distributing fixed cell nucleic acid material in a first droplet of an emulsion or in a hole (e.g., on a well plate). These droplets further include a recombinase site, such as an integrase site or a chimeric end, optionally modified to an anti-nuclease exonuclease as described herein, and an integrase or transposase and a ligase. Separately, a linker nucleic acid molecule can be configured for delivery to the first droplet of an emulsion. The linker nucleic acid can be optionally distributed in a droplet or a second hole of a second emulsion and optionally amplified (e.g., using rolling circle amplification), and processed to produce multiple copies of a given linker molecule in each second emulsion droplet.

[0108] The second emulsion droplets can then be merged in pairs with the first emulsion droplets to assemble the integrase or transposase-ligated nucleic acid fragments with integrase or transposase-compatible adapters, typically with each droplet exhibiting uniform labeling. However, droplets with two or more identifiers per nucleic acid sample can still generate meaningful data, particularly when data analysis indicates that more than one type of tag is present in the droplet.

[0109] As an alternative to pairwise merging, in some cases, the integrase or transposase compatible adapters can be delivered as a colony of solid particles in a reagent stream that contacts the first emulsion droplets via droplets to the merging stream, such as described in US20170335369A1, published on November 23, 2017, which is incorporated herein by reference in its entirety. The adapter nucleic acid can optionally be amplified in a solid particle or gel. The first emulsion droplets can be merged into the stream, and the stream can be segmented or partitioned to recover the second emulsion droplets to obtain a desired ratio of nucleic acid clusters to adapter particles, such as 1:1, greater than 1:1, or less than 1:1.

[0110] Alternatively, some systems and methods involve distributing fixed cellular nucleic acid material into wells of a chip or plate and then delivering adaptor nucleic acid into the partitions, either unamplified or amplified as discussed above.

[0111] Alternatively, in some cases, the delivery of the linker nucleic acid is not temporarily separated from the partition. Instead, the linker nucleic acid or the enzymatic activity or the factor necessary for the enzymatic activity is isolated until a specific treatment (such as heating, electromagnetic activation or other application) is performed to temporarily activate the enzymatic activity, resulting in the linker being covalently bound to the nucleic acid sample (such as via a linker) at the exposed end.

[0112] Various integrases are consistent with the disclosure herein. Integrase (such as commercially available from ThermoFisher Integrase) exhibits many benefits to the practice of the methods, operation of the systems, and use of the compositions herein. Some of the benefits of the integrase are as follows. It uses a small integration site (attB / attP). The enzyme itself is a small single polypeptide. Integration is irreversible if a separate enzyme is not used to excise the integration event. The activity is high, and the enzyme is easily engineered to alter activity. Nevertheless, its use need not exclude other enzymes, as many integration systems are consistent with the disclosure herein. Aspects of the disclosure may be related to Integrase is described, but any compatible enzyme is contemplated for use.

[0113] A variety of transposases are consistent with the disclosure herein. Tn5 transposase (such as Tn5 transposase commercially available from Lucigen) exhibits many benefits to the practice of the methods herein, the operation of the systems, and the use of the compositions. Some of the benefits of this transposase are as follows: Tn5 uses a 19 bp chimeric end identification sequence, the insertion bias is small and stable, and Tn5 can be delivered to cells for in vivo transposition or delivered to isolated nucleic acids for in vitro reactions. Nevertheless, its use need not exclude other enzymes, as many transposase systems (such as Tn3, Tn7, or Sleeping Beauty transposases) are consistent with the disclosure herein. Aspects of the present disclosure may be described with respect to Tn3, Tn5, Tn7, or Sleeping Beauty transposases, but any compatible enzyme is contemplated for use.

[0114] Sequence information obtained from library components was evaluated by various methods, such as Hi-C, Chicago Methods in the context of in vitro proximity connection or other three-dimensional conformation analysis. Importantly, cell-specific read pairs can be obtained so that the frequency of terminal adjacent sequences mapped to a specific region of the genome or a specific overlapping group can be evaluated on the basis of cell specificity. That is, the cell-specific occurrence of possible three-dimensional conformations can be evaluated. In some cases, the cell-specific intensity of the signal can also be evaluated, which is related to the cell-specific distance in the three-dimensional conformation, so that it can be concluded that some regions of the nucleic acid have a relatively close proximity in one cell relative to the second cell, wherein they are quite but "weaker" or farther proximity, and there is no signal indicating proximity in the third cell. That is, the qualitative and quantitative evaluation of the three-dimensional configuration is consistent with the disclosure of this article. In some cases, the proximity of a region to a second region is evaluated at least in part by calculating the number of cluster components of the first cluster, which appear simultaneously with the cluster components of the second cluster in the paired end reading, particularly in the library components of the sequences (such as unique partition labels) that share a common partition distinction.

[0115] Conformation information need not be obtained by multiple occurrences of the same end-neighboring sequence in multiple library components. Rather, in some cases, when two members of a cluster are mapped to different regions of a second cluster on a second region of a nucleic acid reference (such as a genome), the end-neighboring sequence that maps to the second end-neighboring sequence mapping site (mapped to the common "cluster") can re-enhance the three-dimensional conformational assessment.

[0116] In some cases, the methods disclosed herein are used to label and / or associate polynucleotides or sequence segments thereof, and use the data for various applications. In some cases, the disclosure provides methods for generating a highly contiguous and accurate human genome assembly having less than about 10,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, about 1 million, about 2 million, about 5 million, about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 70 million, about 80 million, about 90 million, about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 700 million, about 800 million, about 900 million, or about 1 billion read pairs. In some cases, the disclosure provides methods for phasing or assigning physical linkage information for about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more heterozygous variants in a human genome with an accuracy of about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0117] In some embodiments, the compositions and methods described herein allow for the study of metagenomes, such as those found in the human intestine. Thus, partial or complete genome sequences of some or all organisms that inhabit a given ecological environment can be studied. Examples include random sequencing of all intestinal microorganisms, microorganisms found in certain areas of the skin, and microorganisms living in toxic waste sites. The composition of the microbial populations in these environments can be determined using the compositions and methods described herein and aspects of the associated biochemistry encoded by their respective genomes. The methods described herein can enable metagenomic studies from complex biological environments, for example, those environments containing more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 or more organisms and / or organism variants.

[0118] Thus, the methods disclosed herein can be applied to intact human genomic DNA samples, but can also be applied to a wide variety of nucleic acid samples, such as reverse transcribed RNA samples, circulating free DNA samples, cancer tissue samples, crime scene samples, archaeological samples, non-human genomic samples, or environmental samples (such as environmental samples that contain genetic information from more than one organism (such as organisms that are not easily cultured under laboratory conditions)).

[0119] The high accuracy required for cancer genome sequencing can be achieved using the methods and systems described herein. When sequencing cancer genomes, inaccurate reference genomes can present base recognition challenges. Heterogeneous samples and small starting materials, such as samples obtained by biopsy, introduce additional challenges. In addition, the detection of large-scale structural variants and / or loss of heterozygosity is often crucial for cancer genome sequencing, as well as the ability to distinguish somatic variants from base recognition errors.

[0120] The systems and methods described herein can generate accurate long sequences from complex samples containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more different genomes. Mixed samples of normal, benign and / or tumor origin can be analyzed (optionally without normal controls). In some embodiments, accurate long sequences are generated using only 100ng or even only hundreds of genome equivalent starting samples. The systems and methods described herein can allow for the detection of large-scale structural variants and rearrangements, and phased variant recognition can be obtained on long sequences of nucleotides spanning about 1kbp, about 2kbp, about 5kbp, about 10kbp, about 20kbp, about 50kbp, about 100kbp, about 200kbp, about 500kbp, about 1Mbp, about 2Mbp, about 5Mbp, about 10Mbp, about 20Mbp, about 50Mbp or about 100Mbp or more. For example, phase variant recognition can be obtained on long sequences spanning about 1Mbp or about 2Mbp.

[0121] In certain aspects, the methods disclosed herein are used to assemble multiple overlapping groups derived from a single DNA molecule. In some cases, the method includes generating multiple read pairs from a single DNA molecule cross-linked with multiple nanoparticles and assembling overlapping groups using the read pairs. In some cases, a single DNA molecule is cross-linked outside a cell. In some cases, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the read pairs are cross-linked on a single DNA molecule. A distance greater than 1 kB, 2 kB, 3 kB, 4 kB, 5 kB, 6 kB, 7 kB, 8 kB, 9 kB, 10 kB, 15 kB, 20 kB, 30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, 100 kB, 150 kB, 200 kB, 250 kB, 300 kB, 400 kB, 500 kB, 600 kB, 700 kB, 800 kB, 900 kB or 1 MB. In some cases, at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the read pairs span a distance of greater than 5kB, 6kB, 7kB, 8kB, 9kB, 10kB, 15kB, 20kB, 30kB, 40kB, 50kB, 60kB, 70kB, 80kB, 90kB, 100kB, 150kB, or 200kB on a single DNA molecule. In further cases, at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% of the read pairs span a distance greater than 20 kB, 30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, or 100 kB on a single DNA molecule. In specific cases, at least 1% or 5% of the read pairs span a distance greater than 50 kB or 100 kB on a single DNA molecule. In some cases, the read pairs are generated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or 60 days. In some cases, the read pairs are generated within 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In further cases, the read pairs are generated within 7, 8, 9, 10, 11, 12, 13, or 14 days. In specific cases, the read pairs are generated within 7 or 14 days.

[0122] Haplotypes determined using the methods and systems described herein can be assigned to computing resources, such as computing resources on a network, such as a cloud system. If necessary, relevant information stored in the computing resources can be used to correct short variant identification. Structural variants can be detected based on the combined information from short variant identification and the information stored in the computing resources. Problematic parts of the genome, such as segmental duplications, regions prone to structural variation, highly variable and medically relevant MHC regions, centromere and telomeric regions, and other heterochromatin regions (including those heterochromatin regions with repeated regions, low sequence accuracy, high mutation rates, ALU repeats, segmental duplications), or any other relevant problematic parts, can be reassembled to improve accuracy.

[0123] Sample types can be assigned to sequence information in local or networked computing resources (such as the cloud). Where the source of the information is known, for example, when the source of the information is from cancer or normal tissue, the source can be assigned to the sample as part of the sample type. Other sample type examples typically include, but are not limited to, tissue type, sample collection method, presence of infection, type of infection, treatment method, size of the sample, etc. Where all or part of a comparative genome sequence is available, such as a comparison of a normal genome with a cancer genome, the difference between the sample data and the comparative genome sequence can be determined and optionally output.

[0124] The method of the present disclosure can be used to analyze the genetic information of the selective genomic region of interest and the genomic region that can interact with the selective region of interest. The amplification method disclosed herein can be used in the device, kit and method of genetic analysis, such as but not limited to those found in U.S. Patent Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117, 6,225,109 and 6,110,709. In some cases, the amplification method of the present disclosure can be used to amplify the target nucleic acid for DNA hybridization research to determine the presence or absence of polymorphism. Polymorphism or allele can be associated with a disease or condition such as a genetic disease. In some other cases, polymorphism can be associated with susceptibility to a disease or condition, for example, polymorphisms associated with addiction, degenerative and age-related conditions, cancer, etc. In other cases, polymorphisms can be associated with beneficial traits, such as enhanced coronary artery health, or resistance to diseases such as HIV or malaria, or resistance to degenerative diseases such as osteoporosis, Alzheimer's disease, or dementia.

[0125] The compositions and methods of the present disclosure can be used for diagnosis, prognosis, treatment, patient stratification, drug development, treatment selection and screening purposes. The present disclosure provides an advantage that many different target molecules can be analyzed from a single biomolecule sample at one time using the methods of the present disclosure. For example, this allows multiple diagnostic tests to be performed on one sample.

[0126] The method provided herein can greatly advance the field of genomics by overcoming the substantial obstacles caused by these repeat regions, and thus can make important progress in many fields of genome analysis. To assemble from scratch using previous technology, it is necessary to either be satisfied with assembling fragments into many small scaffolds, or invest a lot of time and resources to produce large insertion libraries or use other methods to generate more continuous assemblies. Such methods can include obtaining very deep sequencing coverage, building BAC or Fox plasmid libraries, optical mapping, or most likely some combinations of these and other technologies. A large amount of resources and time requirements make it impossible for most small laboratories to use such methods, and prevent the study of non-model organisms. Because the method described herein can produce a very long range of read sets, it is possible to realize de novo assembly by a single sequencing run. This reduces the assembly cost by several orders of magnitude, and shortens the required time from months or years to weeks. In some cases, the methods disclosed herein allow for the generation of multiple read sets in less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, or in a range between any two of the above specified time periods. In some cases, the methods allow for the generation of multiple read sets in about 10 to 14 days. Genome construction will become routine even for organisms of the smallest ecological niches, phylogenetic analyses will not lack for comparison, and projects such as Genome 10k can be realized.

[0127] The methods described herein allow previously provided, previously generated or de novo synthesized contig information to be assigned to physical linkage groups (such as chromosomes or shorter continuous nucleic acid molecules). Similarly, the methods disclosed herein allow the contigs to be positioned relative to each other in linear order along the physical nucleic acid molecules. Similarly, the methods disclosed herein allow the contigs to be oriented relative to each other in linear order along the physical nucleic acid molecules.

[0128] Similarly, the methods disclosed herein can provide progress in structural and phasing analysis for medical purposes. There is surprising heterogeneity between cancers, individuals with the same type of cancer, or even within the same tumor. Sorting out the cause effects from the result effects requires very high precision and throughput at low cost for each sample. In the field of personalized medicine, one of the gold standards for genomic therapy is a sequenced genome with all variants of complete characterization and phasing, including large and small structural rearrangements and novel mutations. In order to achieve this standard with previous technology, efforts similar to those required for de novo assembly are needed, which are currently too expensive and laborious to become routine medical procedures. In some cases, the methods disclosed herein quickly produce complete and accurate genomes at low cost, and thus produce many highly sought-after capabilities in the research and treatment of people's diseases.

[0129] In addition, applying the methods disclosed herein to phasing can combine the convenience of statistical methods with the accuracy of family analysis, providing more savings (money, labor and samples) than using either method alone. De novo variant phasing is a highly desirable phasing analysis that is prohibited by previous technologies and can be easily performed using the methods disclosed herein. This is particularly important because the vast majority of human variants are rare (minor allele frequency is less than 5%). Relative to non-joined genotypes, phasing information is valuable for population genetic studies, which gain significant advantages from highly connected haplotype networks (sets of variants assigned to a single chromosome). Haplotype information can enable higher resolution studies of historical changes in population size, migration, and inter-subpopulation exchanges, and enables us to trace specific variants back to specific parents and grandparents. This in turn clarifies the genetic transmission of variants associated with the disease, as well as the interactions of variants when they are together in a single individual. In further cases, the methods of the present disclosure are capable of preparing, sequencing and analyzing extremely long range read sets (XLRS) or extremely long range read pairs (XLRP) libraries.

[0130] In some embodiments of the present disclosure, a tissue or DNA sample from a subject is provided, and the method returns an assembled genome, an alignment with identified variants (including large structural variants), phased variant calls, or any other analysis. In other embodiments, the methods disclosed herein provide an XLRP library directly to an individual.

[0131] In various embodiments, method disclosed herein generates extremely long ranges separated by large distances and reads pairs. The upper limit of this distance can be improved by the ability to collect large-size DNA samples. In some cases, read to span 50,60,70,80,90,100,125,150,175,200,225,250,300,400,500,600,700,800,900,1000,1500,2000,2500,3000,4000,5000kbp or more genome distance. In some cases, read to span the genome distance of 500kbp at most. In other cases, read to span the genome distance of 2000kbp at most. Method disclosed herein can be integrated and constructed based on the standard techniques in molecular biology, and is further very suitable for improving efficiency, specificity and genome coverage. In some cases, the read pairs are generated in less than about 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, 60, or 90 days. In some cases, the read pairs are generated in less than about 14 days. In further cases, the read pairs are generated in less than about 10 days. In some cases, the methods of the present disclosure provide greater than about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% read pairs in correctly ordering and / or orienting multiple contigs with an accuracy of at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%. In some cases, the methods provide about 90% to 100% accuracy in correctly ordering and / or orienting multiple contigs.

[0132] In other embodiments, the methods disclosed herein are used together with currently used sequencing technologies. In some cases, the methods are used in combination with well-tested and / or widely deployed sequencing instruments. In further embodiments, the methods disclosed herein are used together with techniques and methods derived from currently used sequencing technologies.

[0133] Method disclosed herein can significantly simplify the de novo genome assembly of numerous organisms.Using previous technology, such assembly is currently limited to the short insertion of economical paired (mate-pair) libraries.Although it is possible to generate read pairs on the genome distance of up to 40-50kbp accessible to Fox plasmid, this is expensive, cumbersome, and too short to span the longest repeat extension, including repeat extension in centromere, which is 300kbp to 5Mbp in size in people.In some cases, method disclosed herein provides read pairs that can span large distances (e.g., megabases or longer), thereby overcoming these scaffold integrity challenges.Therefore, by utilizing method disclosed herein, it can be conventional to produce chromosome-level assembly.Similarly, obtaining long-range phasing information can provide huge additional power for population genome, phylogeny and disease research.In some cases, method disclosed herein can accurately phase a large number of individuals, thereby expanding the breadth and depth of our ability to detect genomes at population and deep time levels.

[0134] In the field of personalized medicine, the XLRS read sets generated from the methods disclosed herein represent a meaningful step towards accurate, low-cost, phased, and rapidly generated personal genomes. Previous methods have fallen short in their ability to phase variants over long distances, preventing the characterization of phenotypic effects of compound heterozygous genotypes. In addition, structural variants of major significance for genomic disease have been difficult to accurately identify and characterize with previous techniques due to their large size compared to the reads and read inserts used to study them. Read sets spanning tens of thousands of bases to millions of bases or longer can help alleviate this difficulty, allowing for highly parallel and personalized analysis of structural variants.

[0135] The progress of high-throughput sequencing technology can promote basic evolution and biomedical research. The cost of generating a large amount of DNA sequence data is relatively low now. However, it is difficult to produce high-quality, highly continuous genome sequences with previous technologies in theory and practice. In addition, many organisms including people are diploid, and each individual has two haploid copies of genome. At the site of heterozygosity (for example, the allele provided by the mother is different from the allele provided by the father), it is difficult to know which group of alleles comes from which parent (called haplotype phasing). This information is crucial for performing many evolutionary and biomedical studies (such as disease and trait association studies).

[0136] The present disclosure provides a method for genome assembly, which combines DNA preparation technology with labeled sequence reading to find short-term, medium-term and long-term connections corresponding to sequence readings from single physical nucleic acid molecules bound to complexes (such as chromatin complexes within a given genome) at high throughput. The present disclosure further provides methods for using these connections to assist genome assembly, for haplotype phasing and / or for metagenomic research. Although the methods proposed herein can be used to determine the assembly of a subject's genome, it should also be understood that in some cases, the methods proposed herein can be used to determine the assembly of a portion of a subject's genome (such as a chromosome), or the assembly of chromatin of different lengths of a subject. It should also be understood that in some cases, the methods proposed herein are used to determine or guide the assembly of non-chromosomal nucleic acid molecules. In fact, the methods disclosed herein can be used to promote any nucleic acid that is complicated by the presence of repeated regions that separate non-repetitive overlapping groups.

[0137] In further cases, the methods disclosed herein allow accurate and predictive results for genotype assembly, haplotype phasing, and metagenomics to be obtained using small amounts of material. In some cases, the methods disclosed herein use less than about 100 picograms (pg), about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1.0 nanogram (ng), about 2.0 ng, about 3.0 ng, about 4.0 ng, about 5.0 ng, about 6.0 ng, about 7.0 ng, about 8.0 ng, about 9.0 ng, about 10 ng, about 15 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 90 ng, about 100 ng, about 200 ng, about 300 ng, about 400 ng, about 500 ng, about 600 ng, about 700 ng, about 800 ng g, about 900ng, about 1.0 microgram (μg), about 1.2μg, about 1.4μg, about 1.6μg, about 1.8μg, about 2.0μg, about 2.5μg, about 3.0μg, about 3.5μg, about 4.0μg, about 4.5μg, about 5.0μg, about 6.0μg, about 7.0μg, about 8.0μg, about 9.0μg, about 10μg, about 1 5μg, about 20μg, about 30μg, about 40μg, about 50μg, about 60μg, about 70μg, about 80μg, about 90μg, about 100μg, about 150μg, about 200μg, about 300μg, about 400μg, about 500μg, about 600μg, about 700μg, about 800μg, about 900μg or about 1000μg of DNA. In some cases, the DNA used in the methods disclosed herein is extracted from less than about 10,000,000, about 5,000,000, about 4,000,000, about 3,000,000, about 2,000,000, about 1,000,000, about 500,000, about 200,000, about 100,000, about 50,000, about 20,000, about 10,000, about 5,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, about 20, or about 10 cells.

[0138] In diploid genomes, it is usually important to understand which allelic variants are physically connected on the same chromosome, rather than being mapped to homologous positions on chromosome pairs. Mapping alleles or other sequences to specific physical chromosomes of diploid chromosome pairs is referred to as haplotype phasing. Short reads from high-throughput sequence data rarely allow people to directly observe which allelic variants are connected, particularly in the most common case, if the distance between the allelic variants is greater than the longest single read. The computational inference of haplotype phasing may be unreliable over long distances. Methods disclosed herein allow the use of allelic variants read on pairs to determine which allelic variants are physically connected.

[0139] In many cases, the methods and compositions of the present disclosure can perform haplotype phasing on a diploid or polyploid genome with respect to a plurality of allele variants. Therefore, the methods described herein provide for determining the allele variants of the connection based on variant information from the marker sequence segments and / or the assembled contigs using the marker sequence segments. Examples of allele variants include, but are not limited to, allele variants known from 1000genomes, UK10K, HapMap, and other projects for finding genetic variants in humans. In some cases, it is easier to reveal the association of a disease with a specific gene by obtaining haplotype phased data, such as shown by the following findings: unlinked inactivating mutations in both copies of SH3TC2 cause Charcot-Marie-Tooth neuropathy (Lupski JR, Reid JG, Gonzaga-Jauregui C et al. N. Engl. J. Med. 362: 1181–91, 2010) and unlinked inactivating mutations in both copies of ABCG5 cause hypercholesterolemia 9 (Rios J, Stein E, Shendure J et al. Hum. Mol. Genet. 19: 4313–18, 2010).

[0140] On average, 1 site is heterozygous for every 1,000 sites in people. In some cases, single-channel data using high-throughput sequencing methods generate at least about 150,000,000 reads. In further cases, a single read is about 100 base pairs long. If we assume that the average size of the input DNA fragments is 150kbp and we obtain 100 paired-end reads per fragment, then we expect to observe 30 heterozygous sites in each set (i.e., every 100 reads are in phase (i.e., molecular connection) with all other reads in the same set in a set. In some cases, contrary to a single read, this attribute provides greater ability for phasing using a set. There are approximately 3 billion bases in the human genome, and one thousandth is heterozygous, with approximately 3 million heterozygous sites in the average human genome. Using approximately 45,000,000 read pairs comprising heterozygous sites, using a typical high-throughput sequencer, the average coverage of each heterozygous site phased using a single channel of a high-throughput sequencing method is about (15X). Therefore, a diploid human genome can be reliably and completely phased with a channel of high-throughput sequence data related to the sequence variants of a sample prepared using the method disclosed herein. In some cases, a data channel is a set of DNA sequence read data. In further cases, a data channel is a set of DNA sequence read data from a single run of a high-throughput sequencing instrument.

[0141] Since the human genome consists of two sets of homologous chromosomes, it is necessary to describe the maternal and paternal copies or haplotypes of genetic material to understand the true genetic composition of an individual. Obtaining the haplotype of an individual can be used in several aspects. For example, haplotypes can be used clinically to predict the results of donor-host matching in organ transplantation. Haplotypes are increasingly used to detect disease associations. In genes showing compound heterozygosity, haplotypes provide information about whether two harmful variants are located in the same allele (i.e., "cis", using genetic terms) or in two different alleles ("trans"), greatly affecting the prediction of whether the inheritance of these variants is harmful, and affecting the conclusion that whether an individual carries a functional allele and a non-functional allele with two harmful variant sites or the individual carries two non-functional alleles, each of which has different defects. Haplotypes from individual groups provide epidemiologists and anthropologists with information about the structure of the target population and provide information on the history of human evolution. In addition, extensive allelic imbalances in gene expression have been reported, and it has been shown that genetic or epigenetic differences between allele phases can lead to quantitative differences in expression. An understanding of haplotype structure will describe the mechanisms of variants leading to allelic imbalance.

[0142] In certain embodiments, the methods disclosed herein include in vitro techniques to fix and capture associations between distant regions of the genome according to the needs of long-distance linkage and phasing. In some cases, the method includes constructing and sequencing one or more read sets to deliver very far read pairs on the genome. In further cases, each read set contains two or more reads labeled by a common barcode, which can represent two or more sequence segments from a common polynucleotide. In some cases, the interaction mainly comes from random associations within a single polynucleotide. In some cases, the genomic distance between the inferred sequence segments is because the sequence segments close to each other in the polynucleotide interact more frequently and with higher probability, while the interaction between the distant parts of the molecules is less frequent. Therefore, there is a systematic relationship between the number of pairs connecting the two loci and their proximity on the input DNA.

[0143] In some aspects, the disclosure provides methods and compositions for generating data to achieve extremely high phasing accuracy. Compared with previous methods, the methods described herein can phase a higher proportion of variants. In some cases, phase is achieved while maintaining a high level of accuracy. In further cases, the phase information extends to a longer range, for example, greater than about 200kbp, about 300kbp, about 400kbp, about 500kbp, about 600kbp, about 700kbp, about 800kbp, about 900kbp, about 1Mbp, about 2Mbp, about 3Mbp, about 4Mbp, about 5Mbp or about 10Mbp, or longer than about 10Mbp, until and including the entire length of chromosome. In some embodiments, using less than about 250 million reads (for example, by using only 1 channel of Illumina HiSeq data) with an accuracy of more than 99% more than 90% of heterozygous SNPs in human samples are phased. In other cases, less than about 250 million or about 500 million reads (e.g., by using only 1 or 2 channels of Illumina HiSeq data) are used to phase more than about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the heterozygous SNPs in human samples with an accuracy of more than about 70%, 80%, 90%, 95% or 99%. In some cases, less than about 250 million or about 500 million reads are used to phase more than about 95% or 99% of the heterozygous SNPs in human samples with an accuracy of more than about 95% or 99%. In further cases, additional variants are captured by increasing the read length to approximately 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1500bp, 2kbp, 3kbp, 4kbp, 5kbp, 10kbp, 20kbp, 50kbp or 100kbp.

[0144] The compositions and methods of the present disclosure can be used for gene expression analysis. The methods described herein distinguish nucleotide sequences. The differences between target nucleotide sequences can be, for example, single nucleic acid base differences, nucleic acid deletions, nucleic acid insertions or rearrangements. Such sequence differences involving more than one base can also be detected. The process of the present disclosure can detect infectious diseases, genetic diseases and cancer. It can also be used for environmental monitoring, forensics and food science. Examples of genetic analyses that can be performed on nucleic acids include, for example, SNP detection, STR detection, RNA expression analysis, promoter methylation, gene expression, virus detection, virus subtyping and drug resistance.

[0145] The method can be used to analyze biomolecular samples obtained or derived from a patient to determine the presence of diseased cell types in the sample, the stage of the disease, the patient's prognosis, the patient's ability to respond to a particular treatment, or the patient's optimal treatment. The method can also be used to identify biomarkers for a particular disease.

[0146] In some embodiments, the methods described herein are used to diagnose a condition. As used herein, the term "diagnose" or "diagnosis" of a condition may include predicting or diagnosing a condition, determining susceptibility to a condition, monitoring treatment of a condition, diagnosing a therapeutic response to a disease or a prognosis of a condition, progression of a condition, or a response to a specific treatment for a condition. For example, a blood sample may be assayed according to any of the methods described herein to determine the presence and / or amount of markers of a disease or malignant cell type in the sample, thereby diagnosing a disease or cancer or staging a disease or cancer.

[0147] In some embodiments, the methods and compositions described herein are used for diagnosis and prognosis of a condition.

[0148] Many immunological, proliferative and malignant diseases and conditions are particularly suitable for the methods described herein. Immunological diseases and conditions include allergic diseases and conditions, immune dysfunction and autoimmune diseases and conditions. Allergic diseases and conditions include but are not limited to allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis and food allergies. Immunodeficiency includes but is not limited to severe combined immunodeficiency (SCID), eosinophilic syndrome, chronic granulomatous disease, leukocyte adhesion deficiency I and II, high IgE syndrome, congenital leukocyte granule abnormality syndrome (ChediakHigashi), neutrophilia, neutropenia, dysplasia, agammaglobulinemia, high IgM syndrome, DiGeorge / velocardiofacial syndrome and interferon gamma-TH1 pathway defect. Autoimmune and immune dysregulation conditions 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 lymphomatous goiter (Hashimoto's thyroiditis, lymphadenoid goiter), alopecia areata, autoimmune myocarditis, lichen sclerosus, 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, allograft rejection and tissue destruction caused by allergic reactions to infectious microorganisms or environmental antigens.

[0149] Proliferative diseases and disorders that can be assessed by the methods of the present disclosure include, but are not limited to, neonatal hemangiomatosis; secondary progressive multiple sclerosis; chronic progressive myelodegenerative disease; neurofibromatosis; ganglioneuroma; keloid formation; Paget's disease of bone; fibrocystic disease (e.g., of the breast or uterus); sarcoidosis; Peronies and Duputren fibrosis, cirrhosis of the liver, atherosclerosis, and vascular restenosis.

[0150] Malignant diseases and conditions that may be assessed by the methods of the present disclosure include both hematological malignancies and solid tumors.

[0151] When the sample is a blood sample, hematological malignancies are particularly suitable for the methods of the present disclosure because such malignancies involve changes in blood-borne cells. Such malignancies include non-Hodgkin's lymphoma, Hodgkin's lymphoma, non-B cell lymphoma and other lymphomas, acute or chronic leukemias, polycythemia, thrombocythemia, multiple myeloma, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, atypical immune lymphoproliferations, and plasma cell disorders.

[0152] Plasma cell disorders that may be assessed by the methods of the disclosure include multiple myeloma, amyloidosis, and Waldenstrom's macroglobulinemia.

[0153] Examples 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, melanoma, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian cancers, head and neck tumors, and cervical tumors.

[0154] Genetic diseases can also be detected by the process of the present disclosure. This can be done by prenatal or postnatal screening for chromosomes and genetic aberrations or genetic diseases. Examples of genetic diseases that can be detected include: 21-hydroxylase deficiency, cystic fibrosis, fragile X syndrome, Turner syndrome, Duchenne muscular dystrophy, Down syndrome or other trisomy syndromes, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs disease, thalassemia, Klinefelter syndrome, Huntington's disease, autoimmune diseases, lipid storage diseases, obesity defects, hemophilia, inborn errors of metabolism and diabetes.

[0155] The methods described herein can be used to diagnose pathogen infections, such as infections caused by intracellular bacteria and viruses, by determining the presence and / or amount of bacterial or viral markers, respectively, in a sample.

[0156] A variety of infectious diseases can be detected through the process of the present disclosure. Infectious diseases can be caused by bacterial, viral, parasitic and fungal infectious agents. The present disclosure can also be used to determine the resistance of various infectious agents to drugs.

[0157] Bacterial infectious agents that can be detected by the present disclosure include Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria, Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridium, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumoniae, beta-hemolytic Streptococcus, Corynebacterium, Legionella, Mycoplasma, Ureaplasma urealyticum, Chlamydia, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema pallidum, Borrelia burgdorferi, Relapsing fever spirochetes, Rickettsial pathogens, Nocardia, and Actinomycetes.

[0158] Fungal infectious agents that may be detected by the present disclosure include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigatus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycetes, and Maduramycosis.

[0159] Viral infectious agents that can be detected by the present disclosure include human immunodeficiency virus, human T-cell lymphotropic virus, hepatitis viruses (e.g., hepatitis B virus and hepatitis C virus), Epstein-Barr virus, cytomegalovirus, human papillomavirus, orthomyxovirus, paramyxovirus, adenovirus, coronavirus, rhabdovirus, poliovirus, togavirus, bunyavirus, arenavirus, rubella virus and reovirus.

[0160] Parasites that can be detected by the present disclosure include Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Onchocerca volvulus, Leishmania, Trypanosoma, Schistosoma, Entamoeba histolytica, Cryptosporidium, Giardia, Trichomonas, Balantidium coli, Wuchereria bancrofti, Toxoplasma, Enterobacteriaceae, Ascaris lumbricoides, Trichuris, Dracunculiasis, Trematodes, Diphyllobothrium latum, Taenia, Pneumocystis carinii, and Necator americanus.

[0161] The present disclosure can also be used to detect drug resistance of infectious agents. For example, vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multidrug-resistant Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus can all be identified using the present disclosure.

[0162] Thus, the target molecule detected using the compositions and methods of the present disclosure can be a patient marker (such as a cancer marker) or a marker of exogenous infection (such as a bacterial or viral marker).

[0163] The compositions and methods of the present disclosure can be used to identify and / or quantify target molecules whose abundance is indicative of a biological state or disease condition (eg, a blood marker that is up- or down-regulated due to a disease state).

[0164] 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 will contribute to the early detection of cytokines (e.g., as biomarkers of the condition), the diagnosis or prognosis of diseases (such as cancer), and the identification of subclinical conditions.

[0165] The different samples from which the target polynucleotide is derived can include multiple samples from the same individual, samples from different individuals, or a combination thereof. In some embodiments, the sample comprises multiple polynucleotides from a single individual. In some embodiments, the sample comprises multiple polynucleotides from two or more individuals. An individual is any organism or part thereof from which a target polynucleotide can be derived, and its non-limiting examples include plants, animals, fungi, protists, ankaryotic protozoa, viruses, mitochondria, and chloroplasts. Sample polynucleotides can be separated from an object, such as a cell sample, a tissue sample, or an organ sample derived therefrom, including, for example, a cultured cell line, a biopsy, a blood sample, or a fluid sample containing cells. The object can be an animal, including but not limited to animals, such as cattle, pigs, mice, rats, chickens, cats, dogs, etc., and generally mammals, such as humans. The sample can also be artificially obtained, such as by chemical synthesis. In some embodiments, the sample comprises DNA. In some embodiments, the sample comprises genomic DNA. In some embodiments, the sample comprises mitochondrial DNA, chloroplast DNA, plasmid DNA, bacterial artificial chromosomes, yeast artificial chromosomes, oligonucleotide tags, or a combination thereof. In some embodiments, the sample comprises DNA produced by a primer extension reaction (including but not limited to polymerase chain reaction (PCR), reverse transcription and combinations thereof) using any suitable combination of primers and DNA polymerase. In the case where the template of the primer extension reaction is RNA, the reverse transcription product is referred to as complementary DNA (cDNA). The primers used for the primer extension reaction may include sequences, random sequences, partially random sequences and combinations thereof specific to one or more targets. Reaction conditions suitable for primer extension reactions are known. In general, the sample polynucleotides include any polynucleotides present in the sample, which may or may not include the target polynucleotides.

[0166] In some embodiments, nucleic acid template molecules (e.g., DNA or RNA) are separated from biological samples containing a variety of other components (such as proteins, lipids, and non-template nucleic acids). Nucleic acid template molecules can be obtained from any cell material, and the cell material is obtained from animals, plants, bacteria, fungi, or any other cell organisms. Biological samples used in the present disclosure include viral particles or preparations. Nucleic acid template molecules can be obtained directly from organisms or from biological samples obtained from organisms, for example, from blood, urine, cerebrospinal fluid, semen, saliva, sputum, feces, and tissues. Any tissue or body fluid sample can be used as the source of nucleic acids used in the present disclosure. Nucleic acid template molecules can also be separated from cultured cells (such as primary cell cultures or cell lines). Cells or tissues from which template nucleic acids are obtained can be infected by viruses or other intracellular pathogens. Samples can also be total RNA extracted from biological samples, cDNA libraries, viruses, or genomic DNA. Samples can also be DNA separated from non-cellular sources, such as DNA amplified / separated from a freezer.

[0167] Methods for extracting and purifying nucleic acids are known. For example, nucleic acids can be purified by organic extraction using 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, such as using phenol / chloroform organic reagent (Ausubel et al., 1993), with or without the use of an automated nucleic acid extractor, such as the Model 1000 available from Applied Biosystems (Foster City, Calif.). 341 DNA extractor; (2) stationary phase adsorption method (U.S. Patent No. 5,234,809; Walsh et al., 1991); and (3) salt-induced nucleic acid precipitation method (Miller et al., (1988), such precipitation methods are generally referred to as "salting out" methods. Another example of nucleic acid separation and / or purification includes the use of magnetic particles to which nucleic acids can bind specifically or non-specifically, followed by separation of the beads using a magnet, and washing and eluting the nucleic acids from the beads (see, e.g., U.S. Patent No. 5,705,628). In some embodiments, the above separation methods can be preceded by an enzymatic digestion step to help eliminate unwanted proteins from the sample, such as digestion with proteinase K or other similar proteases. See, e.g., U.S. Patent No. 5,705,628). US Patent No. 7,001,724. If necessary, RNase inhibitors can be added to the lysis buffer. For certain cell or sample types, it may be necessary to add a protein denaturation / digestion step to the protocol. The purification method can be directed to separating DNA, RNA, or both. When DNA and RNA are separated together during or after the extraction process, further steps can be taken to separate one or both from the other for purification. Subfractions of the extracted nucleic acid can also be produced, for example, purified by size, sequence, or other physical or chemical characteristics. In addition to the initial nucleic acid separation step, purification of nucleic acids can be performed after any step in the method of the present disclosure, for example to remove excess or unwanted reagents, reactants, or products.

[0168] The nucleic acid template molecule can be obtained as described in U.S. Patent Application Publication No. US2002 / 0190663A1, published on October 9, 2003. Generally, nucleic acids can be extracted from biological samples by a variety of techniques, such as those described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, pp. 280-281 (1982). In some cases, nucleic acids can be first extracted from biological samples and then cross-linked in vitro. In some cases, natural binding proteins (e.g., histones) can be further removed from nucleic acids.

[0169] In other embodiments, the present disclosure can be readily applied to any high molecular weight double-stranded DNA, including, for example, DNA isolated from tissues, cell cultures, body fluids, animal tissues, plants, bacteria, fungi, viruses, and the like.

[0170] Hi-C method with size selection

[0171] Provided herein is a method comprising obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein; contacting the stabilized biological sample with a DNase to cut the nucleic acid molecule into a plurality of segments; attaching a first segment and a second segment in a plurality of segments at a splicing sequence; and performing size selection on a plurality of segments to obtain a plurality of selection segments. In some cases, a plurality of selection segments are about 145bp to about 600bp. In some cases, a plurality of selection segments are about 100bp to about 2500bp. In some cases, a plurality of selection segments are about 100bp to about 600bp. In some cases, a plurality of selection segments are about 600bp to about 2500bp. In some cases, the plurality of selected segments are between about 100 bp and about 600 bp, between about 100 bp and about 700 bp, between about 100 bp and about 800 bp, between about 100 bp and about 900 bp, between about 100 bp and about 1000 bp, between about 100 bp and about 1100 bp, between about 100 bp and about 1200 bp, between about 100 bp and about 1300 bp, between about 100 bp and about 1400 bp, between about 100 bp and about 1500 bp, or between about 100 bp and about 1600 bp. bp, between about 100bp and about 1600bp, between about 100bp and about 1700bp, between about 100bp and about 1800bp, between about 100bp and about 1900bp, between about 100bp and about 2000bp, between about 100bp and about 2100bp, between about 100bp and about 2200bp, between about 100bp and about 2300bp, between about 100bp and about 2400bp, or between about 100bp and about 2500bp.

[0172] In another aspect of the method for the size selection step provided herein, the method further includes, before the size selection step, preparing a sequencing library from a plurality of sections. In some embodiments, the method further includes carrying out size selection to the sequencing library to obtain a size-selected library. In some cases, the size of the library selected by size is between about 350bp and about 1000bp. In some cases, the size of the library selected by size is between about 100bp and about 2500bp, for example, between about 100bp and about 350bp, between about 350bp and about 500bp, between about 500bp and about 1000bp, between about 1000 and about 1500bp and about 2000bp, between about 2000bp and about 2500bp, between about 350bp and about 1000bp, between about 350bp and about 1500bp, between about 350bp and about 2000bp , between about 350bp and about 2500bp, between about 500bp and about 1500bp, between about 500bp and about 2000bp, between about 500bp and about 3500bp, between about 1000bp and about 1500bp, between about 1000bp and about 2000bp, between about 1000bp and about 2500bp, between about 1500bp and about 2000bp, between about 1500bp and about 2500bp, or between about 2000bp and about 2500bp.

[0173] Size selection used in the methods involving a size selection step provided herein can be performed using gel electrophoresis, capillary electrophoresis, size selection beads, gel filtration columns, other suitable methods, or combinations thereof.

[0174] On the other hand, the method related to the size selection step provided herein can further include analyzing multiple selection segments to obtain QC values. In some cases, QC values ​​are selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI). CDE is calculated as the ratio of the segment with desired length. For example, in some cases, CDE is calculated as the ratio of the segment of 100bp to 2500bp before size selection. In some cases, when the CDE value is at least 65%, select sample for further analysis. In some cases, when the CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, select sample for further analysis. CDI is calculated as the ratio of the segment quantity of mononucleosome size to the segment quantity of dinucleosome size before size selection. For example, the CDI can be calculated as the logarithm of the ratio of fragments of size 600-2500 bp to fragments of size 100-600 bp. In some cases, a sample is selected for further analysis when the CDI value is greater than -1.5 and less than 1. In some cases, a sample is selected for further analysis when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 and less than about 1.5, When the value is greater than about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample is selected for further analysis.

[0175] On the other hand, the stabilized biological sample used in the method for the size selection step of this article includes a biological material treated with a stabilizer. In some cases, the stabilized biological sample includes a stabilized cell lysate. Alternatively, the stabilized biological sample includes a stabilized intact cell. Alternatively, the stabilized biological sample includes a stabilized intact nucleus. In some cases, contacting the stabilized intact cell or intact nucleus sample with DNase is performed before the cracking of the intact cell or intact nucleus. In some cases, the first segment and the second segment in a plurality of segments are attached at the splicing sequence before the lysed cell and / or nucleus.

[0176] In another aspect, the method of the size selection step of this article is carried out on a small sample containing a small amount of cells or a small amount of nucleic acid. For example, in some cases, the stabilized biological sample contains less than 3,000,000 cells. In some cases, the stabilized biological sample contains less than 2,000,000 cells. In some cases, the stabilized biological sample contains less than 1,000,000 cells. In some cases, the stabilized biological sample contains less than 500,000 cells. In some cases, the stabilized biological sample contains less than 400,000 cells. In some cases, the stabilized biological sample contains less than 300,000 cells. In some cases, the stabilized biological sample contains less than 200,000 cells. In some cases, the stabilized biological sample contains less than 100,000 cells. In some cases, the stabilized biological sample contains less than 50,000 cells. In some cases, the stabilized biological sample contains less than 40,000 cells. In some cases, the stabilized biological sample contains less than 30,000 cells. In some cases, the stabilized biological sample comprises less than 20,000 cells. In some cases, the stabilized biological sample comprises less than 10,000 cells. In some cases, the stabilized biological sample may comprise about 10,000 cells. In some cases, the stabilized biological sample comprises less than 10 μg DNA. In some cases, the stabilized biological sample comprises less than 9 μg DNA. In some cases, the stabilized biological sample comprises less than 8 μg DNA. In some cases, the stabilized biological sample comprises less than 7 μg DNA. In some cases, the stabilized biological sample comprises less than 6 μg DNA. In some cases, the stabilized biological sample comprises less than 5 μg DNA. In some cases, the stabilized biological sample comprises less than 4 μg DNA. In some cases, the stabilized biological sample comprises less than 3 μg DNA. In some cases, the stabilized biological sample comprises less than 2 μg DNA. In some cases, the stabilized biological sample comprises less than 1 μg DNA. In some cases, the stabilized biological sample comprises less than 0.5 μg DNA.

[0177] On the other hand, the method involving the size selection step herein can be performed on a separate or single cell. For example, the method herein can be performed on cells distributed into various partitions. Exemplary partitions include, but are not limited to, wells, droplets in emulsions, or surface locations (e.g., array spots, beads, etc.) containing different patches of differential sequence adapter molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with the methods, compositions, and systems disclosed herein.

[0178] In other aspects, the stabilized biological sample used in the method for the size selection step related to this article is treated with a nuclease such as DNase to produce a fragment of DNA. In some cases, DNase is non-sequence specific. In some cases, DNase has activity to both single-stranded DNA and double-stranded DNA. In some cases, DNase has specificity to double-stranded DNA. In some cases, DNase preferentially cuts double-stranded DNA. In some cases, DNase has specificity to single-stranded DNA. In some cases, DNase preferentially cuts single-stranded DNA. In some cases, DNase is DNase I. In some cases, DNase is DNase II. In some cases, DNase is selected from one or more of DNase I and DNase II. In some cases, DNase is micrococcal nuclease. In some cases, DNase is selected from one or more of DNase I and DNase II and micrococcal nuclease. In some cases, DNase can be coupled or fused to immunoglobulin binding protein or its fragment. Immunoglobulin binding protein can be, for example, protein A, protein G, protein A / G or protein L. In some cases, the DNase can be coupled to a fusion protein comprising two or more immunoglobulin binding proteins and / or fragments thereof.Other suitable nucleases are also within the scope of the present disclosure.

[0179] In other aspects, the stabilized biological sample provided herein for use in the method for size selection step is treated with one or more cross-linking agents. In some cases, the cross-linking agent is a chemical fixative. In some cases, the chemical fixative comprises formaldehyde, which has a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length. For example, the cross-linking agent may have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative may comprise ethylene glycol bis (succinimidyl succinate) (EGS), which has a spacer arm of about 16.1A in length. The chemical fixative may comprise disuccinimidyl glutarate (DSG), which has a spacer arm of about 7.7A in length. In some cases, the chemical fixative includes formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In some cases where multiple chemical fixatives are used, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with a large (e.g., >1kb) read pair separation distance. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative comprises psoralen. In some cases, the cross-linking agent is ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample is a paraffin-embedded tissue sample cross-linked.

[0180] In a further aspect, the methods comprising a size selection step provided herein comprise contacting a plurality of selection segments with an antibody.

[0181] In other aspects, the method comprising the size selection step provided herein includes attaching the first and second segments in a plurality of segments at the splicing sequence. In some cases, attachment includes filling the sticky ends with biotin-tagged nucleotides and connecting the flat ends. In some cases, attachment includes contacting at least the first and second segments with a bridging oligonucleotide. In some cases, attachment includes contacting at least the first and second segments with a barcode. In some embodiments, the length of the bridging oligonucleotide herein can be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein can be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide can be 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 35, about 40, about 45 or about 50 nucleotides. In some embodiments, the bridging oligonucleotide herein may comprise a barcode. In some embodiments, the bridging oligonucleotide may comprise a plurality of barcodes. In some embodiments, the bridging oligonucleotide comprises a plurality of bridging oligonucleotides linked together. In some embodiments, the bridging oligonucleotide may be coupled or connected to an immunoglobulin binding protein such as protein A, protein G, protein A / G or protein L or a fragment thereof. In some cases, the coupled bridging oligonucleotide may be delivered to the position of the binding antibody in the sample nucleic acid.

[0182] Splitting and pooling methods can be used to produce bridging oligonucleotides with unique barcodes. The sample population can be split into multiple groups, and the bridging oligonucleotide can be attached to the sample so that the bridging oligonucleotide barcode is different between groups but the same within a group, and the sample group can be brought together again, and the process can be repeated multiple times. Iteration of this process will eventually lead to each sample in the population having a series of unique bridging oligonucleotide barcodes, thereby allowing single sample (e.g., single cell, single cell nucleus, single chromosome) analysis. In an illustrative example, the cross-linked digested nucleus sample attached to the bead solid support is split into 8 test tubes, each test tube containing 1 of 8 unique members of the first adapter group (first iteration), and the first adapter group includes double-stranded DNA (dsDNA) adapters to be connected. Each of the 8 adapters can have the same 5' overhang sequence for connecting to the nucleic acid end of the cross-linked chromatin aggregate in the nucleus, but otherwise has a unique dsDNA sequence. After the first adapter group is connected, the nucleus can be brought back together and washed to remove the ligation reaction components. The scheme of distribution, connection and collection can be repeated 2 more times (2 iterations). After connecting the members of each adapter group, the cross-linked chromatin aggregates can be attached in series to multiple barcodes. In some cases, the sequential connection of multiple members of multiple adapter groups (iterations) leads to barcode combinations. The number of available barcode combinations depends on the number of groups in each iteration and the total number of barcode oligonucleotides used. For example, 3 iterations containing 8 members each time can have 83 possible combinations. In some cases, the barcode combination is unique. In some cases, the barcode combination is redundant. The total number of barcode combinations can be adjusted by increasing or decreasing the number of groups receiving unique barcodes and / or increasing or decreasing the number of iterations. When more than one adapter group is used, iterative adapter attachment can be performed using the distribution, attachment and collection scheme. In some cases, the scheme of distribution, attachment and collection can be repeated at least 3, 4, 5, 6, 7, 8, 9 or 10 times. In some cases, members of the last adaptor set include sequences that are subsequently used to enrich for adaptor-attached DNA, for example, during preparation of a sequencing library by PCR amplification.

[0183] In additional aspects, methods involving a size selection step herein do not comprise a shearing step (eg, the nucleic acids are not sheared).

[0184] In further aspects of the methods involving the size selection step herein, the method includes obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0185] In further aspects of the methods involving the size selection step herein, the methods include mapping a first read pair onto a set of contigs, and determining a path through representatives of the set of contigs to an order and / or orientation of the genome.

[0186] In further aspects of the methods involving the size selection step herein, the methods include mapping the first read pairs onto a set of contigs; and determining the presence of the structural variant or loss of heterozygosity in the stabilized biological sample from the set of contigs.

[0187] In further aspects of the methods involving the size selection step herein, the methods include mapping the first read pairs onto a set of contigs, and assigning variants in the set of contigs to phases.

[0188] In a further aspect of the method involving the size selection step of the present invention, the method includes mapping a first read pair to a set of overlapping groups; determining the presence of a variant in the set of overlapping groups from the set of overlapping groups, and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or treatment course of the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of the drug in the stabilized biological sample.

[0189] Hi-C method including QC calculation

[0190] Additionally, methods are provided herein, including obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein, contacting the stabilized biological sample with DNase to cut the nucleic acid molecule into multiple segments, attaching the first segment and the second segment in the multiple segments at the splicing sequence, and analyzing multiple segments to determine the QC value. In some cases, the QC value is selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI). CDE is calculated as the ratio of the segment with the desired length. For example, in some cases, CDE is calculated as the ratio of the segment with a size of 100bp to 2500bp before size selection. In some cases, when the CDE value is at least 65%, select the sample for further analysis. In some cases, when the CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, select the sample for further analysis. CDI is calculated as the ratio of the number of segments of mononucleosome size to the number of segments of dinucleosome size before size selection. For example, CDI can be calculated as the logarithm of the ratio of fragments of 600-2500 bp to fragments of 100-600 bp in size. In some cases, when the CDI value is greater than -1.5 and less than 1, the sample is selected for further analysis. In some cases, when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 and less than about 1.5, When the value is greater than about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample is selected for further analysis.

[0191] On the other hand, the method of the QC determination step related to this paper may include size selection of multiple segments to obtain multiple selection segments. In some cases, multiple selection segments are about 145bp to about 600bp. In some cases, multiple selection segments are about 100bp to about 2500bp. In some cases, multiple selection segments are about 100bp to about 600bp. In some cases, multiple selection segments are about 600bp to about 2500bp. In some cases, the plurality of selected segments are between about 100 bp and about 600 bp, between about 100 bp and about 700 bp, between about 100 bp and about 800 bp, between about 100 bp and about 900 bp, between about 100 bp and about 1000 bp, between about 100 bp and about 1100 bp, between about 100 bp and about 1200 bp, between about 100 bp and about 1300 bp, between about 100 bp and about 1400 bp, between about 100 bp and about 1500 bp, or between about 100 bp and about 1600 bp. bp, between about 100bp and about 1600bp, between about 100bp and about 1700bp, between about 100bp and about 1800bp, between about 100bp and about 1900bp, between about 100bp and about 2000bp, between about 100bp and about 2100bp, between about 100bp and about 2200bp, between about 100bp and about 2300bp, between about 100bp and about 2400bp, or between about 100bp and about 2500bp.

[0192] In another aspect of the method for QC determination step related to the present invention, the method may further include, before the size selection step, preparing a sequencing library from a plurality of sections. In some embodiments, the method further includes carrying out size selection to the sequencing library to obtain a library selected by size. In some cases, the size of the library selected by size is between about 350bp and about 1000bp. In some cases, the size of the library selected by size is between about 100bp and about 2500bp, for example, between about 100bp and about 350bp, between about 350bp and about 500bp, between about 500bp and about 1000bp, between about 1000 and about 1500bp and about 2000bp, between about 2000bp and about 2500bp, between about 350bp and about 1000bp, between about 350bp and about 1500bp, between about 350bp and about 2000bp , between about 350bp and about 2500bp, between about 500bp and about 1500bp, between about 500bp and about 2000bp, between about 500bp and about 3500bp, between about 1000bp and about 1500bp, between about 1000bp and about 2000bp, between about 1000bp and about 2500bp, between about 1500bp and about 2000bp, between about 1500bp and about 2500bp, or between about 2000bp and about 2500bp.

[0193] The size selection used in the methods involving the QC determination step herein can be performed using gel electrophoresis, capillary electrophoresis, size selection beads, gel filtration columns, or a combination thereof. Other suitable size selection methods are also within the scope of the present disclosure.

[0194] On the other hand, the stabilized biological sample used in the method for determining the QC step of this article includes a biological material treated with a stabilizer. In some cases, the stabilized biological sample includes a stabilized cell lysate. Alternatively, the stabilized biological sample includes a stabilized intact cell. Alternatively, the stabilized biological sample includes a stabilized intact nucleus. In some cases, contacting the stabilized intact cell or intact nucleus sample with DNase is performed before the cracking of the intact cell or intact nucleus. In some cases, the first segment and the second segment in a plurality of segments are attached at the splicing sequence before the lysing cell and / or the nucleus.

[0195] In another aspect, the method of the QC determination step related to this article is carried out on a small sample containing a small amount of cells or a small amount of nucleic acid. In some cases, the stabilized biological sample comprises less than 3,000,000 cells. In some cases, the stabilized biological sample comprises less than 2,000,000 cells. In some cases, the stabilized biological sample comprises less than 1,000,000 cells. In some cases, the stabilized biological sample comprises less than 500,000 cells. In some cases, the stabilized biological sample comprises less than 400,000 cells. In some cases, the stabilized biological sample comprises less than 300,000 cells. In some cases, the stabilized biological sample comprises less than 200,000 cells. In some cases, the stabilized biological sample comprises less than 100,000 cells. In some cases, the stabilized biological sample comprises less than 50,000 cells. In some cases, the stabilized biological sample comprises less than 40,000 cells. In some cases, the stabilized biological sample comprises less than 30,000 cells. In some cases, the stabilized biological sample comprises less than 20,000 cells. In some cases, the stabilized biological sample comprises less than 10,000 cells. In some cases, the stabilized biological sample comprises about 10,000 cells. In some cases, the stabilized biological sample comprises less than 10 μg DNA. In some cases, the stabilized biological sample comprises less than 9 μg DNA. In some cases, the stabilized biological sample comprises less than 8 μg DNA. In some cases, the stabilized biological sample comprises less than 7 μg DNA. In some cases, the stabilized biological sample comprises less than 6 μg DNA. In some cases, the stabilized biological sample comprises less than 5 μg DNA. In some cases, the stabilized biological sample comprises less than 4 μg DNA. In some cases, the stabilized biological sample comprises less than 3 μg DNA. In some cases, the stabilized biological sample comprises less than 2 μg DNA. In some cases, the stabilized biological sample comprises less than 1 μg DNA. In some cases, the stabilized biological sample comprises less than 0.5 μg DNA.

[0196] On the other hand, the method for determining the steps of QC herein can be carried out on a single or individual cell. For example, the method herein can be carried out on cells distributed to each partition. Exemplary partitions include, but are not limited to, droplets or surface positions (e.g., array spots, beads, etc.) in holes, emulsions, which contain different parts of differential sequence adapter molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with methods, compositions, and systems disclosed herein.

[0197] In other aspects, the stabilized biological sample used in the method for determining the QC step of this article is treated with a nuclease such as DNase to produce a fragment of DNA. In some cases, DNase is non-sequence specific. In some cases, DNase has activity to both single-stranded DNA and double-stranded DNA. In some cases, DNase has specificity to double-stranded DNA. In some cases, DNase preferentially cuts double-stranded DNA. In some cases, DNase has specificity to single-stranded DNA. In some cases, DNase preferentially cuts single-stranded DNA. In some cases, DNase is DNase I. In some cases, DNase is DNase II. In some cases, DNase is selected from one or more of DNase I and DNase II. In some cases, DNase is micrococcal nuclease. In some cases, DNase is selected from one or more of DNase I and DNase II and micrococcal nuclease. In some cases, DNase can be coupled or fused with immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L or its fragment. Other suitable nucleases are also within the scope of the present disclosure.

[0198] In other aspects, the stabilized biological sample used in the method for determining the step of QC herein is treated with a cross-linking agent. In some cases, the cross-linking agent is a chemical fixative. In some cases, the chemical fixative comprises formaldehyde, which has a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length. For example, the cross-linking agent may have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative may comprise ethylene glycol bis (succinimidyl succinate) (EGS), which has a spacer arm of about 16.1A in length. The chemical fixative may comprise disuccinimidyl glutarate (DSG), which has a spacer arm of about 7.7A in length. In some cases, the chemical fixative includes formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In some cases where multiple chemical fixatives are used, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with large (e.g., >1kb) read pair separation distances. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative comprises psoralen. In some cases, the cross-linking agent is ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample is a paraffin-embedded tissue sample cross-linked.

[0199] In a further aspect, the methods involving the QC determination steps provided herein comprise contacting a plurality of selection segments with an antibody.

[0200] In other aspects, the method for determining the QC step provided herein includes attaching the first and second segments in a plurality of segments at the splicing sequence. In some cases, attachment includes filling the sticky ends with biotin-tagged nucleotides and connecting flat ends. In some cases, attachment includes contacting at least the first and second segments with a bridging oligonucleotide. In some cases, attachment includes contacting at least the first and second segments with a barcode. In some embodiments, the length of the bridging oligonucleotide herein can be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein can be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide can be 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 35, about 40, about 45 or about 50 nucleotides. In some embodiments, the bridging oligonucleotide herein may comprise a barcode. In some embodiments, the bridging oligonucleotide may comprise a plurality of barcodes. In some embodiments, the bridging oligonucleotide comprises a plurality of bridging oligonucleotides linked together. In some embodiments, the bridging oligonucleotide may be coupled or connected to an immunoglobulin binding protein such as protein A, protein G, protein A / G or protein L or a fragment thereof. In some cases, the coupled bridging oligonucleotide may be delivered to the position of the binding antibody in the sample nucleic acid.

[0201] In further aspects, the methods involving the QC determination step herein do not include a shearing step.

[0202] In further aspects of the methods involving the QC determination step of the present invention, the method includes obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0203] In further aspects of the methods involving the QC determination step herein, the methods include mapping a first read pair onto a set of contigs and determining a path through a representative of the set of contigs to an order and / or orientation of the genome.

[0204] In further aspects of the methods involving the QC determination step herein, the methods may include mapping the first read pair to a set of contigs and determining the presence of the structural variant or loss of heterozygosity in the stabilized biological sample from the set of contigs.

[0205] In further aspects of the methods involving the QC determination step herein, the methods include mapping the first read pairs onto a set of contigs and assigning variants in the set of contigs to phases.

[0206] In a further aspect of the method involving the QC determination step of the present invention, the method includes mapping a first read pair to a set of overlapping groups; determining the presence of a variant in the set of overlapping groups from the set of overlapping groups; and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or treatment course of the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of the drug in the stabilized biological sample.

[0207] Hi-C methods involving whole cell or whole nuclear digestion

[0208] Further provided herein is a method, including obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein; contacting the stabilized biological sample with DNase to cut the nucleic acid molecule into multiple segments; and attaching the first segment and the second segment in the multiple segments at the splicing sequence, wherein the stabilized biological sample includes intact cells and / or intact cell nuclei. In some cases, the stabilized biological sample includes stabilized intact cells. Alternatively or in combination, the stabilized biological sample includes stabilized intact cell nuclei. In some cases, contacting the stabilized intact cell or intact cell nucleus sample with DNase is performed before the cracking of the intact cell or intact cell nucleus. In some cases, the first segment and the second segment in the multiple segments are attached at the splicing sequence before the lysing cell and / or the cell nucleus.

[0209] On the other hand, the method for digesting whole cells or whole cell nuclei herein may include size selection of multiple segments to obtain multiple selection segments. In some cases, the multiple selection segments are about 145bp to about 600bp. In some cases, the multiple selection segments are about 100bp to about 2500bp. In some cases, the multiple selection segments are about 100bp to about 600bp. In some cases, the multiple selection segments are about 600bp to about 2500bp. In some cases, the plurality of selected segments is about 100 bp to about 600 bp, about 100 bp to about 700 bp, about 100 bp to about 800 bp, about 100 bp to about 900 bp, about 100 bp to about 1000 bp, about 100 bp to about 1100 bp, about 100 bp to about 1200 bp, about 100 bp to about 1300 bp, about 100 bp to about 1400 bp, about 100 bp to about 1500 bp, about 100 bp to about 1600 bp, about 100 bp to about 1700 bp, about 100 bp to about 1800 bp, about 100 bp to about 1900 bp, about 100 bp to about 2000 bp, about 100 bp to about 2100 bp, about 100 bp to about 2200 bp, about 100 bp to about 2300 bp, about 100 bp to about 2400 bp, or about 100 bp to about 2500 bp.

[0210] In another aspect of the method for digesting whole cells or whole cell nuclei provided herein, the method further comprises, before the size selection step, preparing a sequencing library from a plurality of segments. In some embodiments, the method further comprises size selection of the sequencing library to obtain a size-selected library. In some cases, the size of the size-selected library is about 350bp to about 1000bp. In some cases, the size of the size-selected library is about 100bp to about 2500bp, for example, about 100bp to about 350bp, about 350bp to about 500bp, about 500bp to about 1000bp, about 1000 to about 1500bp to about 2000bp, about 2000bp to about 2500bp, about 350bp to about 1000bp, about 350bp to about 1500bp, about 350bp to about 2000bp, about 350bp to about 2000bp, about 350bp to about 1500bp, about 350bp to about 2000bp. bp, about 350 bp to about 2500 bp, about 500 bp to about 1500 bp, about 500 bp to about 2000 bp, about 500 bp to about 3500 bp, about 1000 bp to about 1500 bp, about 1000 bp to about 2000 bp, about 1000 bp to about 2500 bp, about 1500 bp to about 2000 bp, about 1500 bp to about 2500 bp or about 2000 bp to about 2500 bp.

[0211] Size selection used in the methods herein involving digestion of whole cells or whole nuclei can be performed using gel electrophoresis, capillary electrophoresis, size selection beads, gel filtration columns, or a combination thereof.

[0212] On the other hand, the method that this article relates to digestion whole cell or whole cell nucleus can further include analyzing multiple selection segments to obtain QC value.In some cases, QC value is selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI).CDE is calculated as the ratio of the segment with desired length.For example, in some cases, CDE is calculated as the ratio of the segment of 100 to 2500bp before size selection.In some cases, when CDE value is at least 65%, select sample for further analysis.In some cases, when CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, select sample for further analysis.CDI is calculated as the ratio of the segment number of mononucleosome size to the segment number of dinucleosome size before size selection. For example, the CDI can be calculated as the logarithm of the ratio of fragments of size 600-2500 bp to fragments of size 100-600 bp. In some cases, a sample is selected for further analysis when the CDI value is greater than -1.5 and less than 1. In some cases, a sample is selected for further analysis when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 and less than about 1.5, When the value is greater than about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample is selected for further analysis.

[0213] On the other hand, the method involving whole cell or whole cell nuclear digestion herein is carried out on a small sample containing a small amount of cells or a small amount of nucleic acid. In some cases, the stabilized biological sample comprises less than 3,000,000 cells. In some cases, the stabilized biological sample comprises less than 2,000,000 cells. In some cases, the stabilized biological sample comprises less than 1,000,000 cells. In some cases, the stabilized biological sample comprises less than 500,000 cells. In some cases, the stabilized biological sample comprises less than 400,000 cells. In some cases, the stabilized biological sample comprises less than 300,000 cells. In some cases, the stabilized biological sample comprises less than 200,000 cells. In some cases, the stabilized biological sample comprises less than 100,000 cells. In some cases, the stabilized biological sample comprises less than 50,000 cells. In some cases, the stabilized biological sample comprises less than 40,000 cells. In some cases, the stabilized biological sample comprises less than 30,000 cells. In some cases, the stabilized biological sample comprises less than 20,000 cells. In some cases, the stabilized biological sample comprises less than 10,000 cells. In some cases, the stabilized biological sample comprises about 10,000 cells. In some cases, the stabilized biological sample comprises less than 10 μg DNA. In some cases, the stabilized biological sample comprises less than 9 μg DNA. In some cases, the stabilized biological sample comprises less than 8 μg DNA. In some cases, the stabilized biological sample comprises less than 7 μg DNA. In some cases, the stabilized biological sample comprises less than 6 μg DNA. In some cases, the stabilized biological sample comprises less than 5 μg DNA. In some cases, the stabilized biological sample comprises less than 4 μg DNA. In some cases, the stabilized biological sample comprises less than 3 μg DNA. In some cases, the stabilized biological sample comprises less than 2 μg DNA. In some cases, the stabilized biological sample comprises less than 1 μg DNA. In some cases, the stabilized biological sample comprises less than 0.5 μg DNA.

[0214] On the other hand, the methods herein related to digestion of whole cells or whole cell nuclei can be performed on separate or single cells. For example, the methods herein can be performed on cells distributed to various partitions. Exemplary partitions include, but are not limited to, wells, droplets in emulsions, or surface locations (e.g., array spots, beads, etc.) comprising different portions of differential sequence adapter molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with the methods, compositions, and systems disclosed herein.

[0215] In other aspects, the stabilized biological sample used in the method for digesting whole cells or whole nuclei is treated with a nuclease such as DNase to produce a fragment of DNA. In some cases, DNase is non-sequence specific. In some cases, DNase has activity to both single-stranded DNA and double-stranded DNA. In some cases, DNase has specificity to double-stranded DNA. In some cases, DNase preferentially cuts double-stranded DNA. In some cases, DNase has specificity to single-stranded DNA. In some cases, DNase preferentially cuts single-stranded DNA. In some cases, DNase is DNase I. In some cases, DNase is DNase II. In some cases, DNase is selected from one or more of DNase I and DNase II. In some cases, DNase is micrococcal nuclease. In some cases, DNase is selected from one or more of DNase I and DNase II and micrococcal nuclease. In some cases, DNase can be coupled or fused with immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L or its fragment. Other suitable nucleases are also within the scope of the present disclosure.

[0216] In other aspects, the stabilized biological sample used in the method for whole cell or whole cell nuclear digestion related to this article is treated with a cross-linking agent. In some cases, the cross-linking agent is a chemical fixative. In some cases, the chemical fixative comprises formaldehyde, which has a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length. For example, the cross-linking agent can have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative can include ethylene glycol bis (succinimidyl succinate) (EGS), which has a spacer arm of about 16.1A in length. The chemical fixative can include disuccinimidyl glutarate (DSG), which has a spacer arm of about 7.7A in length. In some cases, the chemical fixative comprises formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In the case of using multiple chemical fixatives, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with large (e.g., >1kb) read pair separation distances. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative comprises psoralen. In some cases, the cross-linking agent is ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample is a paraffin-embedded tissue sample cross-linked.

[0217] In a further aspect, methods provided herein involving digestion of whole cells or whole cell nuclei comprise contacting a plurality of selected segments with an antibody.

[0218] In other aspects, the method provided herein related to digestion of whole cell or whole cell nucleus includes attaching the first section and the second section in a plurality of sections at the splicing sequence. In some cases, attachment includes filling the sticky end with biotin-tagged nucleotides and connecting the flat ends. In some cases, attachment includes contacting at least the first section and the second section with a bridging oligonucleotide. In some cases, attachment includes contacting at least the first section and the second section with a barcode. In some embodiments, the length of the bridging oligonucleotide herein can be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein can be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide can be 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 35, about 40, about 45 or about 50 nucleotides. In some embodiments, the bridging oligonucleotide herein may comprise a barcode. In some embodiments, the bridging oligonucleotide may comprise a plurality of barcodes. In some embodiments, the bridging oligonucleotide comprises a plurality of bridging oligonucleotides linked together. In some embodiments, the bridging oligonucleotide may be coupled or connected to an immunoglobulin binding protein such as protein A, protein G, protein A / G or protein L or a fragment thereof. In some cases, the coupled bridging oligonucleotide may be delivered to the position of the binding antibody in the sample nucleic acid.

[0219] The splitting and pooling method can be adopted to produce the bridging oligonucleotide with unique barcode. The sample population can be split into multiple groups, the bridging oligonucleotide can be attached to the sample so that the bridging oligonucleotide barcode is different between groups but the same within a group, the sample group can be pooled together again, and the process can be repeated multiple times. Iteration of this process will eventually lead to each sample in the population having a series of unique bridging oligonucleotide barcodes, thereby allowing single sample (e.g., single cell, single cell nucleus, single chromosome) analysis. In an illustrative example, the cross-linked digested nucleus sample attached to the bead solid support is split into 8 test tubes, each test tube containing 1 of 8 unique members of the first adapter group (first iteration), and the first adapter group includes double-stranded DNA (dsDNA) adapters to be connected. Each of the 8 adapters can have the same 5' overhang sequence for connecting to the nucleic acid end of the cross-linked chromatin aggregate in the nucleus, but has a unique dsDNA sequence in other aspects. After the first adapter group is connected, the nucleus can be pooled together and washed to remove the connection reaction components. The scheme of distribution, connection and collection can be repeated 2 more times (2 iterations). After connecting the members of each adapter group, the cross-linked chromatin aggregates can be attached in series to multiple barcodes. In some cases, the sequential connection of multiple members of multiple adapter groups (iterations) leads to barcode combinations. The number of available barcode combinations depends on the number of groups in each iteration and the total number of barcode oligonucleotides used. For example, 3 iterations containing 8 members each time can have 83 possible combinations. In some cases, the barcode combination is unique. In some cases, the barcode combination is redundant. The total number of barcode combinations can be adjusted by increasing or decreasing the number of groups receiving unique barcodes and / or increasing or decreasing the number of iterations. When more than one adapter group is used, iterative adapter attachment can be performed using the distribution, attachment and collection scheme. In some cases, the scheme of distribution, attachment and collection can be repeated at least 3, 4, 5, 6, 7, 8, 9 or 10 times. In some cases, members of the last adaptor set include sequences that are subsequently used to enrich for adaptor-attached DNA, for example, during preparation of a sequencing library by PCR amplification.

[0220] In additional aspects, the methods herein involving digestion of whole cells or whole cell nuclei do not include a shearing step.

[0221] In further aspects of the method of digesting whole cells or whole nuclei herein, the method includes obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0222] In additional aspects of the method of digesting whole cells or whole nuclei herein, the method includes mapping a first read pair to a set of contigs and determining a path through a representative of the set of contigs to the order and / or orientation of the genome.

[0223] In further aspects of the method of digesting whole cells or whole cell nuclei herein, the method includes mapping a first read pair to a set of contigs; and determining the presence of a structural variant or loss of heterozygosity in a stabilized biological sample from the set of contigs.

[0224] In additional aspects herein relating to methods of digesting a whole cell or a whole nucleus, the method includes mapping a first read pair onto a set of contigs and assigning variants in the set of contigs to phases.

[0225] In further aspects of the method of digesting whole cells or whole cell nuclei described herein, the method includes mapping a first read pair to a set of overlapping groups; determining the presence of a variant in the set of overlapping groups from the set of overlapping groups; and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or treatment course of the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of a drug in the stabilized biological sample.

[0226] Hi-C methods with low nucleic acid input requirements

[0227] The present invention further provides a method comprising obtaining a stabilized sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein; contacting the stabilized biological sample with a DNase to cut the nucleic acid molecule into a plurality of segments; and attaching the first segment and the second segment in the plurality of segments at a splicing sequence, wherein the stabilized biological sample comprises less than 3,000,000 cells or less than 10 μg DNA. In some cases, the stabilized biological sample comprises less than 3,000,000 cells. In some cases, the stabilized biological sample comprises less than 2,000,000 cells. In some cases, the stabilized biological sample comprises less than 1,000,000 cells. In some cases, the stabilized biological sample comprises less than 500,000 cells. In some cases, the stabilized biological sample comprises less than 400,000 cells. In some cases, the stabilized biological sample comprises less than 300,000 cells. In some cases, the stabilized biological sample comprises less than 200,000 cells. In some cases, the stabilized biological sample comprises less than 100,000 cells. In some cases, the stabilized biological sample comprises less than 50,000 cells. In some cases, the stabilized biological sample comprises less than 40,000 cells. In some cases, the stabilized biological sample comprises less than 30,000 cells. In some cases, the stabilized biological sample comprises less than 20,000 cells. In some cases, the stabilized biological sample comprises less than 10,000 cells. In some cases, the stabilized biological sample comprises about 10,000 cells. In some cases, the sample comprises at least 10,000 cells. In some cases, the sample comprises at least 20,000 cells. In some cases, the sample comprises at least 30,000 cells. In some cases, the sample comprises at least 40,000 cells. In some cases, the sample comprises from about 10,000 cells to about 50,000 cells. In some cases, the sample comprises about 20,000 cells to about 50,000 cells. In some cases, the sample comprises about 30,000 cells to about 50,000 cells. In some cases, the sample comprises about 40,000 cells to about 50,000 cells. In some cases, the sample comprises about 10,000 cells to about 40,000 cells. In some cases, the sample comprises about 10,000 cells to about 30,000 cells. In some cases, the sample comprises about 10,000 cells to about 20,000 cells. In some cases, the sample comprises about 20,000 cells to about 50,000 cells. In some cases, the sample comprises about 20,000 cells to about 40,000 cells. In some cases, the sample comprises about 20,000 cells to about 30,000 cells. In some cases, the sample contains about 30,000 cells to about 50,000 cells.In some cases, the sample comprises about 30,000 cells to about 40,000 cells. In some cases, the stabilized biological sample comprises less than 10 μg DNA. In some cases, the stabilized biological sample comprises less than 9 μg DNA. In some cases, the stabilized biological sample comprises less than 8 μg DNA. In some cases, the stabilized biological sample comprises less than 7 μg DNA. In some cases, the stabilized biological sample comprises less than 6 μg DNA. In some cases, the stabilized biological sample comprises less than 5 μg DNA. In some cases, the stabilized biological sample comprises less than 4 μg DNA. In some cases, the stabilized biological sample comprises less than 3 μg DNA. In some cases, the stabilized biological sample comprises less than 2 μg DNA. In some cases, the stabilized biological sample comprises less than 1 μg DNA. In some cases, the stabilized biological sample comprises less than 0.5 μg DNA.

[0228] In various aspects of the methods herein, the stabilized sample may include nuclei. In some cases, the stabilized sample includes no more than 50,000 nuclei. In some cases, the sample includes no more than 40,000 nuclei. In some cases, the sample includes no more than 30,000 nuclei. In some cases, the sample includes no more than 20,000 nuclei. In some cases, the sample includes at least 10,000 nuclei. In some cases, the sample includes at least 20,000 nuclei. In some cases, the sample includes at least 30,000 nuclei. In some cases, the sample includes at least 40,000 nuclei. In some cases, the sample includes about 10,000 nuclei to about 50,000 nuclei. In some cases, the sample includes about 20,000 nuclei to about 50,000 nuclei. In some cases, the sample includes about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample comprises about 40,000 nuclei to about 50,000 nuclei. In some cases, the sample comprises about 10,000 nuclei to about 40,000 nuclei. In some cases, the sample comprises about 10,000 nuclei to about 30,000 nuclei. In some cases, the sample comprises about 10,000 nuclei to about 20,000 nuclei. In some cases, the sample comprises about 20,000 nuclei to about 50,000 nuclei. In some cases, the sample comprises about 20,000 nuclei to about 40,000 nuclei. In some cases, the sample comprises about 20,000 nuclei to about 30,000 nuclei. In some cases, the sample comprises about 30,000 nuclei to about 50,000 nuclei. In some cases, the sample comprises about 30,000 nuclei to about 40,000 nuclei.

[0229] On the other hand, methods with low nucleic acid input requirements herein can be performed on individual or single cells. For example, methods herein can be performed on cells distributed to various partitions. Exemplary partitions include, but are not limited to, droplets in wells, emulsions, or surface locations (e.g., array spots, beads, etc.) comprising different portions of differential sequence adapter molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with methods, compositions, and systems disclosed herein.

[0230] On the other hand, the method with low nucleic acid input demand herein includes size selection of multiple segments to obtain multiple selection segments. In some cases, multiple selection segments are about 145bp to about 600bp. In some cases, multiple selection segments are about 100bp to about 2500bp. In some cases, multiple selection segments are about 100bp to about 600bp. In some cases, multiple selection segments are about 600bp to about 2500bp. In some cases, the plurality of selected segments are between about 100 bp and about 600 bp, between about 100 bp and about 700 bp, between about 100 bp and about 800 bp, between about 100 bp and about 900 bp, between about 100 bp and about 1000 bp, between about 100 bp and about 1100 bp, between about 100 bp and about 1200 bp, between about 100 bp and about 1300 bp, between about 100 bp and about 1400 bp, between about 100 bp and about 1500 bp, or between about 100 bp and about 1600 bp. bp, between about 100bp and about 1600bp, between about 100bp and about 1700bp, between about 100bp and about 1800bp, between about 100bp and about 1900bp, between about 100bp and about 2000bp, between about 100bp and about 2100bp, between about 100bp and about 2200bp, between about 100bp and about 2300bp, between about 100bp and about 2400bp, or between about 100bp and about 2500bp.

[0231] In another aspect of the method with low nucleic acid input requirements provided herein, the method further includes, before the size selection step, preparing a sequencing library from a plurality of segments. In some embodiments, the method further includes size selection of the sequencing library to obtain a size-selected library. In some cases, the size of the size-selected library is between about 350bp and about 1000bp. In some cases, the size of the size-selected library is between about 100bp and about 2500bp, for example, between about 100bp and about 350bp, between about 350bp and about 500bp, between about 500bp and about 1000bp, between about 1000 and about 1500bp and about 2000bp, between about 2000bp and about 2500bp, between about 350bp and about 1000bp, between about 350bp and about 1500bp, between about 350bp and about 2000bp , between about 350bp and about 2500bp, between about 500bp and about 1500bp, between about 500bp and about 2000bp, between about 500bp and about 3500bp, between about 1000bp and about 1500bp, between about 1000bp and about 2000bp, between about 1000bp and about 2500bp, between about 1500bp and about 2000bp, between about 1500bp and about 2500bp, or between about 2000bp and about 2500bp.

[0232] Size selection used in the methods herein with low nucleic acid input requirements is typically performed using gel electrophoresis, capillary electrophoresis, size selection beads, gel filtration columns, or a combination thereof.

[0233] On the other hand, the method with low nucleic acid input demand herein can further include analyzing multiple selection segments to obtain QC values. In some cases, QC values ​​are selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI). CDE is calculated as the ratio of the segment with desired length. For example, in some cases, CDE is calculated as the ratio of the segment of 100bp to 2500bp before size selection. In some cases, when the CDE value is at least 65%, select sample for further analysis. In some cases, when the CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, select sample for further analysis. CDI is calculated as the ratio of the segment quantity of mononucleosome size to the segment quantity of dinucleosome size before size selection. For example, the CDI can be calculated as the logarithm of the ratio of fragments of size 600-2500 bp to fragments of size 100-600 bp. In some cases, a sample is selected for further analysis when the CDI value is greater than -1.5 and less than 1. In some cases, a sample is selected for further analysis when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 and less than about 1.5, When the value is greater than about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample is selected for further analysis.

[0234] On the other hand, the stabilized biological sample used in the method with low nucleic acid input demand herein includes biological material treated with a stabilizer. In some cases, the stabilized biological sample includes a stabilized cell lysate. Alternatively, the stabilized biological sample includes a stabilized intact cell. Alternatively, the stabilized biological sample includes a stabilized intact nucleus. In some cases, contacting a stabilized intact cell or intact nucleus sample with DNase is performed before the cracking of the intact cell or intact nucleus. In some cases, the first segment and the second segment in a plurality of segments are attached at the splicing sequence before the lysed cell and / or nucleus.

[0235] In other aspects, the stabilized biological sample used in the method with low nucleic acid input demand herein is treated with a nuclease such as DNase to produce a fragment of DNA. In some cases, DNase is non-sequence specific. In some cases, DNase has activity to both single-stranded DNA and double-stranded DNA. In some cases, DNase has specificity to double-stranded DNA. In some cases, DNase preferentially cuts double-stranded DNA. In some cases, DNase has specificity to single-stranded DNA. In some cases, DNase preferentially cuts single-stranded DNA. In some cases, DNase is DNase I. In some cases, DNase is DNase II. In some cases, DNase is selected from one or more of DNase I and DNase II. In some cases, DNase is micrococcal nuclease. In some cases, DNase is selected from one or more of DNase I and DNase II and micrococcal nuclease. In some cases, DNase can be coupled or fused with immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L or its fragment. Other suitable nucleases are also within the scope of the present disclosure.

[0236] In other aspects, the stabilized biological sample used in the method with low nucleic acid input demand is treated with a cross-linking agent. In some cases, the cross-linking agent is a chemical fixative. In some cases, the chemical fixative comprises formaldehyde, which has a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length. For example, the cross-linking agent can have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative can include ethylene glycol bis (succinimidyl succinate) (EGS), which has a spacer arm of about 16.1A in length. The chemical fixative can include disuccinimidyl glutarate (DSG), which has a spacer arm of about 7.7A in length. In some cases, the chemical fixative comprises formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In some cases where multiple chemical fixatives are used, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with large (e.g., >1kb) read pair separation distances. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative comprises psoralen. In some cases, the cross-linking agent is ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample is a paraffin-embedded tissue sample cross-linked.

[0237] In a further aspect, the methods provided herein comprise contacting a plurality of selection segments with the antibody.

[0238] In other aspects, the method with low nucleic acid input demand provided herein includes attaching the first section and the second section in a plurality of sections at the splicing sequence. In some cases, attachment includes filling the sticky end with biotin-tagged nucleotides and connecting the flat ends. In some cases, attachment includes contacting at least the first section and the second section with a bridging oligonucleotide. In some cases, attachment includes contacting at least the first section and the second section with a barcode. In some embodiments, the length of the bridging oligonucleotide herein can be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein can be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide can be 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 35, about 40, about 45 or about 50 nucleotides. In some embodiments, the bridging oligonucleotide herein may comprise a barcode. In some embodiments, the bridging oligonucleotide may comprise a plurality of barcodes. In some embodiments, the bridging oligonucleotide comprises a plurality of bridging oligonucleotides linked together. In some embodiments, the bridging oligonucleotide may be coupled or connected to an immunoglobulin binding protein such as protein A, protein G, protein A / G or protein L or a fragment thereof. In some cases, the coupled bridging oligonucleotide may be delivered to the position of the binding antibody in the sample nucleic acid.

[0239] The splitting and pooling method can be adopted to produce the bridging oligonucleotide with unique barcode. The sample population can be split into multiple groups, the bridging oligonucleotide can be attached to the sample so that the bridging oligonucleotide barcode is different between groups but the same within a group, the sample group can be pooled together again, and the process can be repeated multiple times. Iteration of this process will eventually lead to each sample in the population having a series of unique bridging oligonucleotide barcodes, thereby allowing single sample (e.g., single cell, single cell nucleus, single chromosome) analysis. In an illustrative example, the cross-linked digested nucleus sample attached to the bead solid support is split into 8 test tubes, each test tube containing 1 of 8 unique members of the first adapter group (first iteration), and the first adapter group includes double-stranded DNA (dsDNA) adapters to be connected. Each of the 8 adapters can have the same 5' overhang sequence for connecting to the nucleic acid end of the cross-linked chromatin aggregate in the nucleus, but has a unique dsDNA sequence in other aspects. After the first adapter group is connected, the nucleus can be pooled back together and washed to remove the connection reaction components. The scheme of distribution, connection and collection can be repeated 2 more times (2 iterations). After connecting the members of each adapter group, the cross-linked chromatin aggregates can be attached in series to multiple barcodes. In some cases, the sequential connection of multiple members of multiple adapter groups (iterations) leads to barcode combinations. The number of available barcode combinations depends on the number of groups in each iteration and the total number of barcode oligonucleotides used. For example, 3 iterations containing 8 members each time can have 83 possible combinations. In some cases, the barcode combination is unique. In some cases, the barcode combination is redundant. The total number of barcode combinations can be adjusted by increasing or decreasing the number of groups receiving unique barcodes and / or increasing or decreasing the number of iterations. When more than one adapter group is used, iterative adapter attachment can be performed using the distribution, attachment and collection scheme. In some cases, the scheme of distribution, attachment and collection can be repeated at least 3, 4, 5, 6, 7, 8, 9 or 10 times. In some cases, members of the last adaptor set include sequences that are subsequently used to enrich for adaptor-attached DNA, for example, during preparation of a sequencing library by PCR amplification.

[0240] In additional aspects, the methods herein having low nucleic acid input requirements do not include a shearing step.

[0241] In further aspects of the methods herein having low nucleic acid input requirements, the method includes obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0242] In additional aspects of the methods herein having low nucleic acid input requirements, the methods include mapping a first read pair onto a set of contigs and determining a path through a representative of the set of contigs to an order and / or orientation of the genome.

[0243] In further aspects of the methods herein having low nucleic acid input requirements, the methods include mapping the first read pairs onto a set of contigs and determining the presence of a structural variant or loss of heterozygosity in the stabilized biological sample from the set of contigs.

[0244] In additional aspects of the methods herein having low nucleic acid input requirements, the methods include mapping the first read pairs onto a set of contigs and assigning variants in the set of contigs to phases.

[0245] In a further aspect of the method having low nucleic acid input requirements herein, the method includes mapping a first read pair to a set of overlapping groups; determining the presence of a variant in the set of overlapping groups from the set of overlapping groups; and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or treatment course of the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of a drug in the stabilized biological sample.

[0246] Hi-C method using micrococcal nuclease (MNase)

[0247] Additionally, the methods provided herein may include obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein; contacting the stabilized biological sample with micrococcal nuclease (MNase) to cut the nucleic acid molecule into multiple segments; and attaching the first segment and the second segment of the multiple segments at the splicing sequence. The use of MNase in the methods herein can provide specific information about where the DNA binding protein binds to chromatin at up to a single base pair resolution, because, for example, MNase can cut all base pairs that are not bound to the DNA binding protein. In addition, the use of MNase digestion can allow the creation of contact maps and topologically related domains to decipher three-dimensional chromatin structure information. In some cases, MNase can be coupled or fused to immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L, or fragments thereof.

[0248] For example, the MNase Hi-C method can provide the location of protein binding or genomic contact interactions at a resolution of less than or equal to about 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 2000bp, 3000bp, 4000bp, 5000bp, 6000bp, 7000bp, 8000bp, 9000bp, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb or 100kb. In some cases, protein binding sites, protein footprints, contact interactions, or other features can be mapped to within 1000 bp, within 900 bp, within 800 bp, within 700 bp, within 600 bp, within 500 bp, within 400 bp, within 300 bp, within 200 bp, within 190 bp, within 180 bp, within 170 bp, within 160 bp, within 150 bp, within 140 bp, within 160 bp, within 170 bp, within 180 bp, within 190 bp, within 18 ... Within 0bp, within 130bp, within 120bp, within 110bp, within 100bp, within 90bp, within 80bp, within 70bp, within 60bp, within 50bp, 40b Within p, within 30bp, within 20bp, within 10bp, within 9bp, within 8bp, within 7bp, within 6bp, within 5bp, within 4bp, within 3bp, within 2bp or within 1bp.

[0249] In some aspects, the method involving the MNase digestion step can further include size selection of multiple segments to obtain multiple selection segments. In some cases, multiple selection segments can be about 145bp to about 600bp. In some cases, multiple selection segments can be about 100bp to about 2500bp. In some cases, multiple selection segments can be about 100 to about 600bp. In some cases, multiple selection segments can be about 600bp to about 2500bp. In some cases, the plurality of selected segments may be from about 100 bp to about 600 bp, from about 100 bp to about 700 bp, from about 100 bp to about 800 bp, from about 100 bp to about 900 bp, from about 100 bp to about 1000 bp, from about 100 bp to about 1100 bp, from about 100 bp to about 1200 bp, from about 100 bp to about 1300 bp, from about 100 bp to about 1400 bp, from about 100 bp to about 1500 bp, from about 100 bp to about 1600 bp, from about 100 bp to about 1700 bp, from about 100 bp to about 1800 bp, from about 100 bp to about 1900 bp, from about 100 bp to about 2000 bp, from about 100 bp to about 2100 bp, from about 100 bp to about 2200 bp, from about 100 bp to about 2300 bp, from about 100 bp to about 2400 bp, or from about 100 bp to about 2500 bp.

[0250] In another aspect of the method relating to the MNase digestion step as provided herein, the method may further include preparing a sequencing library from a plurality of segments. In some embodiments, the method may further include size-selecting the sequencing library to obtain a size-selected library. In some cases, the size of the size-selected library may be from about 350bp to about 1000bp. In some cases, the size of the size selected library can be from about 100 bp to about 2500 bp, for example, from about 100 bp to about 350 bp, from about 350 bp to about 500 bp, from about 500 bp to about 1000 bp, from about 1000 to about 1500 bp, from about 2000 bp to about 2500 bp, from about 350 bp to about 1000 bp, from about 350 bp to about 1500 bp, from about 350 bp to about 2000 bp, from about 350 bp to about 2500 bp, from about 500 bp to about 1500 bp, from about 500 bp to about 2000 bp, from about 500 bp to about 3500 bp, from about 1000 bp to about 1500 bp, from about 1000 bp to about 2000 bp, from about 1000 bp to about 2500 bp, from about 1500 bp to about 2000 bp, from about 1500 bp to about 2500 bp, or from about 2000 bp to about 2500 bp.

[0251] On the other hand, the method relating to the MNase digestion step as provided herein can further include analyzing multiple sections to obtain QC values. In some cases, QC values ​​can be selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI). CDE can be calculated as the ratio of the section with desired length. For example, in some cases, CDE can be calculated as the ratio of the section of 100bp to 2500bp before size selection. In some cases, when the CDE value is at least 65%, sample can be selected for further analysis. In some cases, when the CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, sample can be selected for further analysis.

[0252] CDI can be calculated as the ratio of the number of segments of mononucleosome size to the number of segments of dinucleosome size before size selection. For example, CDI can be calculated as the logarithm of the ratio of the fragments of 600-2500bp to the fragments of 100-600bp. In some cases, when the CDI value is greater than -1.5 and less than 1, the sample can be selected for further analysis. In some cases, when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 ...3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about At about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample can be selected for further analysis.

[0253] On the other hand, the stabilized biological sample used in the method relating to the MNase digestion step as provided herein may include a biological material that has been treated with a stabilizer. In some cases, the stabilized biological sample may include a stabilized cell lysate. Alternatively, the stabilized biological sample may include a stabilized intact cell. Alternatively, the stabilized biological sample may include a stabilized intact nucleus. In some cases, contacting a stabilized intact cell or intact nucleus sample with MNase may be performed before the cracking of the intact cell or intact nucleus. In some cases, the first segment and the second segment in a plurality of segments may be lysed before being attached at the splicing sequence.

[0254] On the other hand, the method involving the MNase digestion step provided herein can be performed on a small sample containing a small amount of cells or a small amount of nucleic acid. For example, in some cases, the stabilized biological sample may contain less than 3,000,000 cells. In some cases, the stabilized biological sample may contain less than 2,000,000 cells. In some cases, the stabilized biological sample may contain less than 1,000,000 cells. In some cases, the stabilized biological sample may contain less than 500,000 cells. In some cases, the stabilized biological sample may contain less than 400,000 cells. In some cases, the stabilized biological sample may contain less than 300,000 cells. In some cases, the stabilized biological sample may contain less than 200,000 cells. In some cases, the stabilized biological sample may contain less than 100,000 cells. In some cases, the stabilized biological sample contains less than 50,000 cells. In some cases, the stabilized biological sample contains less than 40,000 cells. In some cases, the stabilized biological sample contains less than 30,000 cells. In some cases, the stabilized biological sample contains less than 20,000 cells. In some cases, the stabilized biological sample contains less than 10,000 cells. In some cases, the stabilized biological sample contains about 10,000 cells. In some cases, the stabilized biological sample may contain less than 10 μg DNA. In some cases, the stabilized biological sample may contain less than 9 μg DNA. In some cases, the stabilized biological sample may contain less than 8 μg DNA. In some cases, the stabilized biological sample may contain less than 7 μg DNA. In some cases, the stabilized biological sample may contain less than 6 μg DNA. In some cases, the stabilized biological sample may contain less than 5 μg DNA. In some cases, the stabilized biological sample may contain less than 4 μg DNA. In some cases, the stabilized biological sample may contain less than 3 μg DNA. In some cases, the stabilized biological sample may contain less than 2 μg DNA. In some cases, the stabilized biological sample contains less than 1 μg DNA. In some cases, the stabilized biological sample contains less than 0.5 μg DNA.

[0255] In another aspect, the methods herein involving the MNase digestion step can be performed on individual or single cells. For example, the methods herein can be performed on cells distributed into various partitions. Exemplary partitions include, but are not limited to, wells, droplets in emulsions, or surface locations (e.g., array spots, beads, etc.) comprising different portions of differential sequence adapter molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with the methods, compositions, and systems disclosed herein.

[0256] In other aspects, the stabilized biological sample used in the method for the MNase digestion step can be further processed with additional nucleases such as DNase to produce fragments of DNA. In some cases, DNase can be non-sequence specific. In some cases, DNase can be active to both single-stranded DNA and double-stranded DNA. In some cases, DNase can be specific to double-stranded DNA. In some cases, DNase can preferentially cut double-stranded DNA. In some cases, DNase can be specific to single-stranded DNA. In some cases, DNase can preferentially cut single-stranded DNA. In some cases, DNase can be DNase I. In some cases, DNase can be DNase II. In some cases, DNase can be selected from one or more of DNase I and DNase II. In some cases, DNase can be coupled or fused to immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L or their fragments. Other suitable nucleases are also within the scope of the present disclosure.

[0257] In other aspects, the stabilized biological sample as provided herein for use in the method involving the MNase digestion step can be treated with a cross-linking agent. In some cases, the cross-linking agent can be a chemical fixative. In some cases, the chemical fixative comprises formaldehyde, which has a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length, for example, the cross-linking agent can have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative can include ethylene glycol bis (succinimidyl succinate) (EGS), which has a spacer arm of about 16.1A in length. The chemical fixative can include disuccinimidyl glutarate (DSG), which has a spacer arm of about 7.7A in length. In some cases, the chemical fixative comprises formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In the case of using multiple chemical fixatives, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with large (e.g., >1kb) read pair separation distances. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative may comprise psoralen. In some cases, the cross-linking agent can be ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample can be a paraffin-embedded tissue sample cross-linked.

[0258] In a further aspect, the methods provided herein involving a MNase digestion step can include contacting a plurality of selection segments with an antibody. In some cases, an immunoglobulin binding protein or fragment thereof linked to an oligonucleotide adaptor can be targeted to an antibody that binds to a plurality of selection segments.

[0259] In other aspects, the method related to the MNase digestion step provided herein may include attaching the first section and the second section in a plurality of sections at the splicing sequence. In some cases, attachment may include filling the sticky ends with biotin-tagged nucleotides and connecting the flat ends. In some cases, attachment may include contacting at least the first section and the second section with a bridging oligonucleotide. In some cases, attachment may include contacting at least the first section and the second section with a barcode. In some embodiments, the length of the bridging oligonucleotide herein may be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein may be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide may be 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 35, about 40, about 45 or about 50 nucleotides. In some embodiments, the bridging oligonucleotides herein may comprise a barcode.

[0260] In further aspects of the methods herein involving a MNase digestion step, the method can include obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0261] In additional aspects of the methods herein involving a MNase digestion step, the methods can include mapping the first read pairs to a set of contigs, and determining a path through the representatives of the set of contigs to the order and / or orientation of the genome.

[0262] In further aspects of the methods herein involving a MNase digestion step, the methods can include mapping the first read pairs to a set of contigs; and determining the presence of a structural variant or loss of heterozygosity in the stabilized biological sample from the set of contigs.

[0263] In further aspects of the methods herein involving a MNase digestion step, the methods can include mapping the first read pairs onto a set of contigs and assigning variants in the set of contigs to phases.

[0264] In further aspects of the methods herein involving a MNase digestion step, the method can include mapping a first read pair to a set of overlapping groups, determining the presence of a variant in the set of overlapping groups from the set of overlapping groups, and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or treatment course of the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of a drug in the stabilized biological sample.

[0265] Improved methods for HiChIP, HiChIRP and Methyl-HiC

[0266] HiChIP is a method that combines HiC methods with chromatin immunoprecipitation methods, allowing targeted analysis of interactions involving one or more proteins of interest. Proximity-linked nucleic acids can be prepared, and the targeted regions can be immunoprecipitated for further analysis. A related method, HiChIRP, uses chromatin isolation (ChIRP) enrichment by RNA purification combined with HiC methods to enable the study of RNA, such as the scaffolding function of long non-coding RNAs (lncRNAs). Methyl HiC combines methylation analysis with HiC methods, allowing the simultaneous capture of chromosome conformation and DNA methylome information. Methyl HiC can reveal coordinated DNA methylation states between spatially adjacent distal genomic segments in the nucleus, depict the heterogeneity of chromatin structure and DNA methylome in mixed populations, and can simultaneously characterize cell type-specific chromatin organization and epigenomes in complex tissues. These methods and other methods can be improved by using the techniques of the present disclosure, including but not limited to size selection steps, surface binding steps (e.g., binding to beads such as SPRI beads), proximity ligation using bridging oligonucleotides, proximity ligation using recombination, and the like.

[0267] In another aspect, the present invention provides improved methods for HiChIP, HiChIRP and methyl HiC, which may include obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein, for example, by immunoprecipitating nucleic acid bound to nucleic acid binding protein or by immunoprecipitating methylated nucleic acid; contacting the stabilized biological sample with DNase to cut the nucleic acid molecule into multiple segments; attaching the first segment and the second segment of the multiple segments at a splicing sequence; and size-selecting the multiple segments to obtain multiple selected segments. Alternatively or in combination, the methods herein may include obtaining a stabilized biological sample comprising a nucleic acid molecule complexed with at least one nucleic acid binding protein, for example, by immunoprecipitating nucleic acid bound to nucleic acid binding protein or by immunoprecipitating methylated nucleic acid; contacting the stabilized biological sample with micrococcal nuclease (MNase) to cut the nucleic acid molecule into multiple segments; and attaching the first segment and the second segment of the multiple segments at a splicing sequence.

[0268] In some aspects of the improved methods for HiChIP, HiChIRP and methyl HiC herein, the stabilized biological sample may include intact cells and / or intact cell nuclei. In some cases, the stabilized biological sample may include stabilized intact cells. Alternatively or in combination, the stabilized biological sample may include stabilized intact cell nuclei. In some cases, contacting the stabilized intact cell or intact cell nucleus sample with DNase may be performed prior to the cleavage of the intact cell or intact cell nucleus. In some cases, the cells and / or cell nuclei may be lysed before the first segment and the second segment in the plurality of segments are attached at the splicing sequence.

[0269] In another aspect, methods of improved methods of HiChIP, HiChIRP, and Methyl HiC herein may include size selecting a plurality of segments to obtain a plurality of selected segments. In some cases, the plurality of selected segments may be about 145 to about 600 bp. In some cases, the plurality of selected segments may be about 100 to about 2500 bp. In some cases, the plurality of selected segments may be about 100 to about 600 bp. In some cases, the plurality of selected segments may be about 600 to about 2500 bp. In some cases, the plurality of selected segments may be from about 100 bp to about 600 bp, from about 100 bp to about 700 bp, from about 100 bp to about 800 bp, from about 100 bp to about 900 bp, from about 100 bp to about 1000 bp, from about 100 bp to about 1100 bp, from about 100 bp to about 1200 bp, from about 100 bp to about 1300 bp, from about 100 bp to about 1400 bp, from about 100 bp to about 1500 bp, from about 100 bp to about 1600 bp, from about 100 bp to about 1700 bp, from about 100 bp to about 1800 bp, from about 100 bp to about 1900 bp, from about 100 bp to about 2000 bp, from about 100 bp to about 2100 bp, from about 100 bp to about 2200 bp, from about 100 bp to about 2300 bp, from about 100 bp to about 2400 bp, or from about 100 bp to about 2500 bp.

[0270] In another aspect of the methods herein relating to improved methods of HiChIP, HiChIRP, and Methyl HiC, the method may further comprise, prior to the size selection step, preparing a sequencing library from the plurality of segments. In some embodiments, the method may further comprise size selecting the sequencing library to obtain a size selected library. In some cases, the size of the size selected library may be about 350 bp to about 1000 bp. In some cases, the size of the size selected library can be from about 100 bp to about 2500 bp, for example, from about 100 bp to about 350 bp, from about 350 bp to about 500 bp, from about 500 bp to about 1000 bp, from about 1000 to about 1500 bp, from about 2000 bp to about 2500 bp, from about 350 bp to about 1000 bp, from about 350 bp to about 1500 bp, from about 350 bp to about 2000 bp, from about 350 bp to about 2500 bp, from about 500 bp to about 1500 bp, from about 500 bp to about 2000 bp, from about 500 bp to about 3500 bp, from about 1000 bp to about 1500 bp, from about 1000 bp to about 2000 bp, from about 1000 bp to about 2500 bp, from about 1500 bp to about 2000 bp, from about 1500 bp to about 2500 bp, or from about 2000 bp to about 2500 bp.

[0271] Size selection used in the methods herein involving improved methods of HiChIP, HiChIRP, and Methyl HiC can be performed using gel electrophoresis, capillary electrophoresis, size selection beads, gel filtration columns, combinations thereof, or any other suitable method.

[0272] On the other hand, the method for improving the method of HiChIP, HiChIRP and methyl HiC herein may include further analyzing multiple selection segments to obtain QC values. In some cases, the QC value may be selected from chromatin digestion efficiency (CDE) and chromatin digestion index (CDI). CDE can be calculated as the ratio of the segment with the desired length. For example, in some cases, CDE can be calculated as the ratio of the segment with a size of 100 to 2500bp before size selection. In some cases, when the CDE value is at least 65%, the sample can be selected for further analysis. In some cases, when the CDE value is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%, the sample can be selected for further analysis.

[0273] CDI can be calculated as the ratio of the number of segments of mononucleosome size to the number of segments of dinucleosome size before size selection. For example, CDI can be calculated as the logarithm of the ratio of the fragments of 600-2500bp to the fragments of 100-600bp. In some cases, when the CDI value is greater than -1.5 and less than 1, the sample can be selected for further analysis. In some cases, when the CDI value is greater than about -2 and less than about 1.5, greater than about -1.9 and less than about 1.5, greater than about -1.8 and less than about 1.5, greater than about -1.7 and less than about 1.5, greater than about -1.6 and less than about 1.5, greater than about -1.5 and less than about 1.5, greater than about -1.4 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.2 and less than about 1.5, greater than about -1.1 and less than about 1.5, greater than about -2 and less than about 1.5, greater than about -1 and less than about 1.5, greater than about -0.9 ...3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about -1.3 and less than about 1.5, greater than about At about -0.8 and less than about 1.5, greater than about -0.7 and less than about 1.5, greater than about -0.6 and less than about 1.5, greater than about -0.5 and less than about 1.5, greater than about -2 and less than about 1.4, greater than about -2 and less than about 1.3, greater than about -2 and less than about 1.2, greater than about -2 and less than about 1.1, greater than about -2 and less than about 1, greater than about -2 and less than about 0.9, greater than about -2 and less than about 0.8, greater than about -2 and less than about 0.7, greater than about -2 and less than about 0.6, or greater than about -2 and less than about 0.5, the sample can be selected for further analysis.

[0274] In another aspect, methods involving improved methods of HiChIP, HiChIRP and methyl HiC herein can be performed on small samples containing a small number of cells or a small amount of nucleic acid. In some cases, the stabilized biological sample may contain less than 3,000,000 cells. In some cases, the stabilized biological sample may contain less than 2,000,000 cells. In some cases, the stabilized biological sample may contain less than 1,000,000 cells. In some cases, the stabilized biological sample may contain less than 500,000 cells. In some cases, the stabilized biological sample may contain less than 400,000 cells. In some cases, the stabilized biological sample may contain less than 300,000 cells. In some cases, the stabilized biological sample may contain less than 200,000 cells. In some cases, the stabilized biological sample may contain less than 100,000 cells. In some cases, the stabilized biological sample contains less than 50,000 cells. In some cases, the stabilized biological sample contains less than 40,000 cells. In some cases, the stabilized biological sample contains less than 30,000 cells. In some cases, the stabilized biological sample contains less than 20,000 cells. In some cases, the stabilized biological sample contains less than 10,000 cells. In some cases, the stabilized biological sample contains about 10,000 cells. In some cases, the stabilized biological sample may contain less than 10 μg DNA. In some cases, the stabilized biological sample may contain less than 9 μg DNA. In some cases, the stabilized biological sample may contain less than 8 μg DNA. In some cases, the stabilized biological sample may contain less than 7 μg DNA. In some cases, the stabilized biological sample may contain less than 6 μg DNA. In some cases, the stabilized biological sample may contain less than 5 μg DNA. In some cases, the stabilized biological sample may contain less than 4 μg DNA. In some cases, the stabilized biological sample may contain less than 3 μg DNA. In some cases, the stabilized biological sample may contain less than 2 μg DNA. In some cases, the stabilized biological sample can contain less than 1 μg DNA. In some cases, the stabilized biological sample can contain less than 0.5 μg DNA.

[0275] In another aspect, the methods herein relating to improved methods of HiChIP, HiChIRP and Methyl HiC can be performed on individual or single cells. For example, the methods herein can be performed on cells distributed into various partitions. Exemplary partitions include, but are not limited to, wells, droplets in an emulsion, or surface locations (e.g., array spots, beads, etc.) comprising different portions of differential sequence linker molecules as described elsewhere herein. Additional partitions are also contemplated and consistent with the methods, compositions and systems disclosed herein.

[0276] In other aspects, the stabilized biological samples used in the methods for the improved methods of HiChIP, HiChIRP and methyl HiC herein can be treated with nucleases such as DNase to produce fragments of DNA. In some cases, DNase can be non-sequence specific. In some cases, DNase can be active on both single-stranded DNA and double-stranded DNA. In some cases, DNase can be specific to double-stranded DNA. In some cases, DNase can preferentially cut double-stranded DNA. In some cases, DNase can be specific to single-stranded DNA. In some cases, DNase can preferentially cut single-stranded DNA. In some cases, DNase can be DNase I. In some cases, DNase can be DNase II. In some cases, DNase can be selected from one or more of DNase I and DNase II. In some cases, DNase can be micrococcal nuclease. In some cases, DNase can be selected from one or more of DNase I and DNase II and micrococcal nuclease. In some cases, DNase can be coupled or fused to immunoglobulin binding proteins such as protein A, protein G, protein A / G or protein L or fragments thereof. Other suitable nucleases are also within the scope of the present disclosure.

[0277] In other aspects, the stabilized biological sample used in the method for the improved method of HiChIP, HiChIRP and methyl HiC described herein can be treated with a cross-linking agent. In some cases, the cross-linking agent can be a chemical fixative. In some cases, the chemical fixative comprises formaldehyde having a spacer arm length of about 2.3-2.7 angstroms (A). In some cases, the chemical fixative comprises a cross-linking agent with a long spacer arm length. For example, the cross-linking agent may have a spacer arm length of at least about 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A or 20A. The chemical fixative may comprise ethylene glycol bis (succinimidyl succinate) (EGS) having a spacer arm length of about 16.1A. The chemical fixative may comprise disuccinimidyl glutarate (DSG) having a spacer arm length of about 7.7A. In some cases, the chemical fixative comprises formaldehyde and EGS, formaldehyde and DSG, or formaldehyde, EGS and DSG. In the case of using multiple chemical fixatives, each chemical fixative is used sequentially; in other cases, some or all of the multiple chemical fixatives are applied to the sample at the same time. The use of a cross-linking agent with a long spacer arm can increase the fraction of read pairs with large (e.g., >1kb) read pair separation distances. DSG is membrane permeable and can be cross-linked intracellularly. In some applications, DSG can improve cross-linking efficiency compared to disuccinimidyl suberate (DSS). EGS has NHS ester reactive groups at both ends and can react with amino groups (e.g., primary amines). EGS is membrane permeable and can be cross-linked intracellularly. For example, EGS cross-linking can be reversed by treatment with hydroxylamine at pH 8.5 for 3 to 6 hours; in one example, lactose dehydrogenase retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative may comprise psoralen. In some cases, the cross-linking agent can be ultraviolet light, nitrogen mustard, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis (2-chloroethyl) ethylamine, bis (2-chloroethyl) methylamine, tris (2-chloroethyl) amine, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, cisplatin, carboplatin, dicycloplatin, ebplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, seraplatin, tetranitrate triplatin, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetyl aldehyde, doxorubicin, daunorubicin, epirubicin or idarubicin. In some cases, the cross-linking agent comprises an embedding agent, an antibiotic or a minor groove binding agent. In some cases, the stabilization biological sample can be a paraffin-embedded tissue sample cross-linked.

[0278] In other aspects, methods for improved methods of HiChIP, HiChIRP, and methyl HiC herein may include attaching a first segment and a second segment of a plurality of segments at a splicing sequence. In some cases, the attachment may include filling the sticky ends with biotin-tagged nucleotides and connecting the flat ends. In some cases, the attachment may include contacting at least the first segment and the second segment with a bridging oligonucleotide. In some cases, the attachment may include contacting at least the first segment and the second segment with a barcode. In some embodiments, the length of the bridging oligonucleotide herein may be at least about 5 nucleotides to about 50 nucleotides. In some embodiments, the length of the bridging oligonucleotide herein may be about 15 to about 18 nucleotides. In some embodiments, the length of the bridging oligonucleotide can be 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 35, about 40, about 45, or about 50 nucleotides. In some embodiments, the bridging oligonucleotide herein can comprise a barcode.

[0279] In additional aspects, the methods described herein involving improved methods of HiChIP, HiChIRP, and Methyl-HiC do not include a shearing step.

[0280] In further aspects of the methods herein relating to improved methods of HiChIP, HiChIRP, and Methyl HiC, the methods can include obtaining at least some sequence on each side of the spliced ​​sequence to generate a first read pair. For example, the method can include obtaining at least about 50 bp, at least about 100 bp, at least about 150 bp, at least about 200 bp, at least about 250 bp, or at least about 300 bp of sequence on each side of the spliced ​​sequence to generate a first read pair.

[0281] In further aspects of the methods herein relating to improved methods of HiChIP, HiChIRP and Methyl HiC, the methods may include mapping a first read pair onto a set of contigs and determining a path through the representatives of the set of contigs to the order and / or orientation of the genome.

[0282] In further aspects of the methods herein relating to improved methods of HiChIP, HiChIRP and Methyl HiC, the methods may include mapping a first read pair to a set of contigs; and determining the presence of a structural variant or loss of heterozygosity in a stabilized biological sample from the set of contigs.

[0283] In further aspects of the present disclosure relating to methods of digesting whole cells or whole cell nuclei, the methods can include mapping the first read pairs onto a set of contigs and assigning variants in the set of contigs to phases.

[0284] In further aspects of the methods herein relating to improved methods for HiChIP, HiChIRP, and Methyl HiC, the methods can include mapping a first read pair to a set of contigs; determining the presence of a variant in the set of contigs from the set of contigs; and performing one or more steps selected from the following: (1) identifying a disease stage, prognosis, or course of treatment for the stabilized biological sample; (2) selecting a drug based on the presence of the variant; or (3) identifying the efficacy of a drug in the stabilized biological sample.

[0285] Generate long range read pairs

[0286] The present disclosure provides methods for generating extremely long range read pairs and using the data to advance all of the above pursuits. In some embodiments, the present disclosure provides methods for generating highly continuous and accurate human genome assemblies with only ~300 million read pairs. In other embodiments, the present disclosure provides methods for phasing 90% or more of heterozygous variants in the human genome with an accuracy of 99% or higher. In addition, the range of read pairs generated by the present disclosure can be extended to span larger genomic distances. The assembly is produced by a standard shotgun library and an extremely long range read pair library. In yet other embodiments, the present disclosure provides software that can utilize these two sequencing data sets. Phased variants are generated with a single long range read pair library, and the reads from the long range read pair library are mapped to a reference genome and then used to assign variants to one of the two parental chromosomes of an individual. Finally, the present disclosure provides extraction of even larger DNA fragments using known techniques to generate particularly long reads.

[0287] The mechanism that these repeats hinder assembly and alignment processes is quite simple and ultimately an ambiguous result. In the case of larger repeat regions, the difficulty can be one of span. If the length of the read or read pair is not enough to span the repeat region, the border region of the repeat element may not be connected credibly. In the case of smaller repeat elements, the problem may be mainly placement. When the two sides of the region are two common repeat elements in the genome, it becomes difficult or even impossible to determine its exact position because the elements on both sides are similar to all other elements in their same class. In both cases, it is precisely because of the lack of distinguishable information in the repeat that identification and thus placement of specific repeats become challenging. It is necessary to experimentally establish the ability to connect between the unique segments surrounded or separated by the repeat region.

[0288] The method of the present disclosure is to advance the field of genomics by overcoming the substantial obstacles caused by these repeat regions, thereby can make important progress in many fields of genome analysis. To use previous technology to assemble from scratch, it is necessary to either be satisfied with assembling fragments into many small scaffolds, or invest a lot of time and resources to produce large insertion libraries or use other methods to generate more continuous assemblies. Such methods can include obtaining very deep sequencing coverage, building BAC or Fox plasmid libraries, optical mapping or some combination of these and / or other technologies. A large amount of resources and time requirements make it impossible for most small laboratories to use such methods, and prevent the study of non-model organisms. Because the method described herein can produce very long range read pairs, it is possible to achieve de novo assembly by a single sequencing run. This will reduce the assembly cost by several orders of magnitude and shorten the required time from months or years to weeks. In some cases, the methods disclosed herein allow for the generation of multiple read pairs in less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or in a range between any two of the above specified time periods. For example, the method can allow for the generation of multiple read pairs in about 10 to 14 days. Even genome construction for organisms of the smallest ecological niches will become routine, phylogenetic analyses will not lack for comparison, and projects such as Genome 10k can be realized.

[0289] Similarly, structural and phasing analysis for medical purposes remains challenging. There is amazing heterogeneity between cancers, individuals with the same type of cancer, or even within the same tumor. Sorting out the causes from the effects of the results requires very high precision and throughput at a low cost for each sample. In the field of personalized medicine, one of the gold standards for genomic therapy is a sequenced genome with all variants fully characterized and phased, including large and small structural rearrangements and novel mutations. In order to achieve this standard, previous technologies require efforts similar to those required for de novo assembly, which is currently too expensive and laborious to become a routine medical procedure. The method of the present disclosure can quickly produce complete and accurate genomes at a low cost, and thus can produce many highly sought-after capabilities in the study and treatment of human diseases.

[0290] Applying the methods disclosed herein to phasing can combine the convenience of statistical methods with the accuracy of family analysis, providing savings (money, labor, and samples) compared to using either method alone. De novo variant phasing is a highly desirable phasing analysis that is prohibited by previous technologies and can be easily performed using the methods disclosed herein. This is particularly important because the vast majority of human variants are rare (minor allele frequency is less than 5%). Relative to non-joined genotypes, phasing information is valuable for population genetic studies, which gain significant advantages from highly connected haplotype networks (sets of variants assigned to a single chromosome). Haplotype information can enable higher resolution studies of historical changes in population size, migration, and exchange between subpopulations, and enables us to trace specific variants back to specific parents and grandparents. This in turn clarifies the genetic transmission of variants associated with the disease, as well as the interactions of variants when they are together in a single individual. The methods of the present disclosure can ultimately enable the preparation, sequencing, and analysis of ultra-long range read pairs (XLRP) libraries.

[0291] In some embodiments of the present disclosure, a tissue or DNA sample from a subject can be provided, and the method can return an assembled genome, an alignment with identified variants (including large structural variants), phased variant identification, or any other analysis. In other embodiments, the methods disclosed herein can provide an XLRP library directly to an individual.

[0292] Extremely long range read pairs

[0293] In various embodiments of the present disclosure, methods disclosed herein can generate extremely long ranges separated by large distances and read pairs. The upper limit of this distance can be improved by the ability to collect large-size DNA samples. In some cases, reading 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 genome distance. In some examples, reading can span up to 500 kbp genome distance. In other examples, reading can span up to 2000 kbp genome distance. Methods disclosed herein can be integrated and constructed based on standard techniques in molecular biology, and are further very suitable for improving efficiency, specificity and genome coverage. In some cases, the read pairs can be generated in less than about 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, 60, or 90 days. In some examples, the read pairs can be generated in less than about 14 days. In further examples, the read pairs can be generated in less than about 10 days. In some cases, the methods of the present disclosure can provide greater than about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% read pairs in correctly ordering and / or orienting multiple contigs with an accuracy of at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%. For example, the method can provide an accuracy of about 90% to 100% in correctly ordering and / or orienting multiple contigs.

[0294] In other embodiments, the methods disclosed herein can be used with currently used sequencing technologies. For example, the methods can be used in combination with well-tested and / or widely deployed sequencing instruments. In further embodiments, the methods disclosed herein can be used with techniques and methods derived from currently used sequencing technologies.

[0295] The method of the present disclosure can significantly simplify the de novo genome assembly of numerous organisms. Using previous technology, such assembly is currently limited to short insertions of economical mate-pair libraries. Although it is possible to generate read pairs on genome distances of up to 40-50kbp accessible to the Fox plasmid, this is expensive, cumbersome, and too short to span the longest repeat extension, including repeat extensions within the centromere, which can be 300kbp to 5Mbp in size in humans. The method disclosed herein can provide read pairs that can span large distances (e.g., megabases or longer), thereby overcoming these scaffold integrity challenges. Therefore, by utilizing the method of the present disclosure, it can be conventional to produce chromosome-level assembly. More laborious assembly approaches (currently spending a lot of time and money in research laboratories, and prohibiting the expansion of genome catalogs) can become unnecessary, thereby freeing up resources for more meaningful analysis. Similarly, obtaining long-range phasing information can provide huge additional power for population genomes, phylogeny, and disease research. The method disclosed herein can accurately phase a large number of individuals, thereby expanding the breadth and depth of our ability to detect genomes at population and deep time levels.

[0296] In the field of personalized medicine, the XLRP read pairs generated from the methods disclosed herein represent a meaningful step towards accurate, low-cost, phased, and rapidly generated personal genomes. Current methods fall short in their ability to phase variants over long distances, preventing the characterization of phenotypic effects of compound heterozygous genotypes. In addition, structural variants of major significance for genomic disease are difficult to accurately identify and characterize with current technology due to their large size compared to the reads and read pair inserts used to study them. Read pairs spanning tens of kilobases to millions of bases or longer can help alleviate this difficulty, allowing for highly parallel and personalized analysis of structural variation.

[0297] Advances in high-throughput sequencing technology are driving basic evolutionary and biomedical research. Although whole genome sequencing and assembly were once the origins of large genome sequencing centers, commercially available sequencers are now cheap enough that most research universities own one or more such machines. The cost of generating large amounts of DNA sequence data is now relatively low. However, it is still difficult to produce high-quality, highly continuous genome sequences with current technology in theory and practice. In addition, because most organisms (including humans) that people are willing to analyze are diploid, each individual has two haploid copies of the genome. At heterozygous sites (for example, the alleles provided by the mother are different from the alleles provided by the father), it is difficult to know which group of alleles comes from which parent (called haplotype phasing). This information can be used to perform many evolutionary and biomedical studies (such as disease and trait association studies).

[0298] In various embodiments, the present disclosure provides a genome assembly method that combines DNA preparation technology with paired-end sequencing technology for high-throughput discovery of short-term, intermediate, and long-term connections within a given genome. The present disclosure further provides methods for using these connections to assist in genome assembly for haplotype phasing and / or for metagenomic studies. Although the methods presented herein can be used to determine the assembly of a subject's genome, it should also be understood that the methods presented herein can also be used to determine the assembly of a portion of a subject's genome (such as a chromosome), or the assembly of chromatin of different lengths of a subject.

[0299] In some embodiments, the present disclosure provides one or more methods disclosed herein, which include the step of generating multiple contigs from sequencing fragments of target DNA obtained from an object. Long extended target DNA can be fragmented by cutting DNA with one or more nucleases (e.g., DNase I, DNase II, micrococcal nuclease, etc.). The obtained fragments can be sequenced using a high-throughput sequencing method to obtain multiple sequencing reads. Examples of high-throughput sequencing methods that can be used with the method of the present disclosure include, but are not limited to, the 454 pyrophosphate sequencing method developed by Roche Diagnostics, the "cluster (cluster)" sequencing method developed by Illumina, the SOLiD and Ion semiconductor sequencing methods developed by Life Technologies, and the DNA nanoball sequencing method developed by Complete Genomics. The overlapping ends of different sequencing reads can then be assembled to form contigs. Alternatively, the fragmented target DNA can be cloned into a vector. Cells or organisms are then transfected with a DNA vector to form a library. After replicating the transfected cells or organisms, the vectors are separated and sequenced to generate multiple sequencing reads. The overlapping ends of different sequencing reads can then be assembled to form contigs.

[0300] Genome assembly, especially using high-throughput sequencing technology, can be problematic. Typically, the assembly consists of tens of thousands of short overlapping groups. The order and orientation of these overlapping groups are usually unknown, which limits the effectiveness of genome assembly. There are technologies for ordering or orienting these scaffolds, but they are typically expensive, labor-intensive, and often fail to discover very long-range interactions.

[0301] A sample containing the target DNA for generating overlapping groups can be obtained from a subject in a variety of ways, including by collecting body fluids (e.g., blood, urine, serum, lymph, saliva, oral swabs, anal and vaginal secretions, sweat and semen, etc.), collecting tissues, or by collecting cells / organisms. The sample obtained can be composed of a single type of cell / organism, or can be composed of multiple types of cells / organisms. DNA can be extracted and prepared from the sample of the subject. For example, the sample can be treated with known lysis buffers, ultrasonic treatment techniques, electroporation, etc. to lyse cells containing polynucleotides. The target DNA can be further purified to remove contaminants, such as proteins, by using alcohol extraction, cesium gradients and / or column chromatography.

[0302] In other embodiments of the present disclosure, methods for extracting very high molecular weight DNA are provided. In some cases, data from the XLRP library can be improved by increasing the fragment size of the input DNA. In some examples, extracting megabase-sized DNA fragments from cells can produce read pairs separated by megabases in the genome. In some cases, the read pairs produced can provide sequence information greater than about 10kB, about 50kB, about 100kB, about 200kB, about 500kB, about 1Mb, about 2Mb, about 5Mb, about 10Mb or about 100Mb spans. In some examples, read pairs can provide sequence information greater than about 500kB spans. In a further example, read pairs can provide sequence information greater than about 2Mb spans. In some cases, very high molecular weight DNA can be extracted by very mild cell lysis (Teague, B. et al. (2010) Proc. Nat. Acad. Sci. USA 107(24), 10848-53) and agarose plugs (Schwartz, DC and Cantor, CR (1984) Cell, 37(1), 67-75). In other cases, very high molecular weight DNA can be extracted using commercially available machines that can purify DNA molecules up to megabases in length.

[0303] Probing the physical layout of chromosomes

[0304] In various embodiments, the present disclosure provides one or more methods disclosed herein, which include the step of detecting the physical layout of chromosomes in living cells. Examples of techniques for detecting the physical layout of chromosomes by sequencing include "C" class technologies, 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 ChIP-loop, ChIA-PET, and HiChIP. These technologies utilize the fixation of chromatin in living cells to consolidate spatial relationships in the nucleus. Subsequent processing and sequencing of the product allow researchers to restore a matrix of neighboring associations between genomic regions. Through further analysis, these associations can be used to generate a three-dimensional geometric map of chromosomes because they are physically arranged in the nucleus of living cells. Such technology describes the discrete spatial organization of chromosomes in living cells and provides an accurate view of the functional interactions between chromosome sites. One problem that plagues these functional studies is the presence of nonspecific interactions, and the associations present in the data are attributed only to chromosome proximity. In the present disclosure, these nonspecific intrachromosomal interactions are captured by the method proposed herein to provide valuable information for assembly.

[0305] In some embodiments, intrachromosomal interactions are related to chromosomal connectivity. In some cases, intrachromosomal data can help genome assembly. In some cases, chromatin is reconstructed in vitro. This can be advantageous because chromatin, particularly histones (the main protein component of chromatin), is important for fixation under the most common "C" class technologies 3C, 4C, 5C and Hi-C for detecting chromatin conformation and structure by sequencing. Chromatin is highly non-specific with respect to sequence, and will usually be assembled uniformly across the genome. In some cases, the genome of species that do not use chromatin can be assembled on reconstructed chromatin, thereby expanding the scope of the present disclosure to all life domains.

[0306] Chromatin conformation capture technology is summarized. In short, crosslinking is created between physically very adjacent genomic regions. The crosslinking of proteins (such as histones) and DNA molecules (such as genomic DNA) in chromatin can be completed according to other suitable methods further described in detail elsewhere herein or known in the art. In some cases, two or more nucleotide sequences can be crosslinked via proteins bound to one or more nucleotide sequences. One method is to expose chromatin to ultraviolet light irradiation (Gilmour et al., Proc.Nat'l.Acad.Sci.USA 81:4275-4279,1984). Other methods, such as chemical or physical (e.g., light) crosslinking, can also be used to crosslink polynucleotide segments. Suitable chemical crosslinking agents include but are not limited to formaldehyde and psoralen (Solomon et al., Proc.Nat'l.Acad.Sci.USA 82:6470-6474,1985; Solomon et al., Cell 53:937-947,1988). For example, crosslinking can be performed by adding 2% formaldehyde to a mixture containing DNA molecules and chromatin proteins. Other examples of agents that can be used to crosslink DNA include, but are not limited to, ultraviolet light, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis-diamine dichloroplatinum (II), and cyclophosphamide. Suitably, the crosslinking agent will form a bridge over a relatively short distance, such as about of cross-links, thereby selecting for close interactions that can be reversed.

[0307] In some embodiments, the DNA molecule can be immunoprecipitated before or after cross-linking. In some cases, the DNA molecule can be fragmented. The fragment can be contacted with a binding partner, such as an antibody that specifically identifies and binds to acetylated histones, such as H3. Examples of such antibodies include, but are not limited to, anti-acetylated histone H3, available from Upstate Biotechnology, Lake Placid, NY. The polynucleotides from the immunoprecipitate can then be collected from the immunoprecipitate. Before the chromatin is fragmented, the acetylated histones can be cross-linked to adjacent polynucleotide sequences. The mixture is then processed to grade the polynucleotides in the mixture. The classification techniques herein include the use of deoxyribonucleases (DNases). DNases suitable for the methods herein include, but are not limited to, DNase I, DNase II, and micrococcal nucleases. The size of the resulting fragments can be different. The resulting fragments may also contain single-stranded overhands at the 5' or 3' ends.

[0308] In some embodiments, a fragment of about 145bp to about 600bp can be obtained. Alternatively, a fragment of about 100bp to about 2500bp, about 100bp to about 600bp or about 600 to about 2500 can be obtained. Samples can be prepared for sequencing the coupled sequence segments of crosslinks. In some cases, a single short extended polynucleotide can be produced, for example, by connecting two sequence segments of intramolecular crosslinks. Any suitable sequencing technology or other suitable method (such as high-throughput sequencing method) described in further detail elsewhere herein can be used to obtain sequence information from the sample. For example, paired end sequencing can be performed on the connection product to obtain sequence information from each end of the fragment. Paired sequence segments can be represented in the sequence information obtained, and haplotype information is associated on the linear distance separating the two sequence segments along the polynucleotide.

[0309] A feature of the data generated by Hi-C is that most read pairs are found to be closely linearly adjacent when mapped back to the genome. In other words, most read pairs are found to be close to each other in the genome. In the resulting data set, the probability of intrachromosomal contact is on average much higher than the probability of interchromosomal contact, as expected if the chromosomes occupy different regions. In addition, although the probability of interaction decays rapidly with linear distance, even loci separated by> 200Mb on the same chromosome are more likely to interact than loci on different chromosomes. When detecting long-range intrachromosomal (especially interchromosomal) contacts, this "background" of short-range and mid-range intrachromosomal contacts is background noise that needs to be excluded using Hi-C analysis.

[0310] It is worth noting that Hi-C experiments in eukaryotes show that in addition to species-specific and cell-type-specific chromatin interactions, there are two typical interaction patterns. One pattern, distance-dependent decay (DDD), is a general trend of interaction frequency decay as a function of genomic distance. The second pattern, the cis-trans ratio (CTR), is that the interaction frequency between loci located on the same chromosome is significantly higher than that of loci located on different chromosomes, even for sequences separated by tens of megabases. These patterns can reflect general polymer dynamics, in which adjacent loci have a higher probability of random interaction, and specific nuclear organization features, such as the formation of chromosome regions, and the phenomenon that interphase chromosomes tend to occupy different volumes in the nucleus without almost any mixing. Although the exact details of these two patterns may differ between species, cell types, and cell conditions, they are ubiquitous and prominent. These patterns are so powerful and consistent that they are used to evaluate experimental quality and are usually standardized from the data to reveal detailed interactions. However, in the method disclosed herein, genome assembly can utilize the three-dimensional structure of the genome. The features that make typical Hi-C interaction patterns such obstacles to analyzing specific circular interactions, namely their prevalence, strength, and consistency, can be used as a powerful tool to estimate the genomic position of contigs.

[0311] In a specific implementation, the physical distance between the read pairs in the chromosome is checked to indicate several useful features of the data in terms of genome assembly. First, shorter range interactions are more common than longer range interactions. That is, each read in the read pair is more likely to be paired with a region close to the actual genome, rather than paired with a region far away. Secondly, there is a long tail of medium and long range interactions. That is, read pairs carry information about the arrangement in the chromosome of kilobases (kB) or even megabases (Mb) distances. For example, read pairs can provide sequence information greater than about 10kB, about 50kB, about 100kB, about 200kB, about 500kB, about 1Mb, about 2Mb, about 5Mb, about 10Mb or about 100Mb spans. These characteristics of the data simply indicate that the genomic regions close on the same chromosome are more likely to be physically close-this is an expected result, because they are chemically connected to each other by the DNA backbone. It is speculated that a genome-wide chromatin interaction data set (such as a data set generated by Hi-C) will provide long range information about sequence grouping and linear organization along the entire chromosome.

[0312] Although the experimental method of Hi-C is simple and relatively low cost, current genome assembly and haplotype analysis protocols require 3-5 million cells, which is a considerable amount of material that may not be available, especially from certain human patient samples. In contrast, the methods disclosed herein include methods that allow accurate and predictive results of genotype assembly, haplotype phasing, and metagenomics to be obtained using significantly less material from cells. For example, the methods disclosed herein can use less than about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, about 1.2 μg, about 1.4 μg, about 1.6 μg, about 1.8 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 6.0 μg, about 7. 0μg, about 8.0μg, about 9.0μg, about 10μg, about 15μg, about 20μg, about 30μg, about 40μg, about 50μg, about 60μg, about 70μg, about 80μg, about 90μg, about 100μg, about 150μg, about 200μg, about 300μg, about 400μg, about 500μg, about 600μg, about 700μg, about 800μg, about 900μg, about 1000μg, about 1200μg, about 1400μg, about 1 600μg, about 1800μg, about 2000μg, about 2200μg, about 2400μg, about 2600μg, about 2800μg, about 3000μg, about 3200μg, about 3400μg, about 3600μg, about 3800μg, about 4000μg, about 4200μg, about 4400μg, about 4600μg, about 4800μg, about 5000μg, about 5200μg, about 5400μg, about 5600μg, about 5800μg, About 6000 μg, about 6200 μg, about 6400 μg, about 6600 μg, about 6800 μg, about 7000 μg, about 7200 μg, about 7400 μg, about 7600 μg, about 7800 μg, about 8000 μg, about 8200 μg, about 8400 μg, about 8600 μg, about 8800 μg, about 9000 μg, about 9200 μg, about 9400 μg, about 9600 μg, about 9800 μg or about 10,000 μg of DNA. In some examples, the DNA used in the methods disclosed herein is extracted from less than about 3,000,000, about 2,500,000, about 2,000,000, about 1,500,000, about 1,000,000, about 500,000, about 100,000, about 50,000, about 10,000, about 5,000, about 1,000, about 500, or about 100 cells.

[0313] Typically, procedures for probing the physical layout of chromosomes, such as Hi-C-based techniques, utilize chromatin formed in cells / organisms, such as chromatin isolated from cultured cells or primary tissues. The present disclosure not only provides the use of such techniques using chromatin isolated from cells / organisms, but also provides the use of such techniques using reconstructed chromatin. Reconstructed chromatin differs from chromatin formed in cells / organisms in a variety of characteristics. First, for many samples, the collection of naked DNA samples can be achieved using a variety of non-invasive to invasive methods, such as by collecting body fluids, swabbing the oral or rectal area, collecting epithelial samples, etc. Second, reconstructed chromatin substantially prevents the formation of inter-chromosomal and other long-range interactions (generating artifacts for genome assembly and haplotype phasing). In some cases, according to the methods and compositions of the present disclosure, the sample may have less than about 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less of interchromosomal or intermolecular crosslinks. In some examples, the sample may have less than about 5% of interchromosomal or intermolecular crosslinks. In some examples, the sample may have less than about 3% of interchromosomal or intermolecular crosslinks. In other examples, there may be less than about 1% of interchromosomal or intermolecular crosslinks. Third, the frequency of sites capable of crosslinking can be adjusted, and therefore the frequency of intramolecular crosslinks within polynucleotides can be adjusted. For example, the ratio of DNA to histones can be varied so that the nucleosome density can be adjusted to a desired value. In some cases, the nucleosome density is reduced to below physiological levels. Therefore, the distribution of crosslinks can be changed to facilitate longer range interactions. In some embodiments, subsamples with different crosslink densities can be prepared to cover short-range and long-range associations. For example, the crosslinking conditions can be adjusted so 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 crosslink density is at least about 1%. % of cross-links occur between DNA segments that are at least about 50 kb, about 60 kb, about 70 kb, about 80 kb, about 90 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 molecules.

[0314] Contact Mapping and Topology

[0315] The reading generated by the method of the present disclosure is to be used for analyzing the three-dimensional structure of genome and chromosome and nucleic acid molecule therein.As discussed herein, each reading in the reading pair can be mapped to the different regions in the genome.It can be inferred that for a given reading pair, the two different regions in the genome to which they are mapped will be adjacent to each other in space, so that they can be connected together.By drawing the reading pair from the sample according to the coordinates of the two readings in the reading pair, a contact map can be created for the sample.

[0316] Contact analysis of the entire sample can allow analysis of chromosome and genome structure. The organization of genomes into A and B compartments, active and inactive compartments, chromosome compartments, euchromatin and heterochromatin, topologically associated domains (TADs) (including TAD subtypes) and other structures can be analyzed on a kilobase or megabase scale. Contact mapping analysis can also allow detection of genomic features, such as structural variants, such as rearrangements, translocations, copy number variations, inversions, deletions and insertions.

[0317] The methods of the present disclosure can provide the location of protein binding, structural variation, or genomic contact interaction at a resolution of less than or equal to about 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 2000bp, 3000bp, 4000bp, 5000bp, 6000bp, 7000bp, 8000bp, 9000bp, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb or 100kb. In some cases, protein binding sites, protein footprints, contact interactions, or other features can be mapped to within 1000 bp, within 900 bp, within 800 bp, within 700 bp, within 600 bp, within 500 bp, within 400 bp, within 300 bp, within 200 bp, within 190 bp, within 180 bp, within 170 bp, within 160 bp, within 150 bp, within 140 bp, within 160 bp, within 170 bp, within 180 bp, within 190 bp, within 18 ... In one example, the method of the present disclosure can achieve the resolution of sites (e.g., protein binding sites, such as CTCF sites) that are within 10,000 bp, 5,000 bp, 2,000 bp or 1,000 bp of each other on the genome. In some cases, resolution or mapping can be improved by using MNase or other endonucleases that degrade unprotected nucleic acids (e.g., nucleic acids not within the bound protein footprint), resulting in proximity ligation events occurring at the edge of the protected region (e.g., protein footprint).

[0318] Contig mapping

[0319] In various embodiments, the present disclosure provides various methods capable of mapping multiple read pairs to multiple contigs. There are several publicly available computer programs for mapping reads to contig sequences. These read mapping program data also provide data describing the uniqueness of specific read mappings in the genome. From the read population uniquely mapped with high confidence within the contig, we can infer the distance distribution between reads in each read pair. For the read pairs whose reads are confidently mapped to different contigs, the mapping data means the connection between the two contigs in question. It also means that the distance between the two contigs is proportional to the distance distribution learned from the above analysis. Therefore, each read pair that reads mapping to different contigs means the connection between the two contigs in the correct assembly. The connection inferred from all such mapped read pairs can be summarized in an adjacency matrix, wherein each contig is represented by both rows and columns. The read pairs connecting the contigs are marked as non-zero values ​​in the corresponding rows and columns, indicating that the reads in the read pairs are mapped to the contigs. Most read pairs will map in contigs, and the distribution of distances between read pairs can be known from this, and the adjacency matrix of the contigs can be constructed from this using the read pairs that map to different contigs.

[0320] In various embodiments, the present disclosure provides methods including using the read mapping data from the read pair data to construct the adjacency matrix of the contig. In some embodiments, the adjacency matrix uses a weighting scheme for the read pair, which combines the trend of short-range interactions into the long-range interactions. Read pairs spanning shorter distances are usually more common than read pairs spanning longer distances. The read pair data mapped to a single contig can be used to fit the function describing the probability of a specific distance to learn the distribution. Therefore, an important feature of the read pairs mapped to different contigs is that they are mapped to the position of the contig. For the read pairs that are all mapped near one end of the contig, the inferred distance between these contigs can be very short, and the distance between the reads of the joint is therefore very small. Since the shorter distance between the read pairs is more common than the longer distance, this configuration provides stronger evidence that the two contigs are adjacent compared to the read mapping away from the edge of the contig. Therefore, the connection in the adjacency matrix is ​​further weighted by reading the distance to the edge of the contig. In a further embodiment, the adjacency matrix can be further rescaled to reduce the weight of a large number of contacts on some contigs representing the promiscuous region of the genome. These regions of the genome (identifiable by having a high proportion of reads mapped to them) are inherently more likely to contain spurious read mappings that can mislead assembly. In yet further embodiments, the scaling can be guided by finding one or more conserved binding sites for one or more agents that modulate scaffold interactions of chromatin (such as the transcriptional repressor CTCF, endocrine receptors, cohesin, or covalently modified histones).

[0321] In some embodiments, present disclosure provides one or more methods disclosed herein, it comprises the step of analyzing the adjacency matrix to determine the path of the order and / or orientation of the representatives of them to genome by overlapping groups.In other embodiments, the path through overlapping groups can be selected so that each overlapping group is only accurately visited once.In a further embodiment, the path through overlapping groups is selected so that the sum of the edge weights visited by the path through the adjacency matrix is ​​maximized.In this way, the most probable overlapping group connection is proposed for correct assembly.In a further embodiment again, the path through overlapping groups can be selected so that each overlapping group is only visited once and the edge weights of the adjacency matrix are maximized.

[0322] Haplotype phasing

[0323] In diploid genomes, it is often important to understand which allelic variants are connected on the same chromosome. This is called haplotype phasing. Short reads from high-throughput sequence data rarely allow direct observation of which allelic variants are connected. The computational inference of haplotype phasing may be unreliable over long distances. The present disclosure provides one or more methods that allow the use of allelic variants on read pairs to determine which allelic variants are connected. In some cases, the phasing of the method of the present disclosure is performed without imputation.

[0324] In various embodiments, the methods and compositions of the present disclosure can perform haplotype phasing on a diploid or polyploid genome with respect to a plurality of allele variants. Therefore, the methods described herein can determine the allele variants of the connection based on the variant information from reading pairs and / or using the assembled contigs of the reading pairs. The examples of allele variants include, but are not limited to, allele variants known from 1000genomes, UK10K, HapMap, and other projects for finding genetic variants among people. Obtaining haplotype-phased data makes it easier to reveal disease associations with specific genes, as shown, for example, by the findings that unlinked inactivating mutations in both copies of SH3TC2 cause Charcot-Marie-Tooth neuropathy (Lupski JR, Reid JG, Gonzaga-Jauregui C et al. N. Engl. J. Med. 362:1181–91, 2010) and unlinked inactivating mutations in both copies of ABCG5 cause hypercholesterolemia 9 (Rios J, Stein E, Shendure J et al. Hum. Mol. Genet. 19:4313–18, 2010).

[0325] People have 1 site that is heterozygous for every 1,000 sites on average. In some cases, single-channel data using high-throughput sequencing methods can generate at least about 150,000,000 read pairs. The length of the read pair can be about 100 base pairs. According to these parameters, it is estimated that one-tenth of all reads in human samples cover heterozygous sites. Therefore, it is estimated that the average one percent of all reads in human samples cover heterozygous site pairs. Therefore, about 1,500,000 read pairs (one percent of 150,000,000) use a single channel to provide phased data. There are about 3 billion bases in the human genome and one thousandth is heterozygous, and there are about 3 million heterozygous sites in the average human genome. Using about 1,500,000 read pairs representing heterozygous site pairs, using a typical high-throughput sequencer, the average coverage of each heterozygous site phased using a single channel of a high-throughput sequencing method is about (1X). Therefore, a diploid human genome can be reliably and completely phased with one channel of high-throughput sequence data associated with sequence variants from a sample prepared using the methods disclosed herein. In some examples, the data channel can be a collection of DNA sequence reads. In a further example, the data channel can be a collection of DNA sequence reads from a single run of a high-throughput sequencing instrument.

[0326] Since the human genome consists of two sets of homologous chromosomes, understanding the true genetic composition of an individual requires describing the maternal and paternal copies of genetic material, or haplotypes. Obtaining an individual's haplotype can be used in several ways. First, haplotypes can be used clinically to predict the outcome of donor-host matching in organ transplantation and are increasingly used to detect disease associations. Second, in genes that show compound heterozygosity, haplotypes provide information about whether two harmful variants are located on the same allele, greatly affecting the prediction of whether the inheritance of these variants is harmful. Third, haplotypes from groups of individuals provide information about population structure and human evolutionary history. Finally, the recent description of widespread allelic imbalance in gene expression suggests that genetic or epigenetic differences between alleles can lead to quantitative differences in expression. Understanding the haplotype structure will describe the mechanism of variants that cause allelic imbalance.

[0327] In certain embodiments, the methods disclosed herein include in vitro techniques to fix and capture associations between distant regions of the genome according to the needs of long-distance linkage and phasing. In some cases, the method includes constructing and sequencing XLRP libraries to deliver very far read pairs on the genome. In some cases, the interaction mainly comes from random associations within a single DNA fragment. In some examples, it can be inferred that the genomic distance between segments is because the segments close to each other in the DNA molecule interact more frequently and with higher probability, while the interaction between distant parts of the molecule is less frequent. Therefore, there is a systematic relationship between the number of pairs connecting two loci and their proximity on the input DNA. The present disclosure can produce read pairs that can span the largest DNA fragment in the extract. The maximum length of the input DNA of the library is 150kbp, which is the longest meaningful read pair observed from the sequencing data. This shows that if a larger input DNA fragment is provided, the present method can also connect loci farther away on the genome. Complete genome assembly is possible by applying improved assembly software tools that are particularly suitable for processing the data type generated by the present method.

[0328] The data produced using the method and composition of the present disclosure can achieve extremely high phasing accuracy.Compared with previous methods, the method described herein can phase a high proportion of variants.Phasing can be achieved while maintaining a high level of accuracy.The technology herein can allow phasing with an accuracy greater than about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999%.The technology herein can allow accurate phasing with less than about 500x sequencing depth, 450x sequencing depth, 400x sequencing depth, 350x sequencing depth, 300x sequencing depth, 250x sequencing depth, 200x sequencing depth, 150x sequencing depth, 100x sequencing depth or 50x sequencing depth. The phase information can extend to a longer range, e.g., greater than about 200 kbp, about 300 kbp, about 400 kbp, about 500 kbp, about 600 kbp, about 700 kbp, about 800 kbp, about 900 kbp, about 1 Mbp, about 2 Mbp, about 3 Mbp, about 4 Mbp, about 5 Mbp, or about 10 Mbp. In some embodiments, less than about 250 million reads or read pairs (e.g., by using only 1 channel of Illumina HiSeq data) can be used to phase greater than 90% of heterozygous SNPs in human samples with greater than 99% accuracy. In other cases, less than about 250 million or about 500 million reads or read pairs (e.g., by using only 1 or 2 channels of Illumina HiSeq data) can be used to phase greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of heterozygous SNPs in a human sample with an accuracy of greater than about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.999%. For example, less than about 250 million or about 500 million reads can be used to phase greater than about 95% or 99% of heterozygous SNPs in a human sample with an accuracy of greater than about 95% or 99%. In further cases, additional variants can be captured by increasing the read length to approximately 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1500bp, 2kbp, 3kbp, 4kbp, 5kbp, 10kbp, 20kbp, 50kbp or 100kbp.

[0329] In other embodiments of the present disclosure, data from XLRP libraries can be used to confirm the phasing ability of long-range read pairs. The accuracy of those results is comparable to the best previously available technology, but extends further to significantly longer distances. The sample preparation scheme currently used for specific sequencing methods identifies variants within the target site read length (e.g., 150bp) for phasing. In one example, from the XLRP library constructed for NA12878 (assembly benchmark sample), 44% of the 1,703,909 heterozygous SNPs present were directed with an accuracy greater than 99%. In some cases, this ratio can be extended to almost all variable sites by judiciously selecting enzymes or digestion conditions.

[0330] Haplotype phasing can include phasing of human leukocyte antigen (HLA) regions (e.g., class I HLA-A, B, and C; class II HLA-DRB1 / 3 / 4 / 5, HLA-DQA1, HLA-DQB 1, HLA-DPA1, HLA-DPB1). The HLA region of the genome has dense polymorphisms and may be difficult to sequence or phase using standard sequencing methods. The technology of the present disclosure can provide improved sequencing and phasing accuracy of the HLA region of the genome. Using the technology of the present disclosure, the HLA region of the genome can be accurately phased as part of a larger phased region (e.g., chromosome arm, chromosome, whole genome) or by itself (e.g., by targeted enrichment such as hybrid capture). In one example, the HLA region itself is accurately phased at a sequencing depth of about 300x. These technologies can provide advantages over traditional HLA analysis methods (such as long range PCR); for example, long range PCR can involve complex protocols and many separate reactions. As further discussed herein, samples can be subjected to multiple sequencing for sequencing analysis, such as by including a sample identification barcode in a bridging oligonucleotide or elsewhere, and performing multiple sequencing analysis on the sequence information based on the barcode. In one example, multiple samples are contiguously connected, barcoded using a sample identification barcode (e.g., in a bridging oligonucleotide), targeted to the HLA region (e.g., captured by hybridization), and subjected to multiple sequencing, allowing the HLA region of multiple samples to be phased. In some cases, the HLA region is phased without calculation.

[0331] Haplotype phasing can include phasing the killer cell immunoglobulin-like receptor (KIR) region. Due to transposon-mediated recombination, the KIR region of the genome has a high degree of homology and structural dynamics, and may be difficult to sequence or phase using standard sequencing methods. The technology of the present disclosure can provide improved sequencing and phasing accuracy of the KIR region of the genome. Using the technology of the present disclosure, the KIR region of the genome can be phased as part of a larger region (e.g., chromosome arm, chromosome, whole genome) or accurately phased by itself (e.g., by targeted enrichment such as hybridization capture). These technologies can provide advantages over traditional HLA analysis methods (such as long-range PCR); for example, long-range PCR can involve complex protocols and many separate reactions. As further discussed herein, samples can be multiplexed sequenced for sequencing analysis, such as by including sample identification barcodes in bridging oligonucleotides or elsewhere, and multiple sequencing analysis of sequence information based on barcodes. In one example, multiple samples are proximity ligated, barcoded using sample identification barcodes (e.g., in bridging oligonucleotides), targeted to KIR regions (e.g., by hybridization capture), and multiplexed sequencing, allowing for phasing of KIR regions of multiple samples. Phases can be performed for at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more genes and / or pseudogenes. In some cases, the KIR regions are phased without computation.

[0332] Metagenomic analysis

[0333] In some embodiments, the compositions and methods described herein allow for the study of metagenomes, such as those found in the human intestine. Thus, partial or complete genome sequences of some or all organisms inhabiting a given ecological environment can be studied. Examples include random sequencing of all intestinal microorganisms, microorganisms found in certain areas of the skin, and microorganisms living in toxic waste sites. Compositions and methods described herein can be used to determine the composition of microbial populations in these environments and aspects of the associated biochemistry encoded by their respective genomes. The methods described herein can enable metagenomic studies from complex biological environments, for example, those environments containing more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 or more organisms and / or organism variants.

[0334] The high accuracy required for cancer genome sequencing can be achieved using the methods and systems described herein. When sequencing cancer genomes, inaccurate reference genomes can present base calling challenges. Heterogeneous samples and small starting materials, such as samples obtained by biopsy, introduce additional challenges. In addition, the detection of large-scale structural variants and / or loss of heterozygosity is often critical for cancer genome sequencing, as well as the ability to distinguish somatic variants from base calling errors.

[0335] Improved sequencing accuracy

[0336] The systems and methods described herein can generate accurate long sequences from complex samples containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more different genomes. Mixed samples of normal, benign and / or tumor origin can be analyzed (optionally without normal controls). In some embodiments, accurate long sequences are generated using only 100ng or even only hundreds of genome equivalent starting samples. The systems and methods described herein can allow for the detection of large-scale structural variants and rearrangements. Phased variant recognition can be obtained on long sequences of nucleotides spanning about 1kbp, about 2kbp, about 5kbp, about 10kbp, about 20kbp, about 50kbp, about 100kbp, about 200kbp, about 500kbp, about 1Mbp, about 2Mbp, about 5Mbp, about 10Mbp, about 20Mbp, about 50Mbp or about 100Mbp or more. For example, phase variant recognition can be obtained on long sequences spanning about 1Mbp or about 2Mbp.

[0337] Haplotypes determined using the methods and systems described herein can be assigned to computing resources, such as computing resources via a network, such as a cloud system. If necessary, relevant information stored in the computing resources can be used to correct short variant identification. Structural variants can be detected based on composite information from short variant identification and information stored in the computing resources. Problematic parts of the genome, such as segmental duplications, regions with structural variation tendencies, highly variable and medically relevant MHC regions, centromere and telomeric regions, and other heterochromatic regions (including but not limited to those heterochromatic regions with repeated regions, low sequence accuracy, high variant rates, ALU repeats, segmental duplications, or any other relevant problematic parts), can be reassembled to improve accuracy.

[0338] Sample types can be assigned to sequence information in local or networked computing resources (such as the cloud). Where the source of the information is known, for example, when the source of the information is from cancer or normal tissue, the source can be assigned to the sample as part of the sample type. Other sample type examples typically include, but are not limited to, tissue type, sample collection method, presence of infection, type of infection, treatment method, size of the sample, etc. Where a complete or partial comparative genome sequence is available, such as a comparison of a normal genome with a cancer genome, the difference between the sample data and the comparative genome sequence can be determined and optionally output.

[0339] Clinical Application

[0340] The method of the present disclosure can be used to analyze the genetic information of the selective genomic region of interest and the genomic region that can interact with the selective region of interest. The amplification method disclosed herein can be used in the device, kit and method of genetic analysis, such as but not limited to those found in U.S. Patent Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117, 6,225,109 and 6,110,709. In some cases, the amplification method of the present disclosure can be used to amplify the target nucleic acid for DNA hybridization research to determine the presence or absence of polymorphism. Polymorphism or allele can be associated with a disease or condition such as a genetic disease. In other cases, polymorphism can be associated with susceptibility to a disease or condition, for example, polymorphisms associated with addiction, degenerative and age-related conditions, cancer, etc. In other cases, polymorphisms can be associated with beneficial traits, such as enhanced coronary artery health, or resistance to diseases such as HIV or malaria, or resistance to degenerative diseases such as osteoporosis, Alzheimer's disease, or dementia.

[0341] The compositions and methods of the present disclosure can be used for diagnosis, prognosis, treatment, patient stratification, drug development, treatment selection and screening purposes. The present disclosure provides an advantage that many different target molecules can be analyzed from a single biomolecule sample at one time using the methods of the present disclosure. For example, this allows multiple diagnostic tests to be performed on one sample.

[0342] The compositions and methods of the present disclosure can be used for genomics. The methods described herein can provide answers quickly, which is very ideal for this application. The methods and compositions described herein can be used for the process of finding biomarkers that can be used for diagnosis or prognosis and as health and disease indicators. The methods and compositions described herein can be used for screening drugs, such as drug development, treatment selection, determination of therapeutic efficacy and / or identification of drug development targets. The ability to test gene expression in screening experiments involving drugs is very important because proteins are the final gene products in the body. In some embodiments, the methods and compositions described herein will measure protein and gene expression simultaneously, which will provide the most information about the specific screening performed.

[0343] The compositions and methods of the present disclosure can be used for gene expression analysis. The methods described herein distinguish nucleotide sequences. The differences between target nucleotide sequences can be, for example, single nucleic acid base differences, nucleic acid deletions, nucleic acid insertions or rearrangements. Such sequence differences involving more than one base can also be detected. The process of the present disclosure can detect infectious diseases, genetic diseases and cancer. It can also be used for environmental monitoring, forensics and food science. Examples of genetic analyses that can be performed on nucleic acids include, for example, SNP detection, STR detection, RNA expression analysis, promoter methylation, gene expression, virus detection, virus subtyping and drug resistance.

[0344] The method can be used to analyze biomolecular samples obtained or derived from a patient to determine the presence of diseased cell types in the sample, the stage of the disease, the patient's prognosis, the patient's ability to respond to a particular treatment, or the patient's optimal treatment. The method can also be used to identify biomarkers for a particular disease.

[0345] In some embodiments, the methods described herein are used to diagnose a condition. As used herein, the terms "diagnose" or "diagnosis" of a condition may include predicting or diagnosing a condition, determining susceptibility to a condition, monitoring treatment of a condition, diagnosing a therapeutic response to a disease or a prognosis of a condition, progression of a condition, or a response to a specific treatment for a condition. For example, a blood sample may be assayed according to any of the methods described herein to determine the presence and / or amount of markers of a disease or malignant cell type in the sample, thereby diagnosing a disease or cancer or staging a disease or cancer.

[0346] In some embodiments, the methods and compositions described herein are used for diagnosis and prognosis of a condition.

[0347] Many immunological, proliferative and malignant diseases and conditions are particularly suitable for the methods described herein. Immunological diseases and conditions include allergic diseases and conditions, immune dysfunction and autoimmune diseases and conditions. Allergic diseases and conditions include but are not limited to allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis and food allergies. Immunodeficiency includes but is not limited to severe combined immunodeficiency (SCID), eosinophilic syndrome, chronic granulomatous disease, leukocyte adhesion deficiency I and II, high IgE syndrome, congenital leukocyte granule abnormality syndrome (ChediakHigashi), neutrophilia, neutropenia, dysplasia, agammaglobulinemia, high IgM syndrome, DiGeorge / velocardiofacial syndrome and interferon gamma-TH1 pathway defect. Autoimmune and immune dysregulation conditions 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 lymphomatous goiter (Hashimoto's thyroiditis, lymphadenoid goiter), alopecia areata, autoimmune myocarditis, lichen sclerosus, 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, allograft rejection and tissue destruction caused by allergic reactions to infectious microorganisms or environmental antigens.

[0348] Proliferative diseases and disorders that can be assessed by the methods of the present disclosure include, but are not limited to, neonatal hemangiomatosis; secondary progressive multiple sclerosis; chronic progressive myelodegenerative disease; neurofibromatosis; ganglioneuroma; keloid formation; Paget's disease of bone; fibrocystic disease (e.g., of the breast or uterus); sarcoidosis; Peronies and Duputren fibrosis, cirrhosis of the liver, atherosclerosis, and vascular restenosis.

[0349] Malignant diseases and conditions that may be assessed by the methods of the present disclosure include both hematological malignancies and solid tumors.

[0350] When the sample is a blood sample, hematological malignancies are particularly suitable for the methods of the present disclosure because such malignancies involve changes in blood-borne cells. Such malignancies include non-Hodgkin's lymphoma, Hodgkin's lymphoma, non-B cell lymphoma and other lymphomas, acute or chronic leukemias, polycythemia, thrombocythemia, multiple myeloma, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, atypical immune lymphoproliferations, and plasma cell disorders.

[0351] Plasma cell disorders that may be assessed by the methods of the disclosure include multiple myeloma, amyloidosis, and Waldenstrom's macroglobulinemia.

[0352] Examples 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, melanoma, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian cancers, head and neck tumors, and cervical tumors.

[0353] Genetic diseases can also be detected by the process of the present disclosure. This can be done by prenatal or postnatal screening for chromosomes and genetic aberrations or genetic diseases. Examples of genetic diseases that can be detected include: 21-hydroxylase deficiency, cystic fibrosis, fragile X syndrome, Turner syndrome, Duchenne muscular dystrophy, Down syndrome or other trisomy syndromes, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs disease, thalassemia, Klinefelter syndrome, Huntington's disease, autoimmune diseases, lipid storage diseases, obesity defects, hemophilia, inborn errors of metabolism and diabetes.

[0354] The methods of the present disclosure can be used to detect genetic or genomic features associated with genetic diseases, including but not limited to gene fusions, structural variants, rearrangements, and topological changes, such as TAD boundary deletions or alterations, TAD subtype changes, compartment changes, chromatin type changes, and modification state changes such as methylation state (e.g., CpG methylation, H3K4me3, H3K27me3 or other histone methylation).

[0355] The methods described herein can be used to diagnose pathogen infections, such as infections caused by intracellular bacteria and viruses, by determining the presence and / or amount of bacterial or viral markers, respectively, in a sample.

[0356] A variety of infectious diseases can be detected through the process of the present disclosure. Infectious diseases can be caused by bacterial, viral, parasitic and fungal infectious agents. The present disclosure can also be used to determine the resistance of various infectious agent...

Claims

1. A method for processing nucleic acid, comprising: (a) obtaining a stabilized sample, the stabilized sample comprising nucleic acid molecules complexed with at least one nucleic acid binding protein; (b) cleaving the nucleic acid molecule into a plurality of segments, the plurality of segments comprising at least a first segment and a second segment, wherein the cleavage is effected by a transposase; as well as (c) ligating the first segment to the second segment, thereby generating a nucleic acid comprising a first sequence from the first segment and a second sequence from the second segment that is contiguously linked.

2. The method of claim 1, wherein the transposase is Tn5 transposase.

3. The method of claim 1, further comprising circularizing the proximity-joined nucleic acids by ligating the 5' end of the proximity-joined nucleic acids to the 3' end of the proximity-joined nucleic acids, thereby producing circularized proximity-joined nucleic acids.

4. The method of claim 1, further comprising sequencing at least a portion of the proximity-ligated nucleic acids.

5. The method of claim 4, wherein the sequencing comprises sequencing at least a portion of the first sequence and at least a portion of the second sequence.

6. The method of claim 5, further comprising mapping at least a portion of the first sequence and at least a portion of the second sequence to a genome.

7. The method of claim 4, further comprising performing three-dimensional genome analysis using information from the sequencing.

8. The method of claim 1, wherein the stabilized sample is a cross-linked sample.

9. The method of claim 1, wherein obtaining the stabilized sample comprises obtaining a sample and stabilizing the sample.

10. The method of claim 1, wherein obtaining the stabilized sample comprises obtaining a previously stabilized sample.

11. The method of claim 1, wherein the nucleic acid binding protein comprises chromatin or a component thereof.

12. The method of claim 1, wherein a linker sequence is connected between the first segment and the second segment.

13. The method of claim 12, wherein the adapter sequence comprises a barcode sequence. The method of claim 13 , wherein the barcode sequence indicates a partition of origin.

15. The method of claim 13, wherein the barcode sequence indicates a cell of origin.

16. The method of claim 13, wherein the barcode sequence indicates a cell population of origin.

17. The method of claim 13, wherein the barcode sequence indicates an organism of origin.

18. The method of claim 1, wherein said cleavage occurs in open and closed chromatin compartments.

19. The method of claim 18, wherein at least 10% of the cleavage occurs in closed chromatin compartments.

20. The method of claim 18, wherein at least 20% of the cleavage occurs in closed chromatin compartments.

21. The method of claim 18, wherein at least 30% of the cleavage occurs in closed chromatin compartments.

22. The method of claim 1, wherein the stabilized sample comprises no more than 50,000 cells.

23. The method of claim 22, wherein the stabilized sample comprises at least 10,000 cells.

24. The method of claim 1, wherein the stabilized sample comprises stabilized cell nuclei.

25. The method of claim 24, wherein the stabilized sample comprises no more than 50,000 cell nuclei.

26. The method of claim 24, wherein the stabilized sample comprises at least 10,000 cell nuclei.

27. The method of claim 1, wherein the proximity-bound nucleic acid does not comprise an affinity tag.

28. The method of claim 3, wherein the circularized proximity-ligated nucleic acids do not comprise an affinity tag.

29. The method of claim 3, wherein the circularized proximity-linked nucleic acid is greater than 250 base pairs in length.

30. The method of claim 3, wherein the circularization does not circularize nucleic acids less than 250 base pairs in length.

31. The method of claim 27 or claim 28, wherein the proximity-ligated nucleic acids and / or the circularized proximity-ligated nucleic acids are separated without the use of an affinity tag.

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