Methods for typing and phasing human leukocyte antigens - Patents.com

Abstract

Provided herein are methods for obtaining a phased human leukocyte antigen (HLA) type of a sample from an individual.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 302,812, filed January 25, 2022, which is incorporated by reference in its entirety herein. [Background technology]

[0002] The human leukocyte antigen (HLA) system, also known as the major histocompatibility complex (MHC), is a complex of genes spanning approximately 3 megabases on chromosome 6 that code for cell surface proteins involved in regulating the immune system. HLA presents peptides to T lymphocytes from inside the cell. These peptides are produced from digested proteins that are broken down in the proteosome. Generally, these peptides are small polymers about 8-14 amino acids long. HLA (A, B, and C), which correspond to MHC class I, all of which are in the HLA class I group, present peptides to CD8+ cytotoxic T cells. HLA (DP, DM, DO, DQ, and DR), which correspond to MHC class II, present antigens to CD4+ helper T cells. HLA genes are highly polymorphic, allowing HLA to distinguish between self and non-self cells. Any cells that display other HLA types are "non-self" and are considered invaders by the body's immune system. Therefore, HLA typing is very important in organ transplantation. Even when donors are related, high-resolution HLA typing is necessary to identify perfect matches for transplantation. Summary of the Invention

[0003] In one aspect, a method is provided for obtaining a phased human leukocyte antigen (HLA) type of a sample. Optionally, the method includes aligning a plurality of sequencing reads to a reference genome, where at least a portion of the plurality of sequencing reads corresponds to an HLA locus. Optionally, the method includes identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. Optionally, the method includes classifying the plurality of SNPs and indels into phase blocks. Optionally, the method includes aligning the plurality of sequencing reads to a variation graph to identify a plurality of HLA types. Optionally, the method includes comparing the plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele. Optionally, the method includes comparing the SNP signature of (e) to the phase blocks of (c) to obtain the phased HLA type. Alternatively or in combination, the method includes identifying the SNPs by comparing the plurality of sequencing reads with the variation graph. Optionally, the plurality of sequencing reads are obtained by crosslinking the sample, fragmenting the nucleic acid in the sample to create nucleic acid fragments, ligating the nucleic acid fragments to create linked nucleic acid fragments, reversing the crosslinking, and sequencing the linked nucleic acid fragments. Optionally, the sample is crosslinked by contacting the sample with a crosslinking agent selected from formaldehyde, psoralen, disuccinimidyl glutarate (DSG), ethylene glycol bis(succinimidyl succinate) (EGS), ultraviolet light, or a combination thereof. Optionally, the fragmentation includes contacting the sample with an enzyme. Optionally, the enzyme is a nuclease, a restriction enzyme, a transposase, or a combination thereof. Optionally, the nuclease is micrococcal nuclease. Optionally, the transposase is Tn5. Optionally, the fragmenting comprises non-enzymatic cleavage. Optionally, the method further comprises adding a label to said nucleic acid fragments after said ligation.Optionally, the label comprises biotin. Optionally, the label comprises an oligonucleotide. Optionally, the oligonucleotide comprises a barcode. Optionally, the crosslink binds the nucleic acid to a nucleic acid binding protein in the sample. Optionally, the plurality of SNPs and / or indels are identified by aligning the sequencing reads to the variation graph. Optionally, the phased HLA types comprise HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and / or HLA-DPB1. Optionally, the sample comprises a nucleic acid enriched for HLA sequences. Optionally, the entire HLA region is phased with more than 90% of the phased SNPs in a single phase block. Optionally, major and minor groups of HLA types are phased. Optionally, the method is completed in less than 30 hours of CPU time. In some cases, the method is completed in less than 15 hours, less than 10 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour of CPU time. In some cases, the method is completed in about 30 to about 40 minutes of CPU time. In some cases, the sequencing reads include paired-end reads. In some cases, the sequencing reads include long reads.

[0004] In another aspect, a computer-implemented method is provided for obtaining a phased human leukocyte antigen (HLA) type of a sample. Optionally, the method includes aligning a plurality of sequencing reads to a reference genome or a variation graph, where at least a portion of the plurality of sequencing reads correspond to HLA loci. Optionally, the method includes identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. Optionally, the method includes classifying the plurality of SNPs and indels into phase blocks. Optionally, the method includes aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types. Optionally, the method includes comparing the plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele. Optionally, the method includes comparing the SNP signature to the phase blocks to obtain the phased HLA type. Optionally, the sequencing reads include paired-end reads. Optionally, the sequencing reads include long reads.

[0005] In another aspect, a method is provided for obtaining a phased human leukocyte antigen (HLA) type of a sample. Optionally, the method includes aligning a plurality of sequencing reads to a variation graph, at least a portion of the plurality of sequencing reads corresponding to HLA loci. Optionally, the method includes identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. Optionally, the method includes classifying the plurality of SNPs and indels into phase blocks. Optionally, the method includes aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types. Optionally, the method includes comparing the plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele. Optionally, the method includes comparing the SNP signature of (e) to the phase blocks of (c) to obtain the phased HLA type. Optionally, the sequencing reads include paired-end reads. Optionally, the sequencing reads include long reads.

[0006] In another aspect, a method for obtaining a phased human leukocyte antigen (HLA) type of a sample is provided. In some cases, the method includes aligning a plurality of sequencing reads to a reference genome or a variation graph, and at least a portion of the plurality of sequencing reads corresponds to an HLA locus. In some cases, the method includes identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. In some cases, the method includes classifying the plurality of SNPs and indels into a plurality of phase blocks. In some cases, the method includes comparing the plurality of SNPs to a plurality of SNP signatures of known HLA types to identify a plurality of HLA types. In some cases, the method includes comparing the SNP signatures to the phase blocks to obtain the phased HLA type. In some cases, comparing the SNP signatures to the phase blocks includes assigning the HLA type phase of the plurality of HLA types based on the amount of SNPs of the plurality of SNPs that match the plurality of SNP signatures. In some cases, classifying the plurality of SNPs and indels into a plurality of phase blocks comprises assigning a phase to each of the plurality of phase blocks. In some cases, the method further comprises generating a database comprising a plurality of SNP signatures of known HLA types. In some cases, generating the database comprises aligning a plurality of HLA allele sequences with a reference genome. In some cases, the plurality of sequencing reads comprises reads generated using a proximity ligation technique. In some cases, the proximity ligation technique comprises Hi-C, Chicago, Micro-C, or Omni-C. In some cases, the plurality of sequencing reads comprises contiguous sequences comprising sequences from regions of a chromosome that are separated from each other in the natural sequence of the chromosome.In some cases, the method further comprises, prior to aligning the plurality of sequencing reads to a reference genome or variation graph, (i) subjecting the sample to a proximity ligation reaction, (ii) generating a sequencing library from the nucleic acids in the sample subjected to proximity ligation in (i), and (iii) sequencing the sequencing library to generate a plurality of sequencing reads. In some cases, the sequencing reads include paired-end reads. In some cases, the sequencing reads include long reads.

[0007] In another aspect, a method is provided for obtaining a phased human leukocyte antigen (HLA) type of a sample. In some cases, the method includes aligning a plurality of sequencing reads obtained from proximity ligation of nucleic acids in the sample to a reference genome or a variation graph, and at least a portion of the plurality of sequencing reads corresponds to HLA loci. In some cases, the method includes identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. In some cases, the method includes classifying the plurality of SNPs and / or indels into a plurality of phase blocks. In some cases, the method includes aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types. In some cases, the method includes obtaining a database including a plurality of HLA alleles, and aligning the plurality of HLA alleles to a reference genome to generate a plurality of SNP signatures of known HLA types. In some cases, the method includes comparing the plurality of SNPs to the plurality of SNP signatures of known HLA types to identify a plurality of HLA types. In some cases, the method includes comparing the SNP signature of (e) to the phase block of (c) to obtain the phased HLA type. In some cases, the sequencing reads include paired-end reads. In some cases, the sequencing reads include long reads.

[0008] In various aspects of the method for obtaining a phased HLA type of a sample provided herein, the plurality of sequencing reads are obtained by crosslinking the sample, fragmenting the nucleic acid in the sample to produce nucleic acid fragments, ligating the nucleic acid fragments to produce linked nucleic acid fragments, reversing the crosslinking, and sequencing the linked nucleic acid fragments. Optionally, the sample is crosslinked by contacting the sample with a crosslinking agent selected from formaldehyde, psoralen, disuccinimidyl glutarate (DSG), ethylene glycol bis(succinimidyl succinate) (EGS), ultraviolet light, or a combination thereof. Optionally, the fragmentation comprises contacting the sample with an enzyme. Optionally, the enzyme is a nuclease, a restriction endonuclease, a transposase, or a combination thereof. Optionally, the nuclease is micrococcal nuclease. Optionally, the transposase is Tn5. Optionally, the fragmentation comprises non-enzymatic cleavage. Optionally, the method further comprises adding a label to the nucleic acid fragment after the ligation. Optionally, the label comprises biotin. Optionally, the label comprises an oligonucleotide. Optionally, the oligonucleotide comprises a barcode. Optionally, the crosslink binds the nucleic acid to a nucleic acid binding protein in the sample. Optionally, the plurality of SNPs and / or indels are identified by aligning the sequencing reads to the variation graph. Optionally, the phased HLA types comprise HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and / or HLA-DPB1. Optionally, the sample comprises a nucleic acid enriched for HLA sequences. Optionally, the entire HLA region is phased with more than 90% of the phased SNPs in a single phase block. Optionally, major and minor groups of HLA types are phased. In some cases, the method completes in less than 30 hours of CPU time.In some cases, the method is completed in less than 15 hours, less than 10 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour of CPU time. In some cases, the method is completed in about 30 to about 40 minutes of CPU time. In some cases, the sequencing reads include paired-end reads. In some cases, the sequencing reads include long reads.

[0009] Incorporation by Reference 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]

[0010] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings of which:

[0011] [Figure 1] FIG. 1 shows a flow diagram of one method of phased HLA typing. [Diagram 2] 1 shows a flow diagram of one method for obtaining sequencing reads. [Diagram 3] Read pair linkage for separation of maternal and paternal read pairs is described. [Figure 4] 1 shows an example of phased HLA typing. [Diagram 5] FIG. 1 shows a flow diagram of one method of phased HLA typing. [Figure 6] The use of capture probes in phased HLA haplotypes is illustrated. [Figure 7] Proximity ligation is used to phase the HLA regions. [Figure 8] 1 illustrates a computer system programmed or configured to implement the methods provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] HLA matching is a key component of donor and recipient matching for transplantation. For unrelated donors and recipients, matching is usually performed by typing at specific HLA loci. However, matching at the haplotype level can sometimes result in better clinical outcomes.

[0013] HLA genes have a high degree of polymorphism and complex patterns of association, which creates challenges in determining HLA haplotypes across multiple loci.

[0014] The present disclosure provides an approach for generating phased HLA types. Long-range haplotype phasing techniques can be combined with HLA genotype calling to generate phased HLA regions. For example, as shown in FIG. 1, phased HLA typing can combine two independent pipelines to generate a final phased output. The first pipeline (FIG. 1, top panel) calls and phases single nucleotide polymorphisms (SNPs) from high-throughput proximity ligation reads. The second pipeline (FIG. 1, bottom panel) uses a database of known HLA alleles as well as calling HLA types from the same data, and then aligns the best-matched sequence from each called HLA allele type to a reference genome to generate a SNP signature for that allele. The SNP signature from the second process (FIG. 1, bottom panel) is then matched to the high-quality phased SNPs from the first process (FIG. 1, top panel) to determine whether the SNP signature is more compatible with parent 1 SNPs or parent 2 SNPs. Each allele is then assigned to the parental chromosome of the best-matching phased SNP set.

[0015] A method for obtaining a phased human leukocyte antigen (HLA) type of a sample is provided herein. Such a method may include aligning a plurality of sequencing reads to a reference genome, and at least a portion of the plurality of sequencing reads corresponds to an HLA locus. Then, the method may include identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads. Alternatively, or in combination, the method may include identifying the plurality of SNPs by comparing the plurality of sequencing reads to a variation graph. Then, the method may include classifying the plurality of SNPs and indels into phase blocks. Then, the method may include aligning the plurality of sequencing reads to a variation graph to identify a plurality of HLA types. Then, the method may include comparing the plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele. The method may then include comparing said SNP signature of (e) to said phase block of (c) to obtain said phased HLA type.

[0016] Sequencing reads can be paired-end reads, single short reads, long reads (e.g., by Nanopore, SMRT, HiFi), or other suitable sequence read formats.

[0017] In some cases, the sequencing reads are obtained using any suitable proximity ligation method, such as Hi-C, Chicago, Micro-C, or Omni-C. In some cases, the sequencing reads are obtained using Micro-C. The proximity ligation reads can be paired-end reads, where each read of the pair provides sequence information on one or the other of the two sides of the proximity ligation site. The proximity ligation reads can be long reads, where one long read provides sequence information on both sides of the proximity ligation site, or even around multiple proximity ligation sites in one concatemer. The sequencing reads can be aligned to a reference genome using a read aligner (e.g., BWA aligner). Alternatively, or in combination, the sequencing reads can be aligned to a variation graph, such as the variation graph constructed by Kourami. SNPs and indels are called based on the alignment using any suitable software, such as DeepVariant or Kourami. In some cases, it is desirable to use alignment to a variation graph when alignment to a fixed reference may result in misaligned reads in homologous regions. In certain cases, HLA-B and HLA-C have regions of high similarity that can occasionally result in reads from HLA-C being misaligned to HLA-B, resulting in accurate SNPs and indels not being called.

[0018] Once the SNPs and indels are obtained, they can be grouped into phase-matched blocks using maximum likelihood-based software. In some cases, the maximum likelihood-based software is Hapcut2. The maximum likelihood-based software assigns the SNPs and indels to phase blocks. Each phase block can contain a set of SNPs and indels labeled P1 or P2 for whether they originate from parent chromosome P1 or parent chromosome P2. When using paired-end reads, the size of the reads can vary from a few hundred bases to the chromosome scale, and thus, in many cases, phase blocks are obtained spanning the entire 4 Mbp HLA region with over 90% of the phased SNPs in a single largest phase block.

[0019] In some cases, HLA allele types are called using hybrid capture proximity ligation reads. The reads are aligned to a variation graph constructed from the IPD-IMGT / HLA database of HLA variants. By examining these alignments, ideal HLA type pairs are identified in the variation graph. Each HLA type is a tag of its form, e.g., A, which describes the major and minor groups of HLA types of alleles. * 26:01:02:01, etc. For this purpose, HLA * LA or Kourami can be used, or alternatively, these steps can be assembled independently.

[0020] In some cases, an HLA type signature database can then be constructed by aligning each sequence in a database of sequences of known HLA alleles to the corresponding region of a reference genome assembly, and analyzing the alignment to generate a list of SNPs and indels for each allele, thereby creating a SNP signature for each allele. In some cases, the allele type calls from the alignment to the variation graph can be used to determine the SNP signature of the sample being analyzed to generate a list of SNP signatures for the sample. Each SNP in the SNP signature can then be compared in both sequence and position to the phased SNPs in that region. The phase (P1 or P2) with the best matching SNPs and indels can be assigned to that allele. The result of this analysis performed on all alleles is a phased set of HLA types. In some cases, HLA types are called directly by matching the allele type signature database to the phased SNPs and indels from the maximum likelihood analysis.

[0021] In some cases, a local phased assembly of each gene is performed using the captured reads. In this case, the local phased assembly of each individual gene can be used similarly to the called type sequence (e.g., matching SNPs in the local phased assembly sequence to SNPs called with Deep Variant and phased with Hapcut2). In some cases, this approach is advantageous when individual samples diverge significantly from even the closest match in a database of known HLA alleles. In addition, this can be a performance advantage, since the graph alignment step requires only 30 real CPU hours to compute, while the local phased assembly of each gene is expected to require less time. In some cases, the graph alignment step requires only 30-40 real CPU hours to compute.

[0022] Long-distance haplotype phasing Disclosed herein are methods for generating read sets, including phased read sets, using long-read or short-read sequencing techniques for applications including genome assembly and haplotype phasing. Exemplary techniques include, but are not limited to, proximity ligation techniques such as Hi-C, Chicago, Micro-C, and Omni-C. Nucleic acid molecules can be joined (e.g., in a chromatin structure), cleaved to expose internal ends, rejoined at the junction to other exposed ends, released from the junction, and sequenced. This technique can produce nucleic acid molecules that include multiple sequence segments. Multiple sequence segments within a nucleic acid molecule can have preserved phase information while rearranged relative to their natural or starting positions and orientations. Sequence segments on either side of a junction can be confidently considered to be from the same phase of the sample nucleic acid molecule. In one example, FIG. 2 shows the steps of cross-linking a chromatin structure, fragmenting the nucleic acid in the chromatin, ligating or connecting exposed ends, reversing the cross-linking, and sequencing the resulting nucleic acid. As shown in Figure 3, linkage of reads from two or more connected regions of nucleic acid generated in this manner can indicate that the reads originate from the same original nucleic acid molecule, allowing binning of maternal and paternal reads and even phasing blocks of sequence spanning entire chromosomes.

[0023] Nucleic acid molecules, including high molecular weight DNA, can be bound or immobilized on at least one nucleic acid binding moiety. For example, DNA assembled into in vitro chromatin aggregates and fixed by formaldehyde treatment is consistent with the methods herein. Techniques for binding or immobilizing nucleic acids include, but are not limited to, in vitro or reconstituted chromatin assemblies, natural chromatin, DNA-binding protein aggregates, nanoparticles, DNA-binding beads or beads coated with DNA-binding substances, polymers, synthetic DNA-binding molecules, or other solid or substantially solid affinity molecules. In some cases, the beads are solid-phase reversible immobilization (SPRI) beads (e.g., beads containing negatively charged carboxyl groups, such as Beckman-Coulter Agencourt AMPure XP beads).

[0024] Nucleic acids bound to nucleic acid binding moieties such as those described herein can be held such that nucleic acid molecules with first and second segments separated on the nucleic acid molecule by a distance greater than the read distance on a sequencing device (e.g., 10 kb, 50 kb, 100 kb, or more) are bound together independently of their common phosphodiester bond. Upon cleavage of such bound nucleic acid molecules, the exposed ends of the first and second segments can ligate to each other. In some cases, the nucleic acid molecules are bound at a concentration such that there is little or no overlap between the bound nucleic acid molecules on the solid surface, such that the exposed internal ends of the cleaved molecules are more likely to religate or rebind only to exposed ends from other segments that are in phase on the common nucleic acid source before cleavage. As a result, the DNA molecule is cleaved and the cleaved exposed internal ends can be religated, for example, randomly, without losing phase information.

[0025] The bound nucleic acid molecule can be cleaved by one of any number of enzymatic and non-enzymatic techniques to expose internal ends. For example, the nucleic acid molecule can be digested using a restriction enzyme, such as a restriction endonuclease that leaves a single-stranded overhang. For example, Mbol digestion is suitable for this purpose, but other restriction endonucleases are also contemplated. A list of restriction endonucleases is available, for example, in most molecular biology product catalogs. Other non-limiting techniques for nucleic acid cleavage include the use of transposases, tagmentation enzyme complexes, topoisomerases, non-specific endonucleases, DNA repair enzymes, RNA-guided nucleases, fragmentases, or alternative enzymes. For example, transposases can be used in combination with unbound left and right boundaries to create sequence-independent breaks in the nucleic acid characterized by the binding of transposase-delivered oligonucleotide sequences. Physical means, including mechanical means (e.g., sonication, shearing), thermal means (e.g., temperature change), or electromagnetic means (e.g., irradiation such as UV irradiation), can also be used to effect cleavage.

[0026] Immobilization of nucleic acids at this stage can keep the cleaved nucleic acid molecule fragments in close physical proximity, thus preserving the phase information of the original molecules. For example, the advantage of immobilization to chromatin aggregates is that distinct regions of common nucleic acid molecules can be held together regardless of their phosphodiester backbone, so that their phase information is not lost upon cleavage of the phosphodiester backbone. This advantage is further conveyed by alternative scaffolds to which the nucleic acid molecules are attached prior to cleavage.

[0027] Optionally, the single-stranded "sticky" end overhangs are modified to prevent re-annealing and re-ligation. For example, the sticky ends are partially filled-in, such as by adding one nucleotide and polymerase. In this way, the entire single-stranded end cannot be filled-in, but the end is modified to prevent re-ligation with the previously complementary end. In the example of Mbol digestion leaving a 5'GATC 5-prime overhang, only guanosine nucleotide triphosphate is added. This results in the fill-in of only the "G" of the first complementary base ("C"), resulting in a 5'GAT overhang. This step makes the free sticky ends incompatible for re-ligation with each other, but preserves the sticky ends for downstream use. Alternatively, blunt ends are generated by complete fill-in of the overhang, restriction digestion with a blunt end generating enzyme, treatment with a single-stranded DNA exonuclease, or non-specific cleavage. In some cases, a transposase is used to ligate adapter ends, either blunt or sticky, to exposed internal ends of the DNA molecule.

[0028] Optionally, a "punctuation oligonucleotide" is introduced. This punctuation oligonucleotide marks the cleavage / religation site. Some punctuation oligonucleotides have single-stranded overhangs at both ends that match the partially filled-in overhangs generated on the exposed internal ends of the nucleic acid sample. Examples of punctuation oligonucleotides are shown below. In some cases, the double-stranded oligonucleotide with single-stranded overhangs is modified, such as by removing the 5' phosphate at its 5' end, so that it cannot form a concatemer during ligation. Alternatively, a blunt-end oligonucleotide is used, or a separate punctuation oligonucleotide is not used to mark the cleavage site. In some systems, for example when a transposase is used, cleavage is achieved by the addition of transpososome border sequences, followed by ligation of the border sequences to each other or to the punctuation oligonucleotide. Exemplary punctuation oligonucleotides are shown below. However, alternative end oligos are consistent with the disclosure herein and differ in sequence, length, the presence or sequence of overhangs, or modifications such as 5' dephosphorylation.

[0029] In some cases, the double-stranded region of the end oligonucleotide is changed.The relevant feature of the end oligonucleotide is the sequence of its overhang, which allows it to ligate to the nucleic acid sample, but is optionally modified to prevent self-ligation or concatemer formation.Often, it is preferred that the end oligonucleotide contains a sequence that does not exist or is unlikely to exist in the target nucleic acid molecule, so that it can be easily identified in downstream sequence reaction.The end oligo is optionally barcoded, for example, with a known barcode sequence or a randomly generated unique identifier sequence.The unique identifier sequence can be designed to make it very unlikely that multiple junctions in a nucleic acid molecule or sample are barcoded with the same unique identifier.

[0030] The cut ends can be joined together, for example, using a ligase or similar enzyme, either directly or via an oligo (e.g., a terminal oligo). Ligation can proceed so that the free single-stranded ends of the immobilized high molecular weight nucleic acid molecule are ligated directly or to a terminal oligonucleotide. Since the terminal oligonucleotide, if utilized, can have two ligatable ends, this ligation can effectively chain together a region of the high molecular weight nucleic acid molecule. Alternative methods that result in the addition of an interruption sequence or molecule between the two exposed ends can also be used, as well as methods for directly joining two exposed ends without a terminal end.

[0031] The nucleic acid can then be released from the nucleic acid binding moiety. In the case of in vitro chromatin aggregates, this can be achieved by reversing the crosslinks, or by digesting the protein components, or by both reversing the crosslinks and digesting the protein components. A suitable approach is treatment of the complex with proteinase K, but many alternatives are contemplated. For other binding techniques, suitable methods such as cleavage of the linker molecule or degradation of the substrate can be used.

[0032] The nucleic acid molecules resulting from such techniques can have a variety of related characteristics. Sequence segments within a nucleic acid molecule can be rearranged relative to their natural or starting positions and orientations, but the phase information is preserved. As a result, sequence segments on either side of a junction can be reliably assigned to a common phase of a common sample molecule. Thus, segments that are far apart on a molecule can be brought together or brought close together by such techniques, such that parts or the entirety of each segment can be sequenced in a single run of a single molecule sequencing device, allowing for definitive phase assignment. Alternatively, in some cases, initially adjacent segments can be separated from each other in the resulting nucleic acid. In some cases, nucleic acid molecules may be religated such that at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 100% of the religations are between segments that were in phase on a common nucleic acid source prior to cleavage.

[0033] Another relevant feature of the resulting molecules is that in some cases, most or all of the original molecular sequence, although possibly rearranged, is preserved in the final interrupted or rearranged molecule. For example, in some cases, 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20% or less of the original molecule is lost in generating the resulting molecule(s). Thus, in addition to being useful as a phase determinant, the resulting molecules retain a substantial proportion of the original molecular sequence, such that the resulting molecules are optionally used to simultaneously generate sequence information, such as contig information useful in de novo sequencing, or as an independent validation of previously generated contig information.

[0034] Another feature of some resulting libraries of molecules is that the cleavage junctions are not common to multiple members of the resulting population of molecules. That is, different copies of the same starting nucleic acid molecule can end up with different patterns of junctions and rearrangements. Random cleavage junctions can be generated using non-specific cleavage molecules or through alteration of restriction endonuclease selection or digestion parameters.

[0035] The consequence of having a molecule-specific cleavage site is that, in some cases, the end oligonucleotide is optionally excluded from the process that leads to reshuffling and religation of the "cut molecule" so as not to cause any adverse effects. By aligning the segments of three or more reshuffled molecules, it is observed that the cut sites are easily identified by their absence in the majority of other members of the library. That is, when three or more reshuffled molecules are aligned locally, it is found that the segments are common to all molecules, but the edges of the segments may differ between molecules. By noting where the local sequence similarity of the segments ends, the cut junctions in the "unpunctuated" reorganized nucleic acid molecules can be mapped.

[0036] The resulting nucleic acid molecules can be sequenced, for example, on a long-read sequencer. The resulting sequence reads contain alternating segments between nucleic acid sequences from the original input molecule and, if used, from the sequence of the end oligo. These reads can be processed by a computer to split the sequence data from each read using the end oligonucleotide sequence, or otherwise to identify junctions. The sequence segments within each read can be segments from a single input high molecular weight DNA molecule. The original nucleic acid molecule can include a genomic sequence, such as a chromosome, or a fraction thereof. The set of segment reads reveals long-range haplotype-phased data, which may be discontinuous in the original nucleic acid molecule. These data can be used for de novo genome assembly and for phasing heterozygous positions in the input genome. The sequences between the junctions indicate contiguous nucleic acid sequences in the source nucleic acid sample, and the sequences spanning the junctions indicate nucleic acid segments that are in phase in the nucleic acid sample but may be far away from adjacent segments in the placed scaffold.

[0037] Junctions can be identified by a variety of techniques. If an end oligo is used, the junctions can be identified in the reads containing the end oligo sequence. Alternatively, the junctions can be identified by comparison to a second sequence source (and preferably a third sequence source) for the nucleic acid molecule, such as a previously generated contig sequence dataset, or a second independently generated DNA stranded molecule with an independently derived junction. Once the sequences are aligned, for example, the quality or confidence of the alignment to a particular position can indicate where one segment ends and another begins. If a restriction enzyme is used to effect the cleavage, sequences containing restriction enzyme recognition sites can be evaluated as potentially containing junctions. Note that not all restriction enzyme recognition sites contain junctions, for example, because some restriction enzyme recognition sites may not be physically accessible by the enzyme while the nucleic acid was bound to the support. Statistical information can also be employed in identifying junctions, for example, the length segments between junctions can be predicted to be a certain average value or follow a certain distribution.

[0038] The advantage of the operation herein is that it can preserve molecular phase information while bringing non-adjacent regions of a molecule into close proximity, so that the non-adjacent regions of a molecule are contained in a single nucleic acid molecule at a distance suitable for sequencing in a single read, such as a long read. Thus, regions that are separated by more than the distance of a single long read operation in the starting sample (e.g., 10 kb, 15 kb, 20 kb, 30 kb, 50 kb, 100 kb, or more) are brought into local close proximity so that they are within the distance covered by a single read of a long-range sequencing reaction. Thus, regions that are separated beyond the range of the sequencing technology for a single read in the original sample are phase-preserved and read in a single reaction in the reorganized molecule.

[0039] The resulting reconstituted molecules can be sequenced, and their sequence information can be mapped to independently or simultaneously generated sequence reads or contig information, or to a known reference genome sequence (e.g., the known sequence of the human genome). Adjacent segments on the resulting reconstituted molecular reads are presumed to be in phase. Thus, when these segments are mapped to heterogeneous contigs or long-range sequence reads, the reads are assigned to a common phase of the common molecule in the sequence assembly.

[0040] Alternatively, if multiple independently generated resulting rearranged molecules are sequenced simultaneously, phased sample data is optionally generated from these molecules only, such that segment sequences separated by junctions are presumed to be in phase, while sequences not separated by junctions represent stretches of contiguous nucleic acid in the sample itself, e.g., useful for de novo sequencing and useful for phase determination. Additionally or alternatively, however, multiple independently generated resulting rearranged molecules sequenced simultaneously can still be compared to independently generated scaffold or contig information.

[0041] The methods and compositions presented herein are capable of preserving long-range phase information, particularly for molecular segments separated by distances greater than the length of the reads in the sequencing technology (e.g., 10 kb, 20 kb, 50 kb, 100 kb, 500 kb, or more), but provide for such non-adjacent segments in rearranged or often "interrupted" molecules when the segments are contiguous or close enough to be covered by a single read.

[0042] In some instances, the resulting reorganized molecules are combined with natural molecules for sequencing. Natural molecules, when used, can be informationally recognized and utilized by the lack of interruption sequences. Natural molecules are sequenced using short or long read techniques, and their assembly is guided by phase information and segment sequence information generated through sequencing of reorganized molecules or libraries.

[0043] Haplotype phasing In diploid genomes, it is often important to know which allelic variants are linked on the same chromosome. This is known as haplotype phasing. Short reads from high-throughput sequence data rarely allow direct observation of which allelic variants are linked. Computational inference of haplotype phasing can be uncertain at long distances. The present disclosure provides one or more methods that allow using allelic variants on read pairs to determine which allelic variants are linked. In some cases, phasing by the methods of the present disclosure is performed without imputation.

[0044] In various embodiments, the methods and compositions of the present disclosure allow for haplotype phasing of diploid or polyploid genomes for multiple allelic variants.Thus, the methods described herein can provide for the determination of linked allelic variants based on variant information from read pairs and / or assembled contigs using it.Examples of allelic variants include, but are not limited to, those known from 1000 Genomes, UK10K, HapMap, and other projects for discovering genetic variation among humans. For example, by having haplotype phasing data, disease association to specific genes can be more easily revealed, as demonstrated by the discovery of unlinked inactivating mutations in both copies of SH3TC2 causing 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 causing hypercholesterolemia 9 (Rios J, Stein E, Shendure J, et al. Hum. Mol. Genet. 19:4313-18, 2010).

[0045] Humans are heterozygous at an average of 1 in 1,000 sites. In some cases, a single lane of data using high-throughput sequencing can generate at least about 150,000,000 read pairs. The read pairs can be about 100 base pairs long. From these parameters, it is estimated that one in ten of all reads from a human sample will cover a heterozygous site. Thus, it is estimated that on average one in one hundred of all read pairs from a human sample will cover a pair of heterozygous sites. Thus, about 1,500,000 read pairs (one hundredth of 150,000,000) provide phasing data using a single lane. With about 3 billion bases in the human genome, and 1 in 1000 being heterozygous, there are about 3 million heterozygous sites in the average human genome. With approximately 1,500,000 read pairs representing pairs of heterozygous sites, the average coverage of each heterozygous site phased using a single lane of high-throughput sequencing is approximately (1X) using a typical high-throughput sequencer. Thus, a diploid human genome can be reliably and completely phased with one lane of high-throughput sequence data associated with sequence variants from a sample prepared using the methods disclosed herein. In some examples, the lane of data can be a set of DNA sequence read data. In a further example, the lane of data can be a set of DNA sequence read data from one run of a high-throughput sequencer.

[0046] Because the human genome consists of two homologous sets of chromosomes, understanding the true genetic makeup of an individual requires the delineation of maternal and paternal copies of genetic material, or haplotypes. Obtaining haplotypes in an individual is useful in several ways. First, haplotypes are clinically useful in predicting the outcome of donor-host matching in organ transplantation and are increasingly being used as a means to detect disease associations. Second, in genes that exhibit compound heterozygosity, haplotypes provide information on whether two deleterious variants are located on the same allele, greatly influencing the prediction of whether the inheritance of these variants is deleterious. Third, haplotypes from populations have provided information on the population structure and evolutionary history of races. Finally, the widespread allelic imbalance in gene expression recently described suggests that genetic or epigenetic differences between alleles may contribute to quantitative differences in expression. Understanding of haplotype structure will delineate the mechanisms of variants that contribute to allelic imbalance.

[0047] In certain embodiments, the methods disclosed herein include in vitro techniques for fixing and capturing associations between distant regions of the genome necessary for long-range binding and phasing. In some cases, the methods include constructing and sequencing XLRP libraries to deliver read pairs that are very genomically separated. In some cases, the interactions arise primarily from random associations within a single DNA fragment. In some instances, sequence segments that are close to each other in a DNA molecule interact more frequently and with higher probability, while interactions between distant parts of the molecule are less frequent, so that genomic distances between segments can be inferred. Thus, there is a systematic relationship between the number of pairs that bind two loci and their proximity on the input DNA. The present disclosure can generate read pairs that can span the largest DNA fragments in the extraction. The input DNA for this library has a maximum length of 150 kbp, which is the longest meaningful read pair observed from the sequencing data. This suggests that the method can bind even more genomically separated loci if larger input DNA fragments are provided. By applying improved assembly software tools specifically adapted to handle the type of data generated by this method, complete genome assembly may be possible.

[0048] The data generated using the methods and compositions of the present disclosure can achieve extremely high phasing accuracy. Compared to previous methods, the methods described herein can phase a higher percentage of variants. Phasing can be achieved while maintaining a high level of accuracy. The techniques herein can allow phasing with an accuracy of more than about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.999%. The techniques herein can allow accurate phasing of 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. This phase information can be extended to greater distances, for example, greater than about 200 kbp, 300 kbp, 400 kbp, 500 kbp, 600 kbp, 700 kbp, 800 kbp, 900 kbp, 1 Mbp, 2 Mbp, 3 Mbp, 4 Mbp, 5 Mbp, or 10 Mbp. In some embodiments, greater than 90% of heterozygous SNPs for a human sample can be phased with greater than 99% accuracy using less than about 250 million reads or read pairs, for example, by using only one lane of Illumina HiSeq data. In other cases, about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 99% of the heterozygous SNPs for human samples can be phased with less than about 250 million or less than about 500 million reads or read pairs with an accuracy of more than about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.999% by using only one or two lanes of Illumina HiSeq data. For example, 95% or more than 99% of the heterozygous SNPs for human samples can be phased with an accuracy of more than about 95% or 99% using less than about 250 million or less than about 500 million reads.In further cases, additional variants can be captured by increasing the read length to about 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1500bp, 2kbp, 3kbp, 4kbp, 5kbp, 10kbp, 20kbp, 50kbp, or 100kbp.

[0049] In other embodiments of the present disclosure, data from XLRP libraries can be used to confirm the phasing capabilities of long-distance read pairs. The accuracy of these results is comparable to the best techniques previously available, but further extends to significantly longer distances. Current sample preparation protocols for certain sequencing methods recognize variants located within the read length, e.g., 150 bp, of the target site for phasing. In one example, 44% of the 1,703,909 heterozygous SNPs present from an XLRP library constructed for NA12878, a benchmark sample for assembly, were phased with an accuracy of over 99%. In some cases, this percentage can be extended to nearly all variable sites with judicious selection of enzymes or digestion conditions.

[0050] 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-DQB1, HLA-DPA1, HLA-DPB1). FIG. 4 shows exemplary phased HLA genotypes. HLA regions of the genome are densely polymorphic and can be difficult to sequence or phase with standard sequencing approaches. The techniques of the present disclosure can provide improved sequencing and phasing accuracy of HLA regions of the genome. Using the techniques of the present disclosure, HLA regions of the genome can be accurately phased as part of phasing of larger regions (e.g., chromosome arms, chromosomes, whole genomes) or by themselves (e.g., by targeted enrichment such as hybrid capture). In one example, the HLA region itself was accurately phased at a sequencing depth of about 300x. These techniques can provide advantages over traditional approaches for HLA analysis, such as long-range PCR, which can include complex protocols and many separate reactions. As discussed further herein, samples can be multiplexed for sequencing analysis, for example, by including sample identification barcodes in bridging oligonucleotides or elsewhere and demultiplexing sequence information based on the barcodes. In one example, multiple samples are subjected to proximity ligation, barcoded with sample identification barcodes (e.g., in bridging oligonucleotides), targeted to HLA regions (e.g., by hybrid capture), and multiplexed sequencing is performed, allowing phasing of HLA regions for multiple samples. In some cases, phasing of HLA regions is performed without complementation.

[0051] In one example, as shown in FIG. 5, sequencing reads of a library prepared as disclosed herein can be called for variants (e.g., using Deep Variant or other techniques) and phased (e.g., using Hapcut2 or other techniques). This can generate a phased set of variants, which can be referred to as "paternal" and "maternal" (as well as any remaining unphased variants). The same sequencing reads can also be typed (e.g., using HLA 1 ... * LA (Dilthey et al. * LA--HLA typing from linearly projected graph alignments. Bioinformatics 35(21)2019,4394-4396), Kourami (Lee,et al. Kourami:graph-guided assembly for novel human leukocyte antigen allele discovery. Genome Biology 19(1)2018,16), which is incorporated by reference in its entirety, or other techniques) and aligned HLA. The phased variants and aligned HLA types can then be matched together to provide a phased HLA type.

[0052] In another example, as shown in FIG. 7, a proximity ligation approach is used to generate a phased HLA typing analysis. A Micro-C is used to prepare a proximity ligation library that is target specific to the HLA region using a capture panel (as discussed further herein). Sequencing reads from the library are used to perform phase block mapping and further haplotype calling (e.g., to the closest allele). Phase block mapping and haplotype calling are then combined to generate a phased haplotype of the HLA region. In addition to calling the haplotype to the closest allele, haplotypes that do not match any known allele can be indicated (instead of or in addition to calling the closest known allele to such haplotype).

[0053] target enrichment In certain embodiments, the present disclosure provides a method for enrichment of HLA nucleic acids for determining phased HLA haplotypes. In some cases, the enrichment method is a solution-based format. In some cases, the target nucleic acid can be labeled with a labeling agent. In other cases, the target nucleic acid can be crosslinked to one or more associated molecules that are labeled with a labeling agent. Examples of labeling agents include, but are not limited to, biotin, polyhistidine tags, and chemical tags (e.g., alkyne and azide derivatives used in click chemistry methods). In addition, the labeled target nucleic acid can be captured and thereby enriched by the use of a capture agent. The capture agent can be streptavidin and / or avidin, antibodies, chemical moieties (e.g., alkynes, azides), and any biological, chemical, physical, or enzymatic agent used in affinity purification.

[0054] In some cases, immobilized or non-immobilized nucleic acid probes can be used to capture target nucleic acids. For example, target nucleic acids can be enriched from a sample by hybridization to a probe on a solid support or in solution. In some examples, the sample can be a genomic sample. In some examples, the probe can be an amplicon. The amplicon can include a predetermined sequence. Furthermore, the hybridized target nucleic acid can be washed and / or eluted from the probe. The target nucleic acid can be a DNA, RNA, cDNA, or mRNA molecule.

[0055] In some cases, the enrichment method may include contacting a sample containing the target nucleic acid with a probe and binding the target nucleic acid to a solid support. In some cases, the sample may be fragmented using an enzymatic method to obtain the target nucleic acid. In some cases, the probe may be specifically hybridized to the target nucleic acid. In some cases, the average size of the target nucleic acid may be about 145 bp to about 600 bp, about 100 bp to about 2500 bp, about 600 to about 2500 bp, or about 350 bp to about 1000 bp. The target nucleic acid may be further separated from unbound nucleic acids in the sample. The solid support may be washed and / or eluted to obtain the enriched target nucleic acid. In some examples, the enrichment step may be repeated about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. For example, the enrichment step may be repeated about 1, 2, or 3 times.

[0056] In some cases, the enrichment method can include providing a probe-derived amplicon, the probe for amplification being bound to a solid support. The solid support can include a nucleic acid probe fixed to the support to capture a specific target nucleic acid from the sample. The probe-derived amplicon can be hybridized to the target nucleic acid. After hybridization to the probe amplicon, the target nucleic acid in the sample can be enriched by capturing the hybridized target nucleic acid from the captured probe (e.g., via a capture agent such as biotin, antibody, etc.) and washing and / or eluting. The target nucleic acid sequence can be further amplified, for example, using a PCR method to produce an amplified pool of enriched PCR products.

[0057] In some cases, the solid support may be a microarray, a slide, a chip, a microwell, a column, a tube, a particle, or a bead. In some examples, the solid support may be coated with streptavidin and / or avidin. In other examples, the solid support may be coated with an antibody. Furthermore, the solid support may comprise a glass, metal, ceramic, or polymeric material. In some embodiments, the solid support may be a nucleic acid microarray (e.g., a DNA microarray). In other embodiments, the solid support may be a paramagnetic bead.

[0058] In certain embodiments, the present disclosure provides a method of amplifying enriched DNA. In some cases, the enriched DNA is a read pair. The read pair can be obtained by the method of the present disclosure.

[0059] In some embodiments, one or more amplification and / or replication steps are used to prepare the library to be sequenced. Any suitable amplification method can be used. Examples of amplification techniques that can be used include, but are not limited to, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real-time PCR (RTPCR), single-cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP), PCK-RFLPIRT-PCR-IRFLP, hot start PCR, nested PCR, in situ polony PCR, in situ rolling circle amplification (RCA), bridge PCR, ligation-mediated PCR, Qb replicase amplification, inverse PCR, picotiter PCR, and emulsion PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, selective amplification of target polynucleotide sequence, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR), and nucleic acid-based sequence amplification (NABSA).Other amplification methods that can be used herein include those described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and 6,582,938.

[0060] In certain embodiments, PCR is used to amplify the DNA molecules after they are dispensed into individual partitions. In some cases, one or more specific priming sequences in the amplification adaptors are utilized for PCR amplification. The amplification adaptors can be ligated to the fragmented DNA molecules before or after dispensing into individual partitions. Polynucleotides containing amplification adaptors with appropriate priming sequences on both ends can be PCR exponentially amplified. For example, polynucleotides with only one appropriate priming sequence due to incomplete ligation efficiency of the amplification adaptors containing priming sequences may only undergo linear amplification. Furthermore, if no adaptors containing appropriate priming sequences are ligated, the polynucleotides can be removed altogether from the amplification, e.g., PCR amplification. In some embodiments, the number of PCR cycles varies between 10 and 30, but can be as low as 9, 8, 7, 6, 5, 4, 3, 2, or less, or as high as 40, 45, 50, 55, 60, or more. As a result, exponentially amplifiable fragments carrying amplification adapters with appropriate priming sequences can be present at much higher concentrations (1000-fold or more) after PCR amplification compared to linearly amplifiable or non-amplifiable fragments.Advantages of PCR compared to whole genome amplification techniques (e.g., amplification with randomized primers or multiple displacement amplification with phi29 polymerase) include, but are not limited to, more uniform relative sequence coverage (since each fragment can be copied at most once per cycle and amplification is controlled by the thermocycling program), a substantially lower rate of forming chimeric molecules than, for example, MDA (Lasken et al., 2007, BMC Biotechnology) (since chimeric molecules pose a significant challenge to accurate sequence assembly by presenting non-biological sequences in the assembly graph, which results in high rates of misassembly or highly ambiguous and fragmented assemblies), a reduction in sequence-specific bias that can result from binding of randomized primers typically used in MDA compared to using specific priming sites with specific sequences, a higher reproducibility in the amount of final amplified DNA product that can be controlled by the choice of PCR cycle number, and a higher fidelity in replication by the polymerases typically used in PCR compared to conventional whole genome amplification techniques.

[0061] In some embodiments, the fill-in reaction involves or is performed as part of the amplification of one or more target polynucleotides using a first primer and a second primer, the first primer comprising a sequence capable of hybridizing to at least a portion of one or more complements of a first adaptor oligonucleotide, and the second primer comprising a sequence capable of hybridizing to at least a portion of one or more complements of a second adaptor oligonucleotide. Each of the first and second primers can be of any suitable length, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 or more nucleotides, fewer nucleotides, or more nucleotides, some or all of which can be complementary to the corresponding target sequence (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides, fewer nucleotides, or more nucleotides). For example, about 10 to 50 nucleotides can be complementary to the corresponding target sequence.

[0062] "Amplification" refers to any process in which the number of copies of a target sequence is increased. In some cases, a replication reaction may produce only a single complementary copy / replica of a polynucleotide. Methods for primer-directed amplification of target polynucleotides include, but are not limited to, methods based on the polymerase chain reaction (PCR). Conditions favorable for amplification of a target sequence by PCR are optimized at various steps in the process and depend on characteristics of the elements in the reaction, such as the type of target, the concentration of the target, the length of the sequence to be amplified, the sequence of the target and / or one or more primers, the length of the primers, the concentration of the primers, the polymerase used, the volume of the reaction, the ratio of one or more elements to one or more other elements, some or all of which can be varied. In general, PCR involves the steps of denaturation of the target to be amplified (if double-stranded), hybridization of one or more primers to the target, and extension of the primers by a DNA polymerase, the steps being repeated (or "cycled") to amplify the target sequence. Steps in this process can be optimized for various outcomes, to enhance yield, to reduce the formation of spurious products, and / or to increase or decrease the specificity of primer annealing, etc. Methods of optimization include, but are not limited to, adjustments to the type or number of elements in the amplification reaction and / or to the conditions of a given step in the process, such as the temperature at a particular step, the duration of a particular step, and / or the number of cycles.

[0063] In some embodiments, the amplification reaction may include at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more cycles. In some examples, the amplification reaction may include at least about 20, 25, 30, 35, or 40 cycles. In some embodiments, the amplification reaction includes no more than about 5, 10, 15, 20, 25, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more cycles. A cycle may include any number of steps, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more steps. A step may include any temperature or temperature gradient appropriate to achieve the objectives of a given step, including, but not limited to, 3' end extension (e.g., adapter fill-in), primer annealing, primer extension, and strand denaturation. The steps can be of any duration, including, but not limited to, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 180, 240, 300, 360, 420, 480, 540, 600, 1200, 1800 seconds or more, less than the above, or more than the above, including indefinitely until manually interrupted. Any number of cycles with different steps can be combined in any order. In some embodiments, different cycles with different steps are combined such that the total number of cycles in combination is about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, or 200 or more cycles, fewer than the above, or more than the above. In some embodiments, amplification is performed after the fill-in reaction.

[0064] In some embodiments, the amplification reaction may be performed on at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 800, 1000 ng of target DNA molecules. In other embodiments, the amplification reaction may be performed on less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 800, 1000 ng of target DNA molecules.

[0065] Amplification can be performed before or after pooling the target polynucleotides from independent samples.

[0066] The disclosed method includes a step of determining the amount of amplifiable nucleic acid present in a sample. Any known method can be used to quantify amplifiable nucleic acid, an exemplary method being polymerase chain reaction (PCR), specifically quantitative polymerase chain reaction (qPCR). qPCR is a technology based on polymerase chain reaction, which is used to amplify and simultaneously quantify targeted nucleic acid molecules. qPCR allows both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalization genes) of specific sequences in a DNA sample. The procedure follows the general principles of polymerase chain reaction, with the additional feature that amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. QPCR is described, for example, in Kurnit et al. (U.S. Patent No. 6,033,854), Wang et al. (U.S. Patent Nos. 5,567,583 and 5,348,853), Ma et al. (The Journal of American Science, 2(3), 2006), Heid et al. (Genome Research 986-994, 1996), Sambrook and Russell (Quantitative PCR, Cold Spring Harbor Protocols, 2006), and Higuchi (U.S. Patent Nos. 6,171,785 and 5,994,056), the contents of which are incorporated by reference herein in their entireties.

[0067] Other methods of quantification include the use of fluorescent dyes that intercalate with double-stranded DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with complementary DNA. These methods can be used broadly, but are also specifically adapted for real-time PCR, as described in more detail below by way of example. In the first method, a DNA-binding dye binds to all double-stranded (ds) DNA in the PCR, resulting in fluorescence of the dye. Thus, an increase in DNA product during PCR causes an increase in fluorescence intensity, which is measured at each cycle, allowing quantification of the DNA concentration. The reaction is prepared similarly to a standard PCR reaction, with the addition of a fluorescent (ds)DNA dye. The reaction is run in a thermocycler, and after each cycle, the level of fluorescence is compared in a detector, the dye only fluorescing when bound to (ds)DNA (i.e., PCR product). With respect to standard dilutions, the (ds)DNA concentration in the PCR can be measured. As with other real-time PCR methods, the values ​​obtained do not have absolute units associated with them. Comparison of the measured DNA / RNA sample with the standard dilution solution results in the proportion or ratio of the sample to the standard dilution solution, allowing for relative comparison between different tissues or experimental conditions. Ensuring accuracy in the quantification and / or expression of target genes can be standardized with respect to stably expressed genes. Copy numbers of unknown genes can be similarly normalized to genes of known copy numbers.

[0068] The second method uses sequence-specific RNA or DNA-based probes to quantify only DNA containing the probe sequence, and thus the use of reporter probes significantly increases specificity, allowing quantification even in the presence of some non-specific DNA amplification. This allows multiplexing, i.e., assaying for various genes in the same reaction by using specific probes with differently colored labels, provided that all genes are amplified with similar efficiency.

[0069] This method is commonly performed with DNA-based probes that have a fluorescent reporter (e.g., 6-carboxyfluorescein) at one end of the probe and a quencher (e.g., 6-carboxy-tetramethylrhodamine) at the opposite end of the probe. The proximity of the reporter to the quencher prevents detection of its fluorescence. Destruction of the probe by the 5' to 3' exonuclease activity of a polymerase (e.g., Taq polymerase) destroys the proximity of the reporter-quencher, thus allowing unquenched emission of fluorescence, which can be detected. An increase in the product targeted by the reporter probe at each PCR cycle results in a proportional increase in fluorescence due to destruction of the probe and release of the reporter. The reaction is prepared similarly to a standard PCR reaction and the reporter probe is added. Once the reaction begins, both the probe and primer are annealed to the DNA target during the annealing stage of PCR. Polymerization of a new DNA strand begins at the primer, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence, which is detected and measured in a real-time PCR thermocycler, and the geometric increase in fluorescence, which corresponds to the exponential increase in product, is used to determine the threshold cycle in each reaction.

[0070] The relative concentration of DNA present during the log phase of the reaction is determined by plotting fluorescence against cycle number on a log scale (so that an exponentially increasing amount results in a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. Since the amount of DNA doubles every cycle during the log phase, the relative amount of DNA can be calculated, for example, a sample with a Ct 3 cycles earlier than another has 23=8 times more template. The amount of nucleic acid (e.g., RNA or DNA) is then determined by comparing the results to a standard curve generated by real-time PCR of serial dilutions of known amounts of nucleic acid (e.g., no dilution, 1:4, 1:16, 1:64).

[0071] In certain embodiments, the qPCR reaction involves a dual fluorophore approach utilizing fluorescence resonance energy transfer (FRET), e.g., LIGHTCYCLER hybridization probes, in which two oligonucleotide probes are annealed to the amplicon (see, e.g., U.S. Pat. No. 6,174,670). The oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated by a distance compatible with efficient energy transfer. Other examples of labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: SCORPIONS probes (e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Patent No. 6,326,145), Sunrise (or AMPLIFLOUR) primers (e.g., Nazarenko et al., Nuc. Acids Res. 25:2516-2521,1997, and U.S. Patent No. 6,117,635), as well as LUX primers and MOLECULAR BEACONS probes (e.g., Tyagi et al., Nature Biotechnology 14:303-308, 1996, and U.S. Patent No. 5,989,823).

[0072] In other embodiments, the qPCR reaction uses the fluorescent Taqman method and an instrument capable of measuring fluorescence in real time (e.g., ABI Prism 7700 Sequence Detector). The Taqman reaction uses a hybridization probe that is labeled with two different fluorescent dyes. One dye is a reporter dye (6-carboxyfluorescein) and the other is a quenching dye (6-carboxy-tetramethylrhodamine). When the probe is intact, fluorescence energy transfer occurs and the fluorescent emission of the reporter dye is absorbed by the quenching dye. During the extension phase of the PCR cycle, the fluorescent hybridization probe is cleaved by the 5'-3' nucleolytic activity of DNA polymerase. Upon cleavage of the probe, the reporter dye emission is no longer efficiently transferred to the quenching dye, resulting in an increase in the fluorescence emission spectrum of the reporter dye. Any nucleic acid quantification method, including real-time or single-point detection methods, can be used to quantify the amount of nucleic acid in a sample. Detection can be carried out by a variety of different methods (e.g., staining, hybridization with a labeled probe, incorporation of a biotinylated primer followed by detection of an avidin-enzyme conjugate, incorporation of 32P-labeled deoxynucleotide triphosphates such as dCTP or dATP into the amplified segment), as well as any other suitable detection method for nucleic acid quantification. Quantification may or may not include an amplification step.

[0073] In some embodiments, the present disclosure provides a label for identifying or quantifying the linked DNA segments.In some cases, the linked DNA segments can be labeled to assist downstream applications such as array hybridization.For example, the linked DNA segments can be labeled using random priming or nick translation.

[0074] A wide variety of labels (e.g., reporters) may be used to label the nucleotide sequences described herein, including but not limited to during the amplification process. Suitable labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as ligands, cofactors, inhibitors, magnetic particles, and the like. Examples of such labels are included in U.S. Patent No. 3,817,837, U.S. Patent No. 3,850,752, U.S. Patent No. 3,939,350, U.S. Patent No. 3,996,345, U.S. Patent No. 4,277,437, U.S. Patent No. 4,275,149, and U.S. Patent No. 4,366,241, which are incorporated herein by reference in their entirety.

[0075] Additional labels include, but are not limited to, β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exo-glucanase, and glucoamylase. Fluorescent labels may also be used, as well as fluorescent reagents specifically synthesized with particular chemistries. A wide variety of methods for measuring fluorescence are available. For example, some fluorescent labels exhibit changes in excitation or emission spectra, some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence while a second fluorescent reporter increases fluorescence, some fluorescent labels exhibit quenching or emergence of fluorescence, and some fluorescent labels report rotational motion.

[0076] Furthermore, to obtain sufficient material for labeling, multiple amplifications can be pooled instead of increasing the number of amplification cycles per reaction. Alternatively, labeled nucleotides can be incorporated into the last cycle of the amplification reaction, e.g., 30 cycles of PCR (no label) + 10 cycles of PCR (with label).

[0077] In certain embodiments, the present disclosure provides a probe that can bind to a bound DNA segment. As used herein, the term "probe" refers to a molecule (e.g., an oligonucleotide, whether naturally occurring, such as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification) that can hybridize to another molecule of interest (e.g., another oligonucleotide). When a probe is an oligonucleotide, it can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific targets (e.g., gene sequences). In some cases, a probe can be associated with a label that makes it detectable in any detection system, including, but not limited to, enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.

[0078] With respect to arrays and microarrays, the term "probe" is used to refer to any hybridizable material that is fixed to an array to detect nucleotide sequences hybridized to the probe. In some cases, the probe can be about 10bp to 500bp, about 10bp to 250bp, about 20bp to 250bp, about 25bp to 200bp, about 25bp to 100bp, about 30bp to 100bp, or about 30bp to 80bp. In some cases, the probe can be about 10bp, about 20bp, about 30bp, about 40bp, about 50bp, about 60bp, about 70bp, about 80bp, about 90bp, about 100bp, about 150bp, about 200bp, about 250bp, about 300bp, about 400bp, or about 500bp long. For example, the probe can be about 20 to about 50bp long. Examples and rationale for probe design can be found in WO95 / 11995, EP717,113, and WO97 / 29212.

[0079] The probe, array of probes, or set of probes can be immobilized on a support. The support (e.g., solid support) can be made of various materials such as glass, silica, plastic, nylon, or nitrocellulose. The support can be rigid and have a flat surface. The support can have about 1 to 10,000,000 resolved loci. For example, the support may have about 10-10,000,000, about 10-5,000,000, about 100-5,000,000, about 100-4,000,000, about 1000-4,000,000, about 1000-3,000,000, about 10,000-3,000,000, about 10,000-2,000,000, about 100,000-2,000,000, or about 100,000-1,000,000 separate loci. The density of the separate loci may be at least about 10, about 100, about 1000, about 10,000, about 100,000, or about 1,000,000 separate loci within one square centimeter. In some cases, each of the separate loci may be occupied by more than 95% of a single type of oligonucleotide. In other cases, each of the separate loci may be occupied by a pooled mixture of probes or a set of probes. In further cases, some separate loci are occupied by a pooled mixture of probes or a set of probes, and other separate loci are occupied by more than 95% of a single type of oligonucleotide.

[0080] In some cases, the number of probes for a given nucleotide sequence on an array may be in vast excess relative to the DNA sample hybridized to such an array. For example, an array may have about 10, about 100, about 1000, about 10,000, about 100,000, about 1,000,000, or about 100,000,000 times the number of probes relative to the amount of DNA in the input sample.

[0081] In some cases, the array may have about 10, about 100, about 1000, about 10,000, about 100,000, about 1,000,000, about 10,000,000, or about 1,000,000,000 probes.

[0082] Arrays or sets of probes can be synthesized stepwise on the support or can be attached in presynthesized form. One synthesis method is VLSIPS™ (described in US Pat. No. 5,143,854 and EP 476,014), which involves the use of light to direct the synthesis of oligonucleotide probes in a high-density miniaturized array. Algorithms for the design of masks to reduce the number of synthesis cycles are described in US Pat. No. 5,571,639 and US Pat. No. 5,593,839. Arrays can also be combinatorially synthesized by delivering monomers to cells on the support through mechanically restricted channels, as described in EP 624,059. Arrays can also be synthesized by spotting reagents onto the support using an inkjet printer (see, for example, EP 728,520).

[0083] In some embodiments, the disclosure provides a method for hybridizing DNA segments linked onto an array. A "substrate" or "array" is a deliberately created collection of nucleic acids that can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (e.g., libraries of soluble molecules and libraries of oligonucleotides tethered to resin beads, silica chips, or other solid supports). In addition, the term "array" includes such libraries of nucleic acids that can be prepared by spotting nucleic acids of virtually any length (e.g., from 1 to about 1000 nucleotide monomers long) onto a substrate.

[0084] Array technology, as well as various related techniques and their applications in general, are described in many textbooks and documents. For example, these documents include: Lemieux et al.,1998,Molecular Breeding 4,277-289;Schena and Davis,Parallel Analysis with Biological Chips.in PCR Methods Manual(eds.M.Innis,D.Gelfand,J.Sninsky);Schena and Davis,1999,Genes,Genomes and Chips.In DNA Microarrays:A Practical Approach(ed.M.Schena),Oxford University Press,Oxford,UK,1999);The Chipping Forecast(Nature Genetics special issue;January 1999 Supplement);Mark Schena(Ed.),Microarray Biochip Technology,(Eaton Publishing Company);Cortes,2000,The Scientist 14

[17] :25;Gwynn and Page,Microarray analysis:the next revolution in molecular biology,Science,1999 Aug.6, and Eakins and Chu, 1999, Trends in Biotechnology, 17, 217-218.

[0085] Generally, any library can be arranged in an array by spatially separating the members of the library. Examples of libraries suitable for arraying include nucleic acid libraries (including libraries of DNA, cDNA, oligonucleotides, etc.), peptide, polypeptide, and protein libraries, and libraries containing any molecule, such as, inter alia, ligand libraries.

[0086] The library may be immobilized or fixed onto a solid phase (e.g., a solid substrate) to limit diffusion and mixing of the members. In some cases, a library of DNA-binding ligands may be prepared. In particular, the library may be immobilized onto a substantially flat solid phase, including membranes and non-porous substrates, such as plastic and glass. Furthermore, the library may be arranged in such a way that indexing (i.e., reference or access to a particular member) is easy. In some examples, the members of the library may be applied as spots in a grid format. A general assay system may be adapted for this purpose. For example, an array may be immobilized on the surface of a microplate with multiple members in a well, or with one member in each well. Furthermore, the solid substrate may be a membrane, such as a nitrocellulose or nylon membrane (e.g., a membrane used in blotting experiments). Alternative substrates include glass or silica-based substrates. Thus, the library may be immobilized by any suitable method, such as charge interactions or chemical binding to the walls or bottom of the well, or to the surface of the membrane. Other means of placement and immobilization can be used, such as pipetting, drop-touch, piezoelectric means, inkjet and bubble jet technology, electrostatic application, etc. In the case of silicon-based chips, photolithography can be utilized to place and immobilize the library on the chip.

[0087] Libraries are arranged on a solid substrate by being "spotted", which may be done manually or by using robotics to deposit the members. In general, arrays may be described as macroarrays or microarrays, the difference being the size of the spots. Macroarrays may contain spot sizes of about 300 microns or more and can be easily imaged by existing gel and blot scanners. Spot sizes in microarrays may be less than 200 microns in diameter, and these arrays usually contain thousands of spots. Thus, microarrays may require special robotics and imaging equipment, which may need to be custom made. Instruments are generally described in a review by Cortese, 2000, The Scientist 14

[11] :26.

[0088] Techniques have been described for making fixed libraries of DNA molecules. Usually, most of these methods describe how to synthesize single-stranded nucleic acid molecule libraries, for example, by using masking techniques to build various permutations of sequences at various distinct locations on a solid substrate. US Patent No. 5,837,832 describes an improved method for making DNA arrays fixed on silicon substrates based on ultra-large scale integration technology. In particular, US Patent No. 5,837,832 describes a strategy called "tiling" to synthesize a specific set of probes at spatially defined locations on a substrate, which can be used to make the fixed DNA library of the present disclosure. US Patent No. 5,837,832 further provides references to earlier techniques that can be used. In other cases, the arrays can also be built using photo deposition chemistry.

[0089] Arrays of peptides (or peptidomimetics) can also be synthesized on a surface in a manner that places each distinct library member (e.g., unique peptide sequence) at a distinct predefined location on the array. The identity of each library member is determined by its spatial location on the array. The location on the array where the binding interaction between a predefined molecule (e.g., target or probe) and a reactive library member occurs is determined, thereby identifying the sequence of the reactive library member based on its spatial location. These methods are described in U.S. Patent No. 5,143,854, WO90 / 15070, and WO92 / 10092, Fodor et al. (1991) Science, 251:767; Dower and Fodor (1991) Ann.Rep.Med.Chem., 26:271.

[0090] To aid in detection, labels such as any easily detectable reporter, e.g., fluorescent, bioluminescent, phosphorescent, radioactive, etc. reporters can be used (as discussed above). Such reporters, their detection, binding to targets / probes, etc. are discussed elsewhere in this document. Probe and target labeling is further disclosed in Shalon et al., 1996, Genome Res 6(7):639-45.

[0091] Examples of some commercially available microarray formats are described in Marshall and Hodgson, 1998, Nature Biotechnology, 16(1), 27-31.

[0092] To generate data from array-based assays, signals can be detected to indicate the presence or absence of hybridization between the probe and the nucleotide sequence. Additionally, direct and indirect labeling techniques can also be utilized. For example, direct labeling incorporates a fluorescent dye directly into the nucleotide sequence that hybridizes to the probe associated with the array (e.g., the dye is incorporated into the nucleotide sequence by enzymatic synthesis in the presence of labeled nucleotides or PCR primers). Direct labeling schemes provide robust hybridization signals and are easy to implement, for example, through the use of a family of fluorescent dyes with similar chemical structures and characteristics. In cases involving direct labeling of nucleic acids, cyanine or alexa analogs can be utilized for multiple-fluor comparative array analyses. In other embodiments, indirect labeling schemes can be utilized to incorporate epitopes into the nucleic acid before or after hybridization to the microarray probe. One or more staining procedures and reagents may be used to label the hybridized complex (e.g., a fluorescent molecule that binds to an epitope, thereby resulting in a fluorescent signal upon conjugation of a dye molecule to the epitope of the hybridized species).

[0093] In one example, enrichment of HLA regions can reduce sequencing burden, as shown in Figure 6. In that example, 48 HLA loci were targeted with 2553 capture probes, each 120 bp in length. These capture probes were tiled head-to-tail, every 120 bp, across a roughly 4 Mb region of chromosome 6.

[0094] Sequencing In various embodiments, a suitable sequencing method described herein or otherwise known is used to obtain sequence information from nucleic acid molecules in a sample.Sequencing can be achieved through classical Sanger sequencing methods.Sequencing can also be achieved using high-throughput systems, some of which allow detection of sequenced nucleotides immediately or as they are incorporated into the growing strand, i.e., detection of sequences in real time or near real time. In some cases, high throughput sequencing generates at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, or at least 500,000 sequence reads per hour, where the sequencing reads can be at least about 50, 60, 70, 80, 90, 100, 120, 150, 180, 210, 240, 270, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bases per read.

[0095] Sequencing can be whole genome, with or without enrichment of specific regions of interest. Sequencing can be targeted to specific regions of the genome. Regions of the genome that can be enriched or targeted include, but are not limited to, single genes (or regions thereof), gene panels, gene fusions, human leukocyte antigen (HLA) loci (e.g., class I HLA-A, B, and C; class II HLA-DRB1 / 3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1), exon regions, exomes, and other loci. Genomic regions can be related to immune response, immune repertoire, immune cell diversity, transcription (e.g., exome), cancer (e.g., BRCA1, BRCA2, panels of genes or regions thereof, e.g., hotspot regions, somatic variants, SNVs, amplifications, fusions, tumor mutation burden (TMB), microsatellite instability (MSI)), cardiac disease, genetic disease, and other diseases or conditions. A variety of methods can be used to enrich or target regions of interest, including but not limited to sequence capture. In some cases, capture Hi-C (CHi-C) or CHi-C-like protocols are used, with a sequence capture step (e.g., by target enrichment arrays) before or after library preparation.

[0096] In some embodiments, high throughput sequencing includes the use of technology available from Illumina's Genome Analyzer IIX, MiSeq personal sequencer, or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000 machines. These machines use reversible terminator-based sequencing by synthesis chemistry. These machines can perform over 200 billion DNA reads in 8 days. Smaller systems may be utilized to perform within 3, 2, 1 or less days.

[0097] In some embodiments, high-throughput sequencing includes the use of technology available from ABI Solid Systems. This genetic analysis platform allows for massively parallel sequencing of clonally amplified DNA fragments bound to beads. The sequencing methodology is based on sequential ligation with dye-labeled oligonucleotides.

[0098] Next generation sequencing can include ion semiconductor sequencing (e.g., using technology from Life Technologies (Ion Torrent)). Ion semiconductor sequencing can take advantage of the fact that ions can be released when a nucleotide is incorporated into a strand of DNA. To perform ion semiconductor sequencing, a high density array of microfabricated wells can be formed. Each well can hold a single DNA template. Under the well is an ion sensitive layer, and under the ion sensitive layer can be an ion sensor. When a nucleotide is added to the DNA, H+ is released and can be measured as a change in pH. The H+ ions can be converted to a voltage and recorded by the semiconductor sensor. The array chip can be flooded with nucleotides one after the other. No scanning, light, or camera is required. In some cases, an IONPROTON™ sequencer is used to sequence the nucleic acids. In some cases, an IONPGM™ sequencer is used. Ion Torrent Personal Genome Machine (PGM). The PGM can perform 10 million reads in 2 hours.

[0099] In some embodiments, high throughput sequencing involves the use of technology available from Helicos BioSciences Corporation (Cambridge, Massachusetts), such as the single molecule sequencing by synthesis (SMSS) method. SMSS is unique because it allows for the sequencing of the entire human genome in up to 24 hours. Finally, SMSS is partially described in U.S. Patent Application Publication Nos. 20060024711, 20060024678, 20060012793, 20060012784, and 20050100932.

[0100] In some embodiments, high throughput sequencing involves the use of technology available from 454 Lifesciences, Inc. (Branford, Connecticut), such as the PicoTiterPlate instrument, which contains a fiber optic plate that transmits chemiluminescent signals generated by the sequencing reaction that are recorded by a CCD camera in the instrument. The use of this fiber optic allows for the detection of a minimum of 20 million base pairs in 4.5 hours.

[0101] Methods using bead amplification followed by fiber optic detection are described in Marguiles, M., et al. "Genome sequencing in microfabricated high-density picolitre reactors," Nature, doi:10.1038 / nature03959, and also in U.S. Patent Application Publication Nos. 20020012930, 20030068629, 20030100102, 20030148344, 20040248161, 20050079510, 20050124022, and 20060078909.

[0102] In some embodiments, high throughput sequencing is performed using Clonal Single Molecule Arrays (Solexa, Inc.) or sequencing by synthesis (SBS) utilizing reversible terminator chemistry. These techniques are described in part in U.S. Patent Nos. 6,969,488, 6,897,023, 6,833,246, 6,787,308, and U.S. Patent Application Publication Nos. 20040106110, 20030064398, 20030022207, and Constans, A., The Scientist 2003, 17(13):36.

[0103] Next generation sequencing technologies may include the SMRT™ technology by Pacific Biosciences. In SMRT, each of the four DNA bases may be attached to one of four different fluorescent dyes. These dyes may be phospho linked. A single DNA polymerase may be immobilized with a single molecule of template single stranded DNA at the bottom of a zero mode waveguide (ZMW). The ZMW may be a confinement structure that allows observation of the incorporation of a single nucleotide by the DNA polymerase against a background of fluorescent nucleotides that can rapidly diffuse in and out of the ZMW (in microseconds). It may take several milliseconds to incorporate a nucleotide into the growing strand. During this time the fluorescent label may be excited, producing a fluorescent signal, and the fluorescent tag may be cleaved. The ZMW may be illuminated from below. Attenuated light from the excitation beam may penetrate the bottom 20-30 nm of each ZMW. 20 zeptoliters (20×10 -21 Microscopes can be created with detection limits of 100 liters. The small detection volume can provide a 1000-fold improvement in reducing background noise. Detection of the corresponding fluorescence of the dye can indicate which base has been incorporated. This process can be repeated.

[0104] In some cases, the next generation sequencing is nanopore sequencing (see, for example, Soni GV and Meller A. (2007) Clin Chem 53:1996-2001). A nanopore can be a small hole with a diameter of about 1 nanometer. Immersion of the nanopore in a conductive fluid and application of a potential thereon can result in a small current due to the conduction of ions through the nanopore. The amount of current that flows can be sensitive to the size of the nanopore. When a DNA molecule passes through the nanopore, each nucleotide on the DNA molecule can block the nanopore to a different extent. Thus, the change in current through the nanopore as the DNA molecule passes through the nanopore can represent a readout of the DNA sequence. Nanopore sequencing technology can be from Oxford Nanopore Technologies, for example, the GridlON system. A single nanopore can be inserted into a polymer membrane that spans the top of a microwell. Each microwell can have an electrode for individual sensing. The microwells can be assembled into array chips with 100,000 or more microwells (e.g., more than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000) per chip. An instrument (or node) can be used to analyze the chip. The data can be analyzed in real time. One or more instruments can be operated at a time. The nanopore can be a protein nanopore, e.g., protein alpha hemolysin, a heptameric protein pore. The nanopore can be made of a solid-state nanopore, e.g., a nanometer-sized hole formed in a synthetic membrane (e.g., SiNx, or SiO2). The nanopore can be a hybrid pore (e.g., integration of a protein pore into a solid-state membrane). The nanopore can be a nanopore with an integrated sensor, such as a tunneling electrode detector, a capacitive detector, or a graphene-based nanogap or edge state detector (see, e.g., Garaj et al. (2010) Nature vol. 67, doi:10.1038 / nature09379).Nanopores can be functionalized to analyze specific types of molecules (e.g., DNA, RNA, or proteins). Nanopore sequencing can include "strand sequencing," where an intact DNA polymer can pass through a protein nanopore with real-time sequencing as the DNA translocates through the pore. An enzyme can separate the strands of double-stranded DNA and present the strand through the nanopore. The DNA can have a hairpin at one end and the system can read both strands. In some cases, nanopore sequencing is "exonuclease sequencing," where individual nucleotides can be cleaved from the DNA strand by a processive exonuclease and the nucleotides can pass through the protein nanopore. The nucleotides can be transiently bound to a molecule (e.g., cyclodextran) within the pore. A characteristic discontinuity in the current can be used to identify the base.

[0105] GENIA's nanopore sequencing technology may be used. Engineered protein pores may be embedded in lipid bilayer membranes. An "active control" technique is used to allow efficient nanopore-membrane assembly and control of DNA movement through the channel. In some cases, the nanopore sequencing technology is from NABsys. Genomic DNA may be fragmented into strands of average length about 100 kb. The 100 kb fragments may be single stranded and then hybridized with hexamer probes. The genome fragments with the probes may be threaded through the nanopore to produce a current vs. time trace. The current trace may provide the location of the probes on each genome fragment. The genome fragments may be aligned to produce a probe map for the genome. The process may be done in parallel for a library of probes. A genome length probe map for each probe may be generated. Errors may be corrected with a process called "moving window sequencing by hybridization (mwSBH)". In some cases, the nanopore sequencing technology is from IBM / Roche. An electron beam can be used to create nanopore-sized openings in a microchip. An electric field can be used to pull or screw DNA through the nanopore. The DNA transistor device in the nanopore can contain nanometer-sized layers of alternating metal and dielectric. The separate charges in the DNA backbone can be trapped inside the DNA nanopore by the electric field. The DNA sequence can be read by turning the gate voltage off and on.

[0106] Next generation sequencing can include DNA nanoball sequencing (e.g., as performed by Complete Genomics. See, e.g., Drmanac et al. (2010) Science 327:78-81). DNA can be isolated, fragmented, and size selected. For example, DNA can be fragmented (e.g., by sonication) to an average length of about 500 bp. Adapters (Adl) can be attached to the ends of the fragments. Adapters can be used to hybridize to anchors for sequencing reactions. DNA with adapters attached to each end can be PCR amplified. Adapter sequences can be modified such that complementary single-stranded ends will join together to form circular DNA. DNA can be methylated to protect it from cleavage by type IIS restriction enzymes used in subsequent steps. Adapters (e.g., right adapters) can have restriction recognition sites, which can remain unmethylated. The unmethylated restriction recognition site in the adaptor can be recognized by a restriction enzyme (e.g., Acul), and the DNA can be cleaved 13 bp to the right of the right adaptor by Acul to form a linear double-stranded DNA. The second round right and left adaptors (Ad2) can be ligated to either end of the linear DNA, and all DNA to which both adaptors are attached can be PCR amplified (e.g., by PCR). The Ad2 sequence can be modified to allow them to bind to each other to form a circular DNA. The DNA can be methylated, but the restriction enzyme recognition site can remain unmethylated in the left Ad1 adaptor. A restriction enzyme (e.g., Acul) can be applied, and the DNA can be cleaved 13 bp to the left of Ad1 to form a linear DNA fragment. The third round right and left adaptors (Ad3) can be ligated to the right and left faces of the linear DNA, and the resulting fragments can be PCR amplified. The adaptors can be modified to bind to each other to form a circular DNA.A type III restriction enzyme (e.g., EcoP15) can be added, which can cut the DNA 26 bp to the left for Ad3 and 26 bp to the right for Ad2. This cut can remove a large segment of DNA and linearize the DNA again. A fourth round right and left adaptor (Ad4) can be ligated to the DNA, and the DNA can be amplified (e.g., by PCR) and modified to join together to form the completed circular DNA template.

[0107] Rolling circle replication (e.g., using Phi29 DNA polymerase) can be used to amplify small fragments of DNA. The four adapter sequences can contain palindromic sequences that can hybridize, folding the single strand back on itself to form a DNA nanoball (DNB™), which can be approximately 200-300 nanometers in diameter on average. The DNA nanoball can be attached (e.g., by adsorption) to a microarray (sequencing flow cell). The flow cell can be a silicon wafer coated with silicon dioxide, titanium, and hexamethyldisilazane (HMDS), as well as a photoresist material. Sequencing can be performed by unsequenced sequencing by ligating fluorescent probes to the DNA. The color of the fluorescence at the interrogated positions can be visualized by a high-resolution camera. The identity of the nucleotide sequence between the adapter sequences can be determined.

[0108] In some embodiments, high-throughput sequencing can be performed using AnyDot.chips (Genovoxx, Germany). In particular, AnyDot.chips can enhance nucleotide fluorescent signal detection by 10× to 50×. AnyDot.chips and methods of using same are described in International Publication Nos. WO 02088382, WO 03020968, WO 03031947, WO 2005044836, PCT / EP05 / 05657, PCT / EP05 / 05655, and German Patent Applications Nos. DE 101 49 786, DE 102 14 395, DE 103 56 837, DE 10 2004 009 704, DE 10 2004 025 696, DE 10 2004 025 746, DE 10 2004 025 694, DE 10 2004 025 695, DE 10 2004 025 744, DE 10 2004 025 745, and in part in DE 10 2005 012 301.

[0109] Other high-throughput sequencing systems include those described in Venter, J., et al. Science 16 February 2001; Adams, M. et al. Science 24 March 2000, and MJLevene et al. Science 299:682-686, January 2003, and US Patent Publication Nos. 20030044781 and 2006 / 0078937. Such a system includes sequencing a target nucleic acid molecule having multiple bases by the time-dependent addition of bases by polymerization reaction measured on the nucleic acid molecule, i.e., the activity of nucleic acid polymerase on the template nucleic acid molecule to be sequenced is tracked in real time. The catalytic activity of nucleic acid polymerase at each step in the sequence of base addition can then be used to infer the sequence by identifying which bases are incorporated into the growing complementary strand of the target nucleic acid. A polymerase on the target nucleic acid molecule complex is provided in a suitable position to move along the target nucleic acid molecule and extend the oligonucleotide primer at the active site. A plurality of labeled types of nucleotide analogs are provided proximate the active site, each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by adding a nucleotide analog to the nucleic acid strand at the active site using the polymerase, the added nucleotide analog being complementary to a nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerization step is identified. The steps of providing the labeled nucleotide analog, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated, thereby further extending the nucleic acid strand and determining the sequence of the target nucleic acid.

[0110] Hi-C method using micrococcal nuclease (MNase) Further provided herein is a method of obtaining phased HLA type information, which may include obtaining a stabilized biological sample containing a nucleic acid molecule complexed to at least one nucleic acid binding protein, contacting the stabilized biological sample with micrococcal nuclease (MNase) to cleave the nucleic acid molecule into a plurality of segments, and joining a first segment and a second segment of the plurality of segments at a junction. The use of MNase in the methods herein can provide specific information regarding where a DNA binding protein is bound to chromatin with up to single base pair resolution, for example, because MNase can cleave all base pairs that are not bound to the DNA binding protein. Furthermore, the use of MNase digestion can enable the creation of contact maps and topologically related domains to decipher three-dimensional chromatin structural information. In some cases, MNase can be bound or fused to an immunoglobulin binding protein A or a fragment thereof, such as protein A, protein G, protein A / G, or protein L.

[0111] For example, the MNase Hi-C method can provide locations of protein binding or genomic contact interactions at a resolution of about 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, or 100 kb or less. In some cases, a protein binding site, protein footprint, contact interaction, or other feature can be mapped to within 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 190 bp, 180 bp, 170 bp, 160 bp, 150 bp, 140 bp, 130 bp, 120 bp, 110 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp.

[0112] In certain embodiments, the method comprising a MNase digestion step may comprise a step of subjecting the plurality of segments to size selection to obtain a plurality of selected segments. In some cases, the plurality of selected segments may be from about 145 to about 600 bp. In some cases, the plurality of selected segments may be from about 100 to about 2500 bp. In some cases, the plurality of selected segments may be from about 100 to about 600 bp. In some cases, the plurality of selected segments may be from 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 1 It may be 500 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.

[0113] In another aspect of the method comprising an MNase digestion step provided herein, the method may further comprise preparing a sequencing library from a plurality of segments. In some embodiments, the method may further comprise subjecting the sequencing library to size selection to obtain a size-selected library. In some cases, the size-selected library may be about 350 bp to about 1000 bp in size. In some cases, the size-selected library may be about 100 bp to about 2500 bp in size, for example, about 100 bp to about 350 bp, about 350 bp to about 500 bp, about 500 bp to about 1000 bp, about 1000 bp to about 1500 bp, about 2000 bp to about 2500 bp, about 350 bp to about 1000 bp, about 350 bp to about 1500 bp, about 350 bp to about 2000 bp. p, 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.

[0114] In another embodiment, the method comprising MNase digestion step provided herein can further comprise analyzing a plurality of segments to obtain a QC value. In some cases, the QC value can be selected from chromatin digestion efficiency (CDE) and chromatin digestion index (GDI). CDE can be calculated as the percentage of segments having a desired length. For example, in some cases, CDE can be calculated as the percentage of segments with a size of 100bp to 2500bp before size selection. In some cases, the sample is selected for further analysis when the CDE value is at least 65%. In some cases, the sample can be selected for further analysis 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%.

[0115] CDI can be calculated as the ratio of the number of mononucleosome-sized segments to the number of dinucleosome-sized segments before size selection. For example, CDI can be calculated as the logarithm of the ratio of fragments with a size of 600-2500 bp to fragments with a size of 100-600 bp. In some cases, samples can be selected for further analysis if the CDI value is greater than -1.5 and less than 1. In some cases, the CDI value may be 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, greater than about -0.8 and less than about 1.5 greater than about -2 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.

[0116] In another embodiment, the stabilized biological sample used in the method comprising MNase digestion step provided herein can comprise a biological material treated with a stabilizing agent. In some cases, the stabilized biological sample can comprise a stabilized cell lysate. Alternatively, the stabilized biological sample can comprise a stabilized intact cell. Alternatively, the stabilized biological sample can comprise a stabilized intact nucleus. In some cases, the step of contacting the stabilized intact cell or intact nucleus sample with MNase can be performed before the lysis of the intact cell or intact nucleus. In some cases, the cell and / or nucleus can be lysed before joining the first segment and the second segment of the plurality of segments at the junction.

[0117] In another embodiment, the method comprising MNase digestion step provided herein can be performed on a small sample containing a small number of cells or a small amount of nucleic acid. For example, in some cases, the stabilized biological sample can contain less than 3,000,000 cells. In some cases, the stabilized biological sample can contain less than 2,000,000 cells. In some cases, the stabilized biological sample can contain less than 1,000,000 cells. In some cases, the stabilized biological sample can contain less than 500,000 cells. In some cases, the stabilized biological sample can contain less than 400,000 cells. In some cases, the stabilized biological sample can contain less than 300,000 cells. In some cases, the stabilized biological sample can contain less than 200,000 cells. In some cases, the stabilized biological sample can 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 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 of DNA. In some cases, the stabilized biological sample comprises less than 9 μg of DNA. In some cases, the stabilized biological sample comprises less than 8 μg of DNA. In some cases, the stabilized biological sample comprises less than 7 μg of DNA. In some cases, the stabilized biological sample comprises less than 6 μg of DNA. In some cases, the stabilized biological sample comprises less than 5 μg of DNA. In some cases, the stabilized biological sample comprises less than 4 μg of DNA. In some cases, the stabilized biological sample comprises less than 3 μg of DNA. In some cases, the stabilized biological sample comprises less than 2 μg of DNA. In some cases, the stabilized biological sample comprises less than 1 μg of DNA. In some cases, the stabilized biological sample contains less than 0.5 μg of DNA.

[0118] In another embodiment, the method of the present invention comprising the MNase digestion step may be performed on individual cells or single cells. For example, the method of the present invention may be performed on cells distributed into individual partitions. Exemplary partitions include, but are not limited to, wells, droplets in emulsions, or surface locations (e.g., array spots, beads, etc.) that contain distinct patches of differentially sequenced linker molecules as described elsewhere herein. Additional partitions are also contemplated and are consistent with the methods, compositions, and systems disclosed herein.

[0119] In further embodiments, the stabilized biological sample used in the methods comprising the MNase digestion step herein may be further treated with an additional nuclease, such as DNase, to generate fragments of DNA. In some cases, the DNase may be non-sequence specific. In some cases, the DNase may be active on both single-stranded and double-stranded DNA. In some cases, the DNase may be specific to double-stranded DNA. In some cases, the DNase may preferentially cleave double-stranded DNA. In some cases, the DNase may be specific to single-stranded DNA. In some cases, the DNase may preferentially cleave single-stranded DNA. In some cases, the DNase may be DNase I. In some cases, the DNase may be DNase II. In some cases, the DNase may be selected from one or more of DNase I and DNase II. In some cases, the DNase may be conjugated or fused to an immunoglobulin-binding protein A or a fragment thereof, such as protein A, protein G, protein A / G, or protein L. Other suitable nucleases are within the scope of this disclosure.

[0120] In further aspects, the stabilized biological samples provided herein for use in the methods comprising an MNase digestion step can be treated with a crosslinking agent. In some cases, the crosslinking agent can be a chemical fixative. In some cases, the chemical fixative includes formaldehyde having a spacer arm length of about 2.3 to 2.7 angstroms (A). In some cases, the chemical fixative includes a crosslinking agent having a long spacer arm length, e.g., the crosslinking agent can have a spacer 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) having a spacer arm having a length of about 16.1A. The chemical fixative can include disuccinimidyl glutarate (DSG) having a spacer arm length of about 7.7A. In some cases, the chemical fixatives include 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, and in other cases, some or all of the multiple chemical fixatives are applied to the sample simultaneously. The use of cross-linkers with long spacer arms can increase the proportion of read pairs with large (e.g., >1 kb) read pair separation distances. DSG is membrane permeable and allows intracellular cross-linking. DSG can increase cross-linking efficiency compared to disuccinimidyl suberate (DSS) in some applications. EGS has NHS ester reactive groups at both termini and can be reactive to amino groups (e.g., primary amines). EGS is membrane permeable and allows intracellular cross-linking. EGS cross-linking can be reversed, for example, by treatment with hydroxylamine at pH 8.5 for 3-6 hours, and lactose dehydrogenase, for example, retained 60% of its activity after reversible cross-linking with EGS. In some cases, the chemical fixative may include a psoralen.In some cases, the crosslinker can be ultraviolet light, chlormethine, cyclophosphamide, chlorambucil, uramustine, melphalan, bendamustine, bis(2-chloroethyl)ethylamine, bis(2-chloroethyl)methylamine, tris(2-chloroethyl)amine, isofamide, carmustine, lomustine, streptozocin, busulfan, cisplatin, carboplatin, cicycloplatin, eptaplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, dacarbazine, mitozolomide, temozolomide, mitomycin C, nitrous acid, formaldehyde, acetylaldehyde, doxorubicin, daunorubicin, epirubicin, or idarubicin. Optionally, the crosslinking agent comprises an intercalating agent, an antibiotic, or a minor groove binder. Optionally, the stabilized biological sample can be a crosslinked paraffin-embedded tissue sample.

[0121] In a further aspect, the methods provided herein that include an MNase digestion step can include contacting the plurality of selected segments with an antibody. In some cases, an immunoglobulin binding protein or fragment thereof tethered to an oligonucleotide adaptor can be targeted to an antibody bound to the plurality of selected segments.

[0122] In a further aspect, the method comprising an MNase digestion step provided herein comprises a step of binding a first segment and a second segment of the plurality of segments at a junction. In some cases, the binding step may comprise filling-in the sticky ends using biotin-tagged nucleotides and ligating the blunt ends. In some cases, the binding step may comprise contacting at least the first segment and the second segment with a bridging oligonucleotide. In some cases, the binding step may comprise contacting at least the first segment and the second segment with a barcode. In some embodiments, the bridging oligonucleotide herein may be at least about 5 nucleotides to about 50 nucleotides in length. In some embodiments, the bridging oligonucleotide herein may be about 15 to about 18 nucleotides in length. In some embodiments, the bridging oligonucleotides 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 length. In some embodiments, the bridging oligonucleotides herein can comprise a barcode.

[0123] In further embodiments of the methods herein that include an MNase digestion step, the method can include obtaining at least some sequence on each side of the junction to generate the 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 junction to generate the first read pair.

[0124] In further aspects of the methods herein including an MNase digestion step, the method can include mapping the first read pair to a set of contigs and determining a path through the set of contigs that represents an order and / or orientation relative to the genome.

[0125] In a further aspect of the method comprising an MNase digestion step herein, the method may comprise mapping the first read pair 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.

[0126] In a further aspect of the method herein comprising an MNase digestion step, the method comprises mapping the first read pair to a set of contigs and assigning variants in the set of contigs to a phase.

[0127] In a further aspect of the method comprising an MNase digestion step herein, the method comprises mapping the first read pair to a set of contigs, and determining from the set of contigs the presence of a variant in the set of contigs, and performing a step selected from one or more of: (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 drug efficacy for the stabilized biological sample.

[0128] Computer Systems The present disclosure provides a computer system that is programmed to perform the method of the present disclosure. Figure 8 shows a computer system (801) that is programmed or configured to obtain phased HLA type. The computer system (801) can adjust various aspects of the HLA type phasing of the present disclosure, for example, synthesize SNPs and indels obtained from sequencing read information to generate phase information of HLA genomic loci. The computer system (801) can be an electronic device of a user or a computer system, and is located remotely to the electronic device. The electronic device can be a mobile electronic device.

[0129] The computer system (801) includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") (805), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (801) also includes memory or storage locations (810) (e.g., random access memory, read-only memory, flash memory), electronic storage (815) (e.g., hard disk), communication interfaces (820) (e.g., network adapters) for communicating with one or more other systems, and peripherals (825), such as cache, other memory, data storage devices, and / or electronic display adapters. The memory (810), storage (815), interface (820), and peripherals (825) communicate with the CPU (805) via a communication bus (solid line), such as a motherboard. The storage (815) may be a data storage device (or data repository) for storing data. The computer system (801) may be operatively connected to a computer network ("network") (830) with the aid of a communication interface (820). The network (830) may be the Internet and / or an extranet, an intranet and / or an extranet in communication with the Internet. In some cases, the network (830) is a telecommunications and / or data network. The network (830) may include one or more computer servers, which may enable distributed computing, such as cloud computing. The network (830) may in some cases, with the aid of the computer system (801), implement a peer-to-peer network, which may enable devices coupled to the computer system (801) to act as clients or servers.

[0130] The CPU (805) can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory (810). The instructions may be directed to the CPU (805), which may then be programmed or otherwise configured to perform the methods of the present disclosure. Examples of operations performed by the CPU (805) include fetch, decode, execute, and writeback.

[0131] The CPU 805 may be part of a circuit, such as an integrated circuit. One or more other components of the system 801 may also be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0132] The storage device (815) can store files such as drivers, libraries, and saved programs. The storage device (815) can store user data, such as user preferences and user programs. The computer system (801) may optionally include one or more additional data storage devices external to the computer system (801), such as located on a remote server in communication with the computer system (801) via an intranet or the Internet.

[0133] The computer system (801) can communicate with one or more remote computer systems via a network (830). For example, the computer system (801) can communicate with a remote computer system of a user (e.g., a researcher who desires phased HLA haplotypes for an individual). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a phone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access the computer system (801) via the network (830).

[0134] Methods as described herein can be performed by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system (801), such as, for example, on memory (810) or electronic storage (815). The machine executable or machine readable code can be provided in the form of software. In use, the code can be executed by the processor (805). In some cases, the code can be retrieved from storage (815) and stored in memory (810) for immediate access by the processor (805). In some situations, the electronic storage (815) can be omitted and the machine executable instructions are stored in memory (810).

[0135] The code may be precompiled and configured for use with a machine having a suitable processor to execute the code, or may be compiled during run-time. The code may be provided in a programming language that may be selected to render the code executable in a precompiled or as-compiled fashion.

[0136] Aspects of the systems and methods provided herein, such as the computer system (801), may be integrated in programming. Various aspects of the technology may be considered as an "article of manufacture" or "article of manufacture" in the form of machine (or processor) executable code and / or associated data, typically carried on or embedded in a type of machine-readable medium. The machine executable code may be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer or processor, or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory recording media at any time for programming the software. All or portions of the software may be communicated from time to time over the Internet or various other telecommunications networks. Such communication may enable, for example, loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, another type of media that may have software elements includes light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, over wired and optical terrestrial communications networks and various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing the software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to media that participate in providing instructions to a processor for execution.

[0137] Thus, a machine-readable medium such as a computer executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, any of the storage devices in a computer, such as those that may be used to implement, for example, the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include copper wire and fiber optics, including coaxial cables, the wires that comprise a bus within a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, other magnetic media, CD-ROMs, DVDs or DVD-ROMs, other optical media, punch cards, paper tape, other physical storage media with patterns of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves carrying data or instructions, cables or links which transmit such carrier waves, or other media from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0138] The computer system (801) can include or communicate with an electronic display (835) that includes a user interface (UI) (840) for providing, for example, synthesized SNP and indel information obtained from sequencing reads of the HLA genomic region. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0139] The disclosed method and system can be implemented by one or more algorithms. The one or more algorithms can be implemented by software after execution by a central processing unit (805). The one or more algorithms can, for example, align a plurality of sequencing reads to a reference genome, where at least a portion of the plurality of sequencing reads correspond to HLA loci, identify a plurality of single nucleotide polymorphisms (SNPs) and / or indels in the plurality of sequencing reads, (c) classify the plurality of SNPs and indels into phase blocks, align the plurality of sequencing reads into a variation graph to identify a plurality of HLA types, compare the plurality of HLA types with a plurality of known HLA alleles to obtain a SNP signature for each HLA allele, and (f) compare the SNP signature of (e) to the phase blocks of (c) to obtain the phased HLA type. Alternatively, one or more algorithms can identify the plurality of SNPs by comparing or aligning the plurality of sequencing reads to the variation graph.

[0140] In some cases, the graph alignment step takes less than 30 hours, 25 hours, 20 hours, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes of CPU time. In some cases, the overall computational method takes less than 30 hours, 25 hours, 20 hours, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes of CPU time.

[0141] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods and materials are described herein.

[0142] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "contig" includes a plurality of such contigs, and reference to "probing the physical layout of a chromosome" includes reference to one or more methods of probing the physical layout of a chromosome, and equivalents thereof, known to those of skill in the art.

[0143] Additionally, the use of "and" means "and / or" unless otherwise stated. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0144] Further, where the description of various embodiments uses the term "comprising," it is to be understood that those of skill in the art will understand that in some specific instances the embodiments may alternatively be described using the terms "consisting essentially of" or "consisting of."

[0145] The term "sequencing read," as used herein, refers to a fragment of DNA whose sequence has been determined.

[0146] The term "subject" as used herein can refer to a eukaryotic or prokaryotic organism.

[0147] The term "read pair or read-pair" as used herein can refer to two or more elements that are combined to provide sequence information. In some cases, the number of read pairs can refer to the number of mappable read pairs. In other cases, the number of read pairs can refer to the total number of read pairs generated.

[0148] The term "stabilized" as used herein can describe a sample that is preserved or protected from degradation. In some cases, the stabilized sample is crosslinked or treated with a fixative or crosslinking agent. In some cases, the stabilized sample is treated with formaldehyde, formalin, paraformaldehyde, glutaraldehyde, osmium tetroxide, etc.

[0149] As used herein, the term "about," unless otherwise specified, can describe a number as a range of values ​​that includes that number plus or minus 10% of that number. EXAMPLES

[0150] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any manner. The examples, together with the methods described herein, are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Variations thereof and other uses encompassed within the spirit of the present invention as defined by the scope of the claims will occur to those skilled in the art.

[0151] Example 1: Phased HLA typing Phased HLA typing combines two independent pipelines to result in a final phased output. The first pipeline (Figure 1, top panel) calls and phases single nucleotide polymorphisms (SNPs) from high-throughput proximity ligation reads. The second pipeline (Figure 1, bottom panel) calls HLA types from the same data but using a database of known HLA alleles, then aligns the best-matching sequence from each called HLA allele type to the reference genome to generate a SNP signature for that allele. The SNP signature from the second process (Figure 1, bottom panel) was then matched to the high-quality phased SNPs from the first process (Figure 1, top panel) to determine whether the SNP signature was better matched to parent 1 SNPs or parent 2 SNPs. Each allele was then assigned to the parental chromosome of the best-matching phased SNP set.

[0152] Step 1: Alignment of reads to a reference

[0153] Align the Micro-C captured reads (A) to a reference genome assembly (B, e.g. Grch38) using a read aligner such as the BWA aligner. The output is an alignment in BAM format (C).

[0154] Step 2: Calling SNPs and indels

[0155] SNPs are called from the alignment (C) generated in step (1). SNPs are called with any suitable software package, for example, DeepVariant (Poplin, et al. A universal SNP and small-indel variant caller using deep neural networks. Nat Biotech. 36.2018, 983-987, which is incorporated herein by reference in its entirety). The output of DeepVariant is a list of SNPs and small insertions and deletions (D). Using this data type, SNPs are called with high sensitivity and very high accuracy (>99%). In some cases, it is desirable to use a variation graph, such as the one constructed by Kourami, to call SNPs from sequence reads. For example, in some cases, alignment to a fixed reference may result in misaligned reads in homologous regions, particularly HLA-B and HLA-C, which have highly similar regions and occasionally result in reads from HLA-C misaligned to HLA-B, which can lead to accurate SNPs and indels not being called.

[0156] Step 3: Phasing SNPs and indels

[0157] Maximum likelihood based software such as Hapcut2 is used to group SNPs and indels into phase-matched blocks (Edge, et al. HapCUT2: reobust and accurate haplotype assembly for diverse sequencing technologies. Genome Res. 27(5). 2017, 801-812, which is incorporated herein by reference in its entirety). The output of Hapcut2 is an assignment of SNPs to phase blocks (G). Each phase block contains a set of SNPs labeled P1 or P2 for whether they originate from parent chromosome P1 or parent chromosome P2. Since read pair sizes often vary from hundreds of bases to the chromosome scale, phase blocks are obtained spanning the entire 4 Mbp HLA region with over 90% of the phased SNPs in a single maximum phase block. In some cases, alleles are phased for a single gene by finding two maximum likelihood paths through the graph. In some cases, alleles are phased for multiple genes by finding two maximum likelihood paths through the graph for multiple genes.

[0158] Step 4: Variation graph-based alignment

[0159] HLA allele types are called using hybrid capture proximity ligation reads. The reads are aligned to a variation graph constructed from the IPD-IMGT / HLA database of HLA variants. These alignments are examined to find the best pair of HLA types in the variation graph. Each HLA type is in the form of a tag, e.g., A, which describes the major and minor groups of HLA types of the alleles. * 26:01:02:01, etc. (F). These steps can be assembled independently, but HLA * Software packages such as LA or Kourami are used for this purpose.

[0160] Step 5: Construction of HLA signature database

[0161] The software is used to align each sequence in a database of sequences of known alleles (E, approximately 15,000 sequences) to the corresponding region of the reference genome assembly sequence. The software is used to analyze each of the resulting sequences and generate a list of SNPs for each called allele. This set of SNPs effectively serves as a SNP signature for each allele.

[0162] Step 6: Check the signature

[0163] The allele type calls from the variation graph pipeline in (4) are used to examine the SNP signatures of the samples being analyzed and generate a list of SNP signatures corresponding to the samples.

[0164] Step 7: Type phasing with SNP and indel matches

[0165] Each SNP in the SNP signature for an allele is compared, both in sequence and position, to the phased SNPs in that region (G). The phase (P1 or P2) with the best matching SNPs and indels is assigned to that allele. The result of this run for all called alleles is a phased set of HLA types (I).

[0166] Optionally, step 4 is omitted and HLA types are called directly by matching the allele type signature database (H) generated in step 5 to the phased SNPs (G) from step 3.

[0167] In some cases, the captured reads are used to perform a local phased assembly of each gene. The local phased assembly of each gene is used as well as the called type sequence (matching SNPs in the local phased assembly sequence to SNPs called with Deep Variant and phased with Hapcut2). This approach can be beneficial in cases where individual samples diverge significantly from even the closest match in the database of known HLA alleles. There may also be additional performance benefits, since in some cases the graph alignment step requires approximately 30 real CPU hours to compute, while the local phased assembly of each gene is expected to require less time. In some cases, the graph alignment step requires approximately 30-40 real CPU hours to compute.

[0168] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments described herein may be employed. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of the claims, and their equivalents, be covered thereby.

Claims

1. 1. A method for obtaining a phased human leukocyte antigen (HLA) type of a sample, comprising: (a) aligning a plurality of sequencing reads to a reference genome or a variation graph, wherein at least a portion of the plurality of sequencing reads corresponds to an HLA locus; (b) identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in said plurality of sequencing reads; (c) classifying the plurality of SNPs and indels into phase blocks; (d) aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types; (e) comparing said plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele; (f) comparing the SNP signature of (e) with the phased block of (c) to obtain the phased HLA type; A method comprising:

2. 2. The method of claim 1, wherein the plurality of sequencing reads are obtained by cross-linking the sample, fragmenting nucleic acids in the sample to create nucleic acid fragments, ligating the nucleic acid fragments to create linked nucleic acid fragments, reversing the cross-linking, and sequencing the linked nucleic acid fragments.

3. 3. The method of claim 2, wherein the sample is crosslinked by contacting the sample with a crosslinking agent selected from formaldehyde, psoralen, disuccinimidyl glutarate (DSG), ethylene glycol bis(succinimidyl succinate) (EGS), ultraviolet light, or a combination thereof.

4. The method of claim 2 or 3, wherein the fragmenting comprises contacting the sample with an enzyme.

5. The method of claim 4 , wherein the enzyme is a nuclease, a restriction endonuclease, a transposase, or a combination thereof.

6. The method of claim 5, wherein the nuclease is micrococcal nuclease.

7. The method of claim 5, wherein the transposase is Tn5.

8. The method of claim 2 or 3, wherein the fragmentation comprises non-enzymatic cleavage.

9. The method of any one of claims 2 to 8, further comprising the step of adding a label to the nucleic acid fragments after the ligation.

10. The method of claim 9 , wherein the label comprises biotin.

11. The method of claim 9 , wherein the label comprises an oligonucleotide.

12. The method of claim 11 , wherein the oligonucleotide comprises a barcode.

13. The method of any one of claims 2 to 12, wherein the cross-linking binds nucleic acids to nucleic acid binding proteins in the sample.

14. The method of any one of claims 1 to 13, wherein the plurality of SNPs and / or indels are identified by aligning the sequencing reads to the variation graph.

15. 15. The method of any one of claims 1 to 14, wherein the phased HLA types comprise HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and / or HLA-DPB1.

16. The method of claims 1-15, wherein the sample comprises nucleic acid enriched for HLA sequences.

17. The method of claims 1 to 15, wherein the entire HLA region is phased with more than 90% of the phased SNPs in a single phase block.

18. The method according to any one of claims 1 to 17, wherein major and minor groups of HLA types are phased.

19. The method of any one of claims 1 to 18, wherein the method is completed in less than 30 hours of CPU time.

20. 20. The method of claim 19, wherein the method is completed in less than 15 hours, less than 10 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour of CPU time.

21. The method of claim 19 or 20, which is completed in about 30 to about 40 minutes of CPU time.

22. 1. A computer-implemented method for obtaining a phased human leukocyte antigen (HLA) type of a sample, comprising: (a) aligning a plurality of sequencing reads to a reference genome or a variation graph, wherein at least a portion of the plurality of sequencing reads corresponds to an HLA locus; (b) identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in said plurality of sequencing reads; (c) classifying the plurality of SNPs and indels into phase blocks; (d) aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types; (e) comparing said plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele; (f) comparing the SNP signature of (e) with the phased block of (c) to obtain the phased HLA type; A method comprising:

23. 1. A method for obtaining a phased human leukocyte antigen (HLA) type of a sample, comprising: (a) aligning a plurality of sequencing reads to a variation graph, wherein at least a portion of the plurality of sequencing reads corresponds to an HLA locus; (b) identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in said plurality of sequencing reads; (c) classifying the plurality of SNPs and indels into phase blocks; (d) aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types; (e) comparing said plurality of HLA types to a plurality of known HLA alleles to obtain a SNP signature for each HLA allele; (f) comparing the SNP signature of (e) with the phased block of (c) to obtain the phased HLA type; A method comprising:

24. 1. A method for obtaining a phased human leukocyte antigen (HLA) type of a sample, comprising: (a) aligning a plurality of sequencing reads to a reference genome or a variation graph, wherein at least a portion of the plurality of sequencing reads corresponds to an HLA locus; (b) identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in said plurality of sequencing reads; (c) classifying the plurality of SNPs and indels into a plurality of phase blocks; (d) comparing said plurality of SNPs to a plurality of SNP signatures of known HLA types to identify HLA types; (e) comparing the plurality of SNP signatures of (d) with the phased blocks of (c) to obtain the phased HLA type; A method comprising:

25. 25. The method of claim 24, wherein (e) comprises assigning a phase of the HLA types of the plurality of HLA types based on a SNP quantity of the plurality of SNPs that match with the plurality of SNP signatures.

26. 25. The method of claim 24, wherein (c) comprises assigning a phase to each of the plurality of phase blocks.

27. 25. The method of claim 24, further comprising generating a database comprising a plurality of SNP signatures of known HLA types.

28. 28. The method of claim 27, wherein generating the database comprises aligning a plurality of HLA allele sequences to a reference genome.

29. 25. The method of claim 24, wherein the plurality of sequencing reads comprises reads generated using proximity ligation technology.

30. 30. The method of claim 29, wherein the proximity ligation technology comprises Hi-C, Chicago, Micro-C, or Omni-C.

31. 25. The method of claim 24, wherein the plurality of sequencing reads comprises a contiguous sequence that includes sequences from regions of a chromosome that are separated from one another in the natural sequence of the chromosome.

32. 25. The method of Claim 24, further comprising, prior to (a), (i) subjecting the sample to a proximity ligation reaction; (ii) generating a sequencing library from nucleic acids in the sample subjected to proximity ligation in (i); and (iii) generating a plurality of sequencing reads by sequencing the sequencing library.

33. 1. A method for obtaining a phased human leukocyte antigen (HLA) type of a sample, comprising: (a) aligning a plurality of sequencing reads obtained from proximity ligation of nucleic acids in the sample to a reference genome or variation graph, wherein at least a portion of the plurality of sequencing reads corresponds to an HLA locus; (b) identifying a plurality of single nucleotide polymorphisms (SNPs) and / or indels in said plurality of sequencing reads; (c) classifying the plurality of SNPs and / or indels into a plurality of phase blocks; (d) aligning the plurality of sequencing reads to the variation graph to identify a plurality of HLA types; (e) obtaining a database comprising a plurality of HLA alleles and aligning said plurality of HLA alleles to a reference genome to generate a plurality of SNP signatures of known HLA types; (e) comparing the plurality of SNPs to the plurality of SNP signatures of known HLA types to identify a plurality of HLA types; (f) comparing the SNP signature of (e) with the phased block of (c) to obtain the phased HLA type; A method comprising:

34. 34. The method of any one of claims 24 to 33, wherein the plurality of sequencing reads are obtained by cross-linking the sample, fragmenting nucleic acids in the sample to create nucleic acid fragments, ligating the nucleic acid fragments to create linked nucleic acid fragments, reversing the cross-linking, and sequencing the linked nucleic acid fragments.

35. 35. The method of claim 34, wherein the sample is crosslinked by contacting the sample with a crosslinking agent selected from formaldehyde, psoralen, disuccinimidyl glutarate (DSG), ethylene glycol bis(succinimidyl succinate) (EGS), ultraviolet light, or a combination thereof.

36. 36. The method of claim 34 or 35, wherein the fragmenting comprises contacting the sample with an enzyme.

37. 37. The method of claim 36, wherein the enzyme is a nuclease, a restriction endonuclease, a transposase, or a combination thereof.

38. 38. The method of claim 37, wherein the nuclease is micrococcal nuclease.

39. 38. The method of claim 37, wherein the transposase is Tn5.

40. 36. The method of claim 34 or 35, wherein the fragmentation comprises non-enzymatic cleavage.

41. The method of any one of claims 34 to 40, further comprising the step of adding a label to the nucleic acid fragments after ligation.

42. 42. The method of claim 41 , wherein the label comprises biotin.

43. 43. The method of claim 41 or 42, wherein the label comprises an oligonucleotide.

44. 44. The method of claim 43, wherein the oligonucleotide comprises a barcode.

45. The method of any one of claims 34 to 44, wherein the cross-linking binds nucleic acids to nucleic acid binding proteins in the sample.

46. 46. ​​The method of any one of claims 24 to 45, wherein the plurality of SNPs and / or indels are identified by aligning the sequencing reads to the variation graph.

47. 47. The method of any one of claims 24 to 46, wherein the phased HLA types comprise HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and / or HLA-DPB1.

48. 48. The method of any one of claims 24 to 47, wherein the sample comprises nucleic acid enriched for HLA sequences.

49. 49. The method of any one of claims 24 to 48, wherein the entire HLA region is phased with more than 90% of the phased SNPs in a single phase block.

50. The method according to any one of claims 24 to 49, wherein major and minor groups of HLA types are phased.

51. The method of any one of claims 24 to 50, wherein the method is completed in less than 30 hours of CPU time.

52. 52. The method of claim 51, wherein the method is completed in less than 15 hours, less than 10 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour of CPU time.

53. 53. The method of claim 51 or 52, which is completed in about 30 to about 40 minutes of CPU time.

54. 54. The method of any one of claims 1 to 53, wherein the sequencing reads comprise paired-end reads.

55. The method of any one of claims 1 to 54, wherein the sequencing reads comprise long reads.

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