Patent Application
20220205017
The technology relates in part to sequencing nucleic acids.
Next-generation sequencing (NGS) has emerged as the predominant set of methods for determining nucleic acid sequence for a plethora of research and clinical applications. The typical NGS workflow is as follows: the native genomic DNA, often organized as chromosome(s), is isolated from the nucleic acid source leading to its fragmentation, to produce nucleic acid templates which are subsequently read by a sequencing instrument to generate sequence data.
The technology pertains to methods for preparing DNA molecules in such a way that preserves spatial-proximal contiguity information and provides full genome coverage equivalent to the coverage of whole genome sequencing.
Provided in certain aspects is a method for preparing DNA molecules from a sample comprising: (a) contacting cross-linked DNA molecules of a sample comprising a genome or portion thereof with a set of restriction endonucleases; thereby generating spatial-proximal digested ends of cross-linked DNA molecules; (b) contacting the spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating cross-linked proximity-ligated DNA molecules comprising ligation junctions; (c) contacting the cross-linked proximity-ligated DNA molecules comprising ligation junctions with a reagent that reverses cross-linking, thereby generating proximity-ligated DNA molecules comprising ligation junctions; and (d) fragmenting the proximity-ligated DNA molecules to generate fragments of proximity-ligated DNA molecules comprising fragments spanning the ligation junctions, wherein fragments spanning the ligation junctions and of lengths that can be templates for short range sequencing, comprise sequences of essentially the whole genome or portion thereof.
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising (a) contacting cross-linked DNA molecules of a sample comprising a genome or portion thereof with a first restriction endonuclease, thereby generating first spatial-proximal digested ends of cross-linked DNA molecules; (b) contacting the first spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating first cross-linked proximity-ligated DNA molecules comprising first ligation junctions; (c) contacting the first cross-linked proximity-ligated DNA molecules comprising first ligation junctions with a second restriction endonuclease, thereby generating second spatial-proximal digested ends of cross-linked DNA molecules; (d) contacting the second spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating second cross-linked proximity-ligated DNA molecules comprising first and second ligation junctions; (d) contacting the second spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating second cross-linked proximity-ligated DNA molecules comprising first and second ligation junctions; (e) contacting the second cross-linked proximity-ligated DNA molecules comprising first and second ligation junctions with a third restriction endonuclease, thereby generating third spatial-proximal digested ends of cross-linked DNA molecules; (f) contacting the third spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating third cross-linked proximity-ligated DNA molecules comprising first, second and third ligation junctions; (g) contacting the third cross-linked proximity-ligated DNA molecules comprising first, second and third ligation junctions with a fourth restriction endonuclease, thereby generating fourth spatial-proximal digested ends of cross-linked DNA molecules; (h) contacting the fourth spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating fourth cross-linked proximity-ligated DNA molecules comprising first, second, third and fourth ligation junctions; (i) contacting the fourth cross-linked proximity-ligated DNA molecules comprising first, second, third and fourth ligation junctions with a reagent that reverses cross-linking, thereby generating proximity-ligated DNA molecules comprising first, second, third and fourth ligation junctions; and (j) fragmenting the proximity-ligated DNA molecules to generate fragments of proximity-ligated DNA molecules comprising fragments spanning the first, second, third and fourth ligation junctions, wherein fragments spanning the first, second, third and fourth ligation junctions and of lengths that can be templates for short range sequencing, comprise sequences of essentially the whole genome or portion thereof
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising: (a) contacting cross-linked DNA molecules of a sample comprising a genome or portion thereof with a set of four restriction endonucleases; thereby generating spatial-proximal digested ends of cross-linked DNA molecules; (b) contacting the spatial-proximal digested ends of cross-linked DNA molecules with one or more reagents that incorporate biotin-attached to a nucleotide into the spatially-proximal digested ends, thereby generating cross-linked DNA molecules comprising labelled spatially-proximal digested ends; (c) contacting the cross-linked DNA molecules comprising labelled spatially-proximal digested ends with ligase, thereby generating cross-linked proximity-ligated DNA molecules comprising labelled ligation junctions; (d) contacting cross-linked proximity-ligated DNA molecules comprising labelled ligation junctions with a reagent that reverses cross-linking, thereby generating proximity-ligated DNA molecules comprising labelled ligation junctions; (e) fragmenting the proximity-ligated DNA molecules comprising labelled ligation junctions to generate fragments of proximity-ligated DNA molecules comprising fragments spanning the labelled ligation junctions, wherein fragments spanning the ligation junctions and of lengths that can be templates for short range sequencing, comprise sequences of essentially the whole genome or portion thereof; and (f) enriching for DNA fragments spanning the labelled ligation junctions by affinity purification of labelled ligation junctions using an affinity purification molecule comprising streptavidin.
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising (a) contacting spatially-proximal DNA molecules with stable spatial interactions from a sample, with two or more restriction endonucleases, thereby digesting the DNA molecules and generating spatial-proximal digested ends of DNA molecules; and (b) contacting the spatial-proximal digested ends of DNA molecules with ligase, thereby generating proximity-ligated DNA molecules comprising ligation junctions, wherein the ligation junctions are unmarked.
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising (a) contacting spatially-proximal DNA molecules with stable spatial interactions that are within cells/nuclei from a sample, with two or more restriction endonucleases, thereby digesting the DNA molecules and generating spatial-proximal digested ends of DNA molecules; and (b) contacting the spatial-proximal digested ends of DNA molecules with ligase, thereby generating proximity-ligated DNA molecules comprising ligation junctions, wherein the ligation junctions are unmarked and the contacting steps are in situ.
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising (a) contacting spatially-proximal DNA molecules with stable spatial interactions from a sample, with a first restriction endonucleases, thereby digesting the DNA molecules and generating first spatial-proximal digested ends of DNA molecules; (b) contacting the first spatial-proximal digested ends of DNA molecules with ligase, thereby generating first proximity-ligated DNA molecules comprising first ligation junctions, wherein the ligation junctions are unmarked; (c) contacting the first proximity-ligated DNA molecules comprising first ligation junctions with a second restriction endonuclease, thereby digesting the first proximity-ligated DNA molecules and generating second spatial-proximal digested ends of DNA molecules and (d) contacting the second spatial-proximal digested ends of DNA molecules with ligase, thereby generating second proximity-ligated DNA molecules comprising first and second ligation junctions, wherein the ligation junctions are unmarked.
Also provided in certain aspects is a method wherein (e) the second proximity-ligated DNA molecules comprising first and second ligation junctions are contacted with a third restriction endonuclease, thereby digesting the second proximity-ligated DNA molecules and generating third spatial-proximal digested ends of DNA molecules and (f) contacting the third spatial-proximal digested ends of DNA molecules with ligase, thereby generating third proximity-ligated DNA molecules comprising first, second and third ligation junctions, wherein the ligation junctions are unmarked.
Also provided in certain aspects is a method for preparing DNA molecules from a sample comprising (a) contacting spatially-proximal DNA molecules with stable spatial interactions that are within cells/nuclei from a sample, with a first restriction endonucleases, thereby digesting the DNA molecules and generating first spatial-proximal digested ends of DNA molecules; (b) contacting the first spatial-proximal digested ends of DNA molecules with ligase, thereby generating first proximity-ligated DNA molecules comprising first ligation junctions, wherein the ligation junctions are unmarked and the contacting steps are in situ; (c) contacting the first proximity-ligated DNA molecules comprising first ligation junctions with a second restriction endonuclease, thereby digesting the first proximity-ligated DNA molecules and generating second spatial-proximal digested ends of DNA molecules and (d) contacting the second spatial-proximal digested ends of DNA molecules with ligase, thereby generating second proximity-ligated DNA molecules comprising first and second ligation junctions, wherein the ligation junctions are unmarked and the contacting steps are in situ.
Also provided in certain aspects is a method wherein (e) the second proximity-ligated DNA molecules comprising first and second ligation junctions are contacted with a third restriction endonuclease, thereby digesting the second proximity-ligated DNA molecules and generating third spatial-proximal digested ends of DNA molecules and (f) contacting the third spatial-proximal digested ends of DNA molecules with ligase, thereby generating third proximity-ligated DNA molecules comprising first, second and third ligation junctions, wherein the ligation junctions are unmarked and the contacting steps are in situ.
Also provided in certain aspects are methods utilizing the above-described optimized 3C protocols with applications that benefit from increased coverage uniformity of read-pairs containing ligation junctions such as clustering, ordering, and orienting contigs in a genome, metagenome assemblies and haplotype phasing.
Also provided in certain aspects are methods utilizing the above-described optimized 3C protocols with applications that depend on 1D genome coverage uniformity such as SNV discovery, breakpoint detection, base polishing genome assemblies, and 1D “peak calling”, such as in ChIP-seq.
Also provided in certain aspects are methods utilizing the above-described optimized 3C protocols with applications that benefit from increased ligation events that preserve spatial-proximal contiguity information such as detection of pairwise 3D genome interactions and 3D conformation analysis.
Also provided in certain aspects are libraries prepared utilizing the methods described herein.
Also provided in certain aspects are kits comprising reagents for performing the methods described herein.
Also provided in certain aspects are methods of obtaining spatial positioning of sequence information obtained from a proximity-ligated tissue section 3C or HiC).
Certain embodiments are described further in the following description, examples, claims and drawings.
The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.
Provided herein are methods and compositions for preparing sequencing templates that provide uniform genome coverage and preserve spatial-proximal contiguity information.
Proximity Ligation
PL methods (see
In 3C, the plurality of LPs are fragmented, prepared as short nucleic acid templates and ready for sequencing. In 3C, the nucleic acid template comprises nucleic acids that are proximal to RE cut sites, and distal to RE cut sites. (Dekker et al. Science 295, 1306-1311 (2002))
In HiC, the digested nucleic acid ends are marked (e.g. biotinylated) and then ligated to create marked ligated products (MLPs, MLPs are a manifestation of LPs), bearing an affinity purification marker at the ligation junctions (LJs). After the plurality of MLPs are fragmented, affinity purification is used to enrich for fragments of MLPs comprising Us and such fragments are prepared as nucleic acid templates and are ready for sequencing—i.e. the fragmented nucleic acids from the MLPs that contain at least an LJ are enriched and prepared as a template and sequenced in HiC, to deplete uMLPs (unligated MLPs that do not usually manifest LJs). Because of this enrichment for Us, the nucleic acid template only comprises nucleic acids that are proximal to RE cut sites. (see Lieberman-Aiden et al. US2017/0362649, Lieberman-Aiden et al. Science 326, 289-293 (2009), Dekker et al. (U.S. Pat. No. 9,434,985)).
In some embodiments, of a proximity ligation method, often includes steps: (1) digestion of chromatin of the solubilized and decompacted sample with a restriction enzyme (or fragmentation); (2) blunting the digested or fragmented ends or omission of the blunting procedure; and (3) ligating the spatially-proximal ends, thus preserving spatial-proximal contiguity information. Once spatial-proximal contiguity information is preserved, further steps can include: using size selection to purify and enrich ligated fragments, which represent ligation junction fragments, preparing a library from the enriched fragments and sequencing the library.
In some embodiments, the proximity-ligated nucleic acid molecules are generated in situ. As used herein the term “in situ” refers to within a nucleus (see U.S. Application US2017/0362649).
In some embodiments, proximity-ligated DNA molecules are analyzed in a chromatin conformation assays other than 3C or HiC. In some embodiments, the chromatin conformation assay is Capture-C(Hughes et al. Nature genetics, 46(2), p. 205 (2014) 4C (Simonis et al. Nature Genetics 38, 1348-1354 (2006), De Laat et al. (U.S. Pat. No. 8,642,295)), 5C (Dostie et al. Genome Research 16, 1299-1309 (2006), Dekker et al. (U.S. Pat. No. 9,273,309)), Capture-HiC (Jäger et al. Nature communications, 6, p. 6178 (2015)), HiChIP (Mumbach et al. Nature methods, 13(11), pp. 919-922 (2016)), PLAC-seq (Fang et al. Cell research, 26(12), pp. 1345-1348 (2016)), tethered chromosome capture (TCC) (Kalhor et al. Nature Biotechnology 30, 90-98 (2012), Chen et al. (US20110287947)), HiCulfite (Stamenova et al. bioRxiv, p. 481283 (2018)) Methyl-HiC (Li et al. Nature methods, 16(10), pp. 991-993 (2019)), HiChIRP (Mumbach et al. Nature methods, 16(6), pp. 489-492 (2019)) or combinations thereof.
Regardless of the specific PL method, all PL methods capture spatial-proximal contiguity information in the form of ligation products, whereby a ligation junction is formed between two natively spatially-proximal nucleic acids. Once the LPs are formed, the spatial-proximal contiguity information is detected using next generation sequencing, whereby one or more ligation junctions (either from an entire LP or fragment of an LP) are sequenced (as described herein). With these sequence information, one is informed that the nucleic acid molecules from a given ligation product (or ligation junction) are natively spatially-proximal nucleic acids.
In certain embodiments, wherein the assay is genome-wide (i.e., is directed to the whole genome). In some embodiments, the assay is 3C, HiC, tethered chromosome capture (TCC), HiCulfite, Methy-HiC or combinations thereof.
In certain embodiments, the assay is directed to one or more target regions in the genome. In some embodiments, the assay is Capture-C, 4C, 5C, Capture-HiC, HiChIP, PLAC-seq, HiChIRP or combinations thereof. In some embodiments the targets are single nucleotide variations, insertions, deletions, copy number variations, genomic rearrangements or targets for phasing. In some embodiments, the sample comprises a cancer genome and the target region is associated with a phenotype of the cancer. In some embodiments the target associated with the cancer is a structural variation such as a genomic rearrangement or a copy number variation. In certain embodiments the target is an oncogene or a panel of oncogenes.
Ultra-High Cut Site Density
Restriction Endonucleases
In some embodiments, restriction endonucleases used in the described methods each have a theoretical digestion frequency of about 1 in 256 and when four are combined have a theoretical digestion frequency of about 1 in 64. However, there is a discrepancy between the theoretical digestion frequency, the predicted in silico frequency and the observed fragment size after chromatin digestion. Theoretical digestion frequency and in silico frequency are poor predictors of how a given restriction endonuclease will digest chromatin and particularly cross-linked chromatin.
In some embodiments, cross-linked DNA molecules of a sample are contacted with a set of restriction endonucleases so that each restriction endonuclease functions to digest the cross-linked DNA molecules during approximately the same period of time. In some embodiments, restriction endonucleases of a set each have a high activity level (i.e., approximately 100% of optimum cutting efficiency) in a common buffer. An examples of a common buffer is CutSmart™ (New England Biolabs, Beverly, Mass.).
In some embodiments restriction endonucleases can result in DNA molecules with 5′ overhangs, 3′ overhangs or no overhang (i.e., blunt ends).
In some embodiments, a set of restriction endonuclease can be at least three restriction endonucleases. In certain embodiments, a set of restriction endonucleases consists of four restriction endonucleases. In some embodiments, a sample comprises a genome other than a bacterial genome and a set of restriction endonucleases are selected to digest that genome. In certain embodiments, the four restriction endonucleases are: MboI, HinfI, MseI and DdeI. In some embodiments, a sample comprises one or more bacterial genomes, as in a metagenomics sample, and a set of restriction endonucleases are selected to digest the one or more bacterial genomes. In certain embodiments, the four restriction endonucleases are: HpyCH4IV, HinfI, HinP1I and MseI.
In some embodiments, the restriction endonucleases can be added to a sample sequentially and do not digest the cross-linked DNA molecules in the sample at the same time. In some embodiments, the restriction endonucleases generate DNA molecules with the same type of ends. In some embodiments, two or more of the restriction endonucleases generate DNA molecules with different types of ends (e.g., 5′ overhang, 3′ overhang, no overhang or blunt). In some embodiments, one or more of the restriction endonucleases require a specific buffer for high activity level that is different from a buffer required for high activity level of another of the restriction endonucleases. As the restriction endonucleases are individually contacting the cross-linked DNA molecules in the sample, each restriction endonuclease can be provided with its own unique buffer, if required. In certain embodiments, restriction endonucleases that are sequentially added to a sample can generate digested ends that can incorporate a different labelled nucleotide from a labelled nucleotide incorporated in a digested end generated by a different restriction endonuclease. This is in distinction with the use of restriction endonucleases that simultaneously digest the DNA molecules of a sample, which are limited to incorporating a common labelled nucleotide in the various digested ends.
Sequencing
Nucleic acid template (or “template” for short) refers to the nucleic acid molecule(s) that are read by a sequencing instrument. The process of generating nucleic acid templates often involves nucleic acid fragmentation to a molecular length recommended for a specific sequencing instrument. For example, current Illumina short-read sequencing can accommodate nucleic acid lengths (sequence template molecules) up to approximately 750 bp. Although smaller sequence template molecules can be utilized, as increasing the sequence coverage further away form cut sites should maximize genome coverage, templates molecules up to approximately 750 bp are often used. Templates comprise fragments that span ligation junctions and sequence information on both sides of a ligation junction can be obtained. However, as DNA shearing or fragmentation is random, the ligation junction can occur at any point along the template molecule. In some cases it may be very much towards the end of the molecule, such that there are only ˜20 bp on one side of the junction, and hundreds of bp on the other side of the junction. The junction can also occur in the middle of the template, such that there are a couple/few hundred base pairs on each side of the ligation junction.
Reads lengths can be can any length including but not limited to 2×150 bp, 2×100 bp, 2×75 bp or 2×50 bp.
In some embodiments, in order to maximize the quantity of sequence information obtained that spans a ligation junction the fragmented proximity-ligated molecules are enriched for fragmented proximity-ligated DNA molecules comprising ligation junctions and the fragmented proximity-ligated DNA molecules comprising ligation junctions are used to prepare a library of template molecules for DNA sequencing. In certain embodiments, the ligation junctions are marked with an affinity purification marker. In some embodiments, the affinity purification marker is biotin conjugated to a nucleotide. In some embodiments, spatial-proximal digested ends having a 5′ overhang are filled in by a polymerase such as Klenow Large Fragment using a single labeled-nucleotide (biotin labeled nucleotide) and other unlabeled nucleotides. In some embodiments, spatial-proximal digested ends having a 3′ overhang can be end labelled using an enzyme such as T4 DNA polymerase and all four nucleotides that are biotin labeled. In certain embodiments, enrichment is by affinity purification of the affinity purification marker with an affinity purification molecule. In some embodiments, affinity purification of the affinity purification marker with an affinity purification molecule is used in HiC, Capture-HiC, HiChIP, PLAC-seq, HiCulfite or Methyl-HiC. In some embodiments, the affinity purification molecule is streptavidin. In certain embodiments, the streptavidin comprises streptavidin coated on a magnetic bead.
In certain embodiments, enrichment for fragmented proximity-ligated DNA molecules comprising ligation junctions does not utilize a label incorporated into the ligation junction. In some embodiments, ends of molecules having 5′ or 3′ overhangs could be blunted without labeling and enriched by size selection. After the ligation step any DNA molecule that represents a proximity-ligated molecule with a ligation junction will be larger than a fragment that is unligated but digested. In some embodiments, the enriched by size selection proximity-ligated DNA molecules comprising ligation junctions are used in 3C-seq, 4C-seq 5C or Capture-C.
In some embodiments, the library of template molecules provides uniform genome-wide coverage of a genome or portion thereof. In some embodiments, the library of template molecules is sequenced to generate sequence reads comprising sequence information. In certain embodiments, the sequencing is short read sequencing.
In some embodiments, the sequence information is used in analysis of a genome. In some embodiments, the sequence information is used in analysis of a portion of a genome, for example in a targeted assay. In both analysis of a genome and analysis of a portion of a genome the uniformity and extent of coverage is the same
In some embodiments, the sequence information is utilized in genomic rearrangement analysis, identification of a breakpoint, clustering and ordering of contigs, determining contig orientation, clustering, ordering and orienting contigs, detection of pairwise 3D genome interactions (such as 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences), protein factor location analysis and 3D conformation, protein factor location analysis and 3D conformation analysis comprising PLAC-seq or HiChIP, haplotype phasing, genome assembly and 3D conformation analysis, DNA methylation analysis, DNA methylation analysis and detection of 3D genome interactions, single nucleotide variant (SNV) discovery, base polishing of long-range sequencing information, highly sensitive copy number variation (CNV) analysis (e.g., the copy number variation (CNV) is an amplification, the copy number variation (CNV) is a heterozygous or homozygous deletion), variant discovery, haplotype phasing and genome assembly, haplotype phasing and genome assembly, genome assembly and detection of 3D genome interaction or combinations thereof.
In certain embodiments, the sequence information is utilized for variant discovery and haplotype phasing in a first sample comprising a paternal genome and a second sample comprising a maternal genome and the phased variants of the paternal genome and the maternal genome are used to analyze sequence data of a fetal genome obtained from cfDNA of the mother.
Full genome coverage and spatial-proximal contiguity information obtained by the methods described herein can be used in other methods or combinations of methods that utilize such sequence information.
Samples
In some embodiments, the DNA is obtained from a sample selected from nuclei, cells, tissues, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples or cell-free DNA. In certain embodiments, the DNA is obtained from a single cell. In certain embodiments, the DNA is obtained from two or more cells. In some embodiments, a sample can comprise two or more genomes representing different species, such as in a metagenomics sample.
Genomic Rearrangement Breakpoint Detection
Contig Clustering and Ordering
Contig Orientation
3D Genome Conformation
Maximizing genome coverage in ultra-high RE cut site density (HiCoverage) to uniquely enable the highest resolution and most sensitive detection of pairwise 3D genome interactions also applies to other forms of HiC and its derivatives, particularly Capture-HiC, HiChIP, TCC, and other restriction enzyme-based genome-wide or targeted HiC-based assays.
Protein Factor Localization
Variant Discovery and Haplotype Phasing
Methylation Analysis
Maximizing genome coverage in the HiCoverage method uniquely enables highly sensitive analysis of DNA methylation, and is comparable to traditional whole genome bisulfite sequencing (WGBS). Only cytosines that are proximal to a RE cut site would be present in a nucleic acid template and available for bisulfite conversion and determination of methylation status. Cytosines distal from a RE cut site would be unknown because those nucleic acids would not be present in the nucleic acid template. HiCoverage uniformity via ultra-high RE cut site density enables the methylation status of all cytosine to be detected due to their proximal positioning relative to RE cut sites. Other types of DNA methylation, e.g. hydroxymethylated cytosines, can also be sensitively detected using HiCoverage by virtue of the genome coverage (apply bisulfite conversion to one set of templates and apply TAB-seq to another set of templates and using the two datasets determine mC and hmC status).
In some embodiments the nucleic acids with preserved spatial-proximal contiguity information generated by the methods described herein are contacted with a bisulfite reagent prior to PCR and sequencing to enable the concurrent analysis of spatial proximity and DNA methylation at base resolution. In some embodiments the bisulfite reagent is sodium bisulfite.
In some embodiments HiC ligation products are generated using a HiC protocol as previously described (Rao et al. Cell, 159(7), pp. 1665-1680 (2014), Li et al. Nature methods, 16(10), pp. 991-993 (2018)). Ligation junctions are enriched using streptavidin beads. Illumina library construction ensues while the DNA is attached to the streptavidin bead, as previously described (Rao et al. Cell (2014)). Directly after adapter ligation, DNA is subject to bisulfite conversion, using methods known in the art. Unmethylated lambda DNA is spiked in at 0.5% prior to bisulfite conversion in order to estimate the conversion rate. The bisulfite converted DNA is purified, amplified, and sequenced.
In some embodiments sheared HiC ligation products are treated with a bisulfite reagent and purified (Stamenova et al. bioRxiv, p. 481283 (2018)). Ligation junctions are then enriched using streptavidin beads. DNA is then detached from the beads, and prepared as a sequencing library using techniques known in the art for converting ssDNA into a dsDNA sequencing library. Adapter ligated molecules are then subject to library amplification and sequencing. Similarly, methods known to the art can also be applied to analyze the DNA methylation status (Lister et al. Nature, 462(7271), pp. 315-322 (2009); Shultz et al. Nature, 523(7559), pp. 212-21 (2015)). Additionally, methods known in the art can also be applied to concurrently analyze the DNA methylation status with respect to 3D genome folding (Li et al. Nature methods, 16(10), pp. 991-993 (2018); Stamenova et al. bioRxiv (2018)), revealing DNA chemical modifications properties and DNA folding patterns in parallel. Specifically in the context of applying this method to protein:cfDNA complexes, it is well known in the art that DNA methylation status of cell free nucleic acids can inform tissue of origin analyses as well as several other cfDNA analysis, including but not limited to the non-invasive detection of tumor DNA, prenatal diagnoses, and organ transplantation monitoring (Zeng et al. Journal of Genetics and Genomics, 45(4), pp. 185-192 (2018); Lehmann-Werman et al. Proceedings of the National Academy of Sciences, 113(13), pp. E1826-E1834 (2016)).
SNV Discovery
Maximizing genome coverage (sequence coverage and uniformity) enables highly sensitive small variant sensitivity. A SNV obtains sequence coverage due to its close proximity to a RE cut site. A SNV that is distal to a RE cut site and receive no sequence coverage and therefore cannot be discovered. In the methods described herein, coverage uniformity via ultra-high RE cut site density enables essentially all SNVs to obtain sequence coverage, thus maximizing small variant sensitivity to an equivalent level as demonstrated with shotgun WGS. Standard HiC results in many SNV distal to an RE cut site, thus being undiscoverable. Many types of small variants, including heterozygous SNV (single nucleotide variations), other types of SNVs, and INDELs (insertions and deletions), can be discovered with maximum sensitivity using the described method.
Base Polishing
Maximizing genome coverage in the HiCoverage method uniquely enables highly sensitive base polishing of erroneous genomic bases, comparable to shotgun WGS, originally detected by error-prone sequencing technologies, in additional to the known genomic scaffolding capabilities of HiC. In one scenario, current de novo genome assembly workflows often involve the combination of a relatively error-prone long-read sequencing technology (e.g. Oxford Nanopore, UK) to produce the most contiguous sequences (“contigs”), followed by performing HiC to scaffold contigs into chromosome-scale scaffolds, followed by shotgun WGS (10× Genomics, Pleasanton, Calif.) to “polish” the erroneous base calls produced by the error-prone long-read technology. HiC has not been conceived as a technology capable of sensitive base polishing due to the uneven genomic representation in the nucleic acid template and thus the uneven coverage of the sequencing data. However using the HiCoverage method uniformity via ultra-high RE cut site density enables maximum base polishing sensitivity comparable to that of shotgun WGS. Oher types of erroneous DNA sequence, besides erroneous individual base calls, produced by error-prone sequencing technologies can also be sensitively polished using the HiCoverage method by virtue of the even genome coverage.
CNV Analysis
In some embodiments, Maximizing genome coverage in the HiCoverage method uniquely enables highly sensitive CNV analysis on bar with that of shotgun WGS. CNVs obtains sequence coverage due to its overlap with a RE cut site, while CNVs that are distal to a RE cut sites, receive no sequence coverage and therefore cannot be discovered or analyzed. The HiCoverage method provides coverage uniformity via ultra-high RE cut site density, thus maximizing CNV detection sensitivity. CNVs, such as amplified regions and heterozygous or homozygous deletions can be discovered and analyzed with maximum sensitivity using the described ultra-high RE cut site density method.
Data Analysis/Applications
The following represents a sampling of some data analysis and applications and is not meant to be all inclusive. Using HiC data for contiguity-preservation-enabled analysis and applications, such as haplotype phasing and genomic rearrangement detection is known to the art. For example, Selvaraj et al. BMC genomics, 16(1), p. 900 (2015), Selvaraj et al. Nature biotechnology, 31(12), p. 1111 (2013), and PCT/US2014/047243 described HiC data for haplotype phasing and Engreitz et al. (PLOS ONE September 2012/Volume 7/Issue 9/e44196) has described HiC data for genomic rearrangement analysis in human disease. Several other papers have described using HiC data for genomic rearrangement detection (Dixon et al. Nature genetics, 50(10), pp. 1388-1398 (2018); Chakraborty and Ay, Bioinformatics (2018); Harewood et al. Genome biology, 18(1), p. 125 (2017)). One such analysis tool for rearrangement detection is HiC-Breakfinder tool (https://github.com/dixonlab/hic_breakfinder) from Dixon et al. Nature genetics (2018). Other contiguity-preservation-enabled analyses and applications include but are not limited to de novo genome and metagenome assembly, structural variation detection, and others.
After sequencing, methods known to the art can be used to analyze the data in the context of spatial proximity and long-range sequence contiguity, such as but not limited to using the spatial-proximal contiguity information to inform genome folding patterns (Lieberman-Aiden et al. Science, 326(5950), pp. 289-293 (2009)), and genomic rearrangement analysis (Dixon et al, Nature genetics, (2018)).
Also, because it is known that HiC signal uniquely captures long-range sequence contiguity information to significantly enhance genomic rearrangement analyses (Dixon et al. Nature genetics (2018)), HiC applied to cfDNA could enrich for such genomic rearrangement signal from liquid biopsy samples and greatly benefit early non-invasive cancer diagnoses. And finally, the combination and concurrent analysis of both DNA methylation and DNA spatial proximity and long-range contiguity will synergize to better enable the analyses described herein.
3C Methods
In some embodiments, proximity ligation products are generated using optimized 3C-based methods, rather than a HiC method. 3C-based methods, include but are not limited to, 3C, 4C, 5C, Capture-C, 3C-ChIP or Methyl-3C.
In some embodiments, the 3C methods do not incorporate a label or marker in the ligation junction, as in HiC. For example, a biotinylated nucleotide or biotinylated bridge adaptor.
A sample is typically crosslinked to preserve spatial-proximal information, however crosslinking of a sample may not always be required (Bryant et al. Mol Syst Biol. 12(12): 891(2016)). In some embodiments, the 3C methods described herein are used with samples of tissues, cells, nuclei, that are not crosslinked, but which have spatially-proximal DNA molecules with stable spatial interactions. Embodiments of 3C methods described herein as applicable to crosslinked samples are also intended as applicable to samples that are not crosslinked.
The 3C methods described herein can be performed ex situ or in situ.
In some embodiments, 3C methods are optimized to improve amount of spatial-proximal contiguity information that is preserved. Long-range cis captured spatially-proximal nucleic acids (cSPNAs) (greater than 15 Kb in linear sequence distance) are most informative for contiguity applications and are often used as a proxy for determining the preservation of spatial-proximal contiguity information. Specifically what percent of nucleic acid templates for sequencing are long-range cis molecules. In certain embodiments, 3C methods are optimized to improve the percent of long-range cis molecules.
In some embodiments, the optimized 3C methods also increase genome coverage uniformity of read-pairs containing ligation junctions.
In some embodiments, optimized 3C is based on the use of multiple restriction endonucleases (optimized 3C proximity ligation) (see Examples 4 and 5 and
Restriction Endonucleases
In some embodiments, DNA molecules of a sample are contacted and digested with two or more restriction endonucleases, three or more restriction endonucleases, four or more restriction endonucleases, five or more restriction endonucleases, six or more restriction endonucleases, seven or more restriction endonucleases, eight or more restriction endonucleases, nine or more restriction endonucleases, ten or more restriction endonucleases, or greater; e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 restriction endonucleases. In certain embodiments, a set of restriction endonucleases is two restriction endonucleases. In certain embodiments, a set of restriction endonucleases is three restriction endonucleases. In certain embodiments, a set of restriction endonucleases is two restriction endonucleases and one of the restriction endonucleases is NlaIII. In some embodiments, one of the restriction endonucleases is NlaIII and the other restriction endonuclease is MboI or MseI. In certain embodiments, a set of restriction endonucleases is three restriction endonucleases and one of the restriction endonucleases is NlaIII. In some embodiments, a set of restriction endonucleases is three restriction endonucleases and one of the restriction endonucleases is NlaIII and another of the restriction endonucleases is either MboI or MseI. In some embodiments, the restriction endonucleases are NlaIII, MboI and MseI. Other restriction endonucleases and combinations of restriction endonucleases that enhance the preservation of spatial-proximal contiguity information are encompassed by the methods described herein.
In some embodiments, the restriction enzymes result in the same overhanging sequence. Examples of such enzymes include: AciI, HinP1I, HpalI, HpyCH4IV, MspI, and TaqI—all of which have 3′-CG-5′ overhangs on the 5′ end of the negative DNA strand. Similarly, BfaI, MseI, and CviQI have 3′-TA-5′ overhangs on the 5′ end of the negative DNA strand.
In some embodiments, the restriction enzymes result in different overhanging sequences.
In some embodiments, contact and digestion of DNA molecules with the two or more restriction endonucleases is performed at one time, i.e., simultaneously. In certain embodiments, the resultant spatial-proximal digested ends of the DNA molecules are then contacted with ligase to generate ligation junctions.
In certain embodiments, contact and digestion with the two or more restriction endonucleases is performed sequentially. In some embodiments, each sequential contact and digestion event can be with one or more restriction endonucleases. For example, a contact and digestion event could be a co-digestion with two restriction endonucleases. In some embodiments, the sequential contact and digestion with the two or more restriction endonucleases is performed in a defined order based on the particular restriction endonucleases used. In certain embodiments, at the conclusion of the sequential digestions (whether ordered or not) the resultant spatial-proximal digested ends of the DNA molecules are contacted with ligase to generate ligation junctions.
In certain embodiments, contact and digestion with each restriction endonuclease or combination of restriction endonucleases is performed sequentially and after the conclusion of each digestion event by one or more restriction endonucleases the resultant spatial-proximal digested ends of the DNA molecules are contacted with ligase to generate ligation junctions (see Example 8 and
In certain embodiments, optimized 3C methods encompass other combinations of restriction endonucleases, types of overhanging ends produced (the same, different or a mixture of the same and different), simultaneous or sequential digestion, order of restriction endonucleases, the number of restriction endonucleases at each sequential step and whether ligation is performed once at the conclusion of all digestions or more frequently following each sequential digestion that improve the preservation of spatial-proximal contiguity information and/or the genome coverage of molecules comprising ligation junctions.
Size Selection
In some embodiments, proximity-ligated DNA molecules produced by using two or more restriction endonucleases are enriched for molecules containing ligation junctions that preserve spatial-proximal contiguity. In certain embodiments, enrichment is by size selection. In some embodiments, size selection is for larger fragments having sizes of approximately >5 kb, >10 kb, >20 kb, >30 kb, >40 kb, >50 kb, or >60 kb. Size selection can be carried out by any means known in the art.
In some embodiments, size selection is performed directly after reversal of cross-linking (if proximity-ligated molecules are crosslinked). In certain embodiments, size selection can be by gel extraction using manual or automated methods (e.g. Sage Science BluePippin instrument (Beverly, Mass.) or, using size selective DNA precipitation based methods (e.g. Circulomics Short Read Eliminator kits (Baltimore, Md.)).
In some embodiments, size selection is carried out following fragmentation of proximity-ligated molecules. In certain embodiments, size selection employs magnetic beads coated with carboxyl groups that bind DNA nonspecifically and reversibly, e.g., solid phase reversible immobilization (SPRI) beads, such Ampure Beads (Beckman Coulter; Brea, Calif.). In certain embodiments, the ratio of beads to sample volume can be adjusted to select larger fragments. For example, the ratio can be 0.4× to 0.8× or 0.4×, 0.5×, 0.6×, 0.7×, or 0.8×.
In some embodiments, size selection is carried out during library preparation, for example before or after performing PCR. A variety of size selection means are applicable, including the use of SPRI beads. Size selection of the described methods that is performed prior to construction of a library is not directed to optimization for molecules of a certain size for use with a particular sequencing machine. Rather, size selection as utilized in the described methods is directed to the purpose of enhancing data composition by impacting the proportion of templates containing ligation junctions and preserving spatial-proximal contiguity. For example, a maximum average library insert size of 350-450 bp is recommended for a HiSeq instruments compared to the much larger recommended insert size of ˜700 bp for optimized 3C.
In some embodiments, an optimized 3C protocol can have no size selection step or can have a single size selection step, two size selection steps or three size selection steps.
In certain embodiments, the means utilized for size selection, the size range selected and the applicability of using more than one size selection step can be evaluated for their effect on improving the preservation of spatial-proximal contiguity information by examining the percent of template molecules that represent long-range cis molecules, for example.
Optimization of a 3C method to improve the preservation of spatial-proximal contiguity information can be by utilizing multiple restriction endonucleases or multiple restriction endonucleases and size selection. Any of the described variations of multiple restriction endonuclease digestion can be utilized alone or in combination with any of the described variations of size selection. For example, a very rigorous size-selection following fragmentation of proximity-ligated molecules using a ratio of 0.4×SPRI beads to sample volume could be combined with sequential rounds of co-digestion and ligation.
In some embodiments, optimized 3C methods as described herein result in proximity-ligated DNA molecules that are derived from sequences covering essentially an entire genome.
In some embodiments, DNA molecules are obtained from any sample type where the nuclear architecture can remain intact. In some embodiments, DNA molecules are obtained from a sample selected from nuclei, cells, tissues, cell lines, primary cells, dissociated tissues, ground tissues, formalin-fixed paraffin-embedded (FFPE) samples, FFPE tissue sections or frozen tissue sections, deeply formalin-fixed samples or cell-free DNA. In certain embodiments, the sample is in an aqueous solution. In certain embodiments, the sample is affixed to a solid surface such as a slide. In certain embodiments, the sample is in an aqueous solution. In some embodiments, FFPE tissue is analyzed on a slide. In some embodiments, FFPE tissue removed from a slide (e.g., scrapped off physically, or by using laser capture microdissection) is analyzed. In some embodiments, frozen tissue is analyzed on a slide. In some embodiments, frozen tissue removed from a slide (e.g., scrapped off) is analyzed.
In some embodiments, the DNA molecules are obtained from a single cell, are obtained from two or more cells or are obtained from a tissue sample or a specific portion of a tissue sample. In some embodiments, the DNA molecules of a sample comprise two or more genomes or portions thereof.
In some embodiments, prior to preparation of a library for sequencing the proximity-ligated DNA molecules comprising ligation junctions are purified. In certain embodiments, if a sample was crosslinked, proximity-ligated DNA molecules comprising ligation junctions are contacted with a reagent that reverses crosslinking.
In some embodiments, a library of template molecules for DNA sequencing is prepared from proximity-ligated DNA molecules produced by the optimized 3C methods described herein.
In certain embodiments, the optimized 3C method include one or more steps specific to a 4C, 5C, Capture-C, 3C-ChIP (3C proximity ligation followed by ChIP-seq) or Methyl-3C method.
In some embodiments, a library of template molecules for DNA sequencing is prepared from the product of an optimized 3C method that include one or more steps 4C, 5C, Capture-C, 3C-ChIP or Methyl-3C method.
In some embodiments, a library of template molecules is sequenced to generate sequence reads comprising sequence information reflecting the use of 3C (3C-seq). In some embodiments, a library of template molecules is sequenced to generate sequence reads comprising sequence information that reflects the use of a 4C, 5C, Capture-C, 3C-ChIP or Methyl-3C method.
In certain embodiments, the sequencing is short-read sequencing. In certain embodiments, the optimized 3C method described herein result in at least 30%, at least 40%, at least 50% or at least 60% of the nucleic acid templates that are used to prepare a library for short-read sequencing being long-range cis molecules.
In some embodiments, prior to the preparation of a library that is used for short-read sequencing the proximity-ligated DNA molecules are fragmented to generate fragments of proximity-ligated DNA molecules comprising fragments spanning the ligation junctions.
In certain embodiments, the sequencing is long-read sequencing.
In some embodiments, a library of template molecules prepared by utilizing an optimized 3C protocol and one or more steps specific to a 4C, 5C, Capture-C, 3C-ChIP or Methyl-3C method, as described herein, is sequenced to generate sequence reads comprising sequence information. In certain embodiments, the sequencing is short-read sequencing. In certain embodiments, the sequencing is long-read sequencing.
Library preparation, sequencing and analysis of sequence information are as previously described herein.
In some embodiments, sequence information is utilized in applications that analyze spatial-proximal contiguity. In certain embodiments, sequence information is utilized for detection of pairwise 3D genome interactions of a genome or portion thereof. In certain embodiments, the 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences. In certain embodiments, sequence information is utilized for protein factor location analysis and 3D conformation analysis of a genome or portion thereof. In certain embodiments, protein factor location analysis and 3D conformation analysis comprises 3C-ChIP.
In some embodiments, optimized 3C methods are utilized in applications that benefit from increased coverage uniformity of read-pairs containing ligation junctions. In certain embodiments, sequence information is utilized for clustering and ordering of contigs of a genome or portion thereof. In certain embodiments, sequence information includes sequence information for each contig that is clustered and ordered. In certain embodiments, sequence information is utilized for clustering, ordering and orientating contigs of a genome or portion thereof. In some embodiments, sequence information is utilized for haplotype phasing of the genome or portion thereof. In some embodiments, sequence information is utilized for metagenome assemblies.
In some embodiments, sequence information is utilized in applications that depend on 1D genome coverage. In certain embodiments, sequence information is utilized for genomic rearrangement analysis of the genome or portion thereof. In certain embodiments, genomic rearrangement analysis comprises identification of a breakpoint. In certain embodiments, sequence information of a given sequence read is located upstream and downstream of the breakpoint. In certain embodiments, sequence information is utilized for DNA methylation analysis of a genome or portion thereof. In certain embodiments, sequence information is utilized for single nucleotide variant (SNV) discovery of a genome or portion thereof. In certain embodiments, sequence information is utilized for base polishing of long-range sequencing information of a genome or portion thereof. In certain embodiments, sequence information is utilized for highly sensitive copy number variation (CNV) analysis of a genome or portion thereof. In certain embodiments, a copy number variation (CNV) is an amplification. In certain embodiments, a copy number variation (CNV) is a heterozygous or homozygous deletion.
In certain embodiments, sequence information is utilized for variant discovery, haplotype phasing and genome assembly of a genome or portion thereof. In certain embodiments, sequence information is utilized for variant discovery and haplotype phasing in a first sample comprising a paternal genome and a second sample comprising a maternal genome and the phased variants of the paternal genome and the maternal genome are used to analyze sequence data of a fetal genome obtained from cfDNA of the mother. In certain embodiments, sequence information is utilized for haplotype phasing and genome assembly of a genome or portion thereof. In certain embodiments, sequence information is utilized for genome assembly and 3D conformation analysis of a genome or portion thereof. In certain embodiments, sequence information is utilized for DNA methylation analysis and detection of 3D genome interactions of a genome or portion thereof. In certain embodiments, sequence information is utilized for genome assembly and detection of 3D genome interaction of a genome or portion thereof.
In some embodiments, molecular contiguity information of proximity-ligated DNA molecules is preserved in addition to the spatial-proximal contiguity information preserved in ligation junctions. In certain embodiments, barcodes are used to preserve molecular contiguity information. In certain embodiments, barcodes are introduced into the proximity-ligated DNA molecules by contacting proximally-ligated DNA with a barcoded transposome linked bead prior to library preparation. In certain embodiments, the sequence information is utilized for detection of higher-order 3D genome interactions of a genome or portion thereof, by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules. In certain embodiments, the sequence information is utilized for detection of three or more concurrent 3D genome interactions of the genome or portion thereof, by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules. In certain embodiments, the sequence information is utilized for detection of virtual pairwise 3D genome interactions by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules. In certain embodiments, a virtual pairwise 3D genome interaction is between restriction fragments that are not directly ligated to one another within a given proximity-ligated DNA molecule of the genome or portion thereof.
In certain embodiments, the pairwise interactions, virtual pairwise interactions, and/or higher order interactions obtained by leveraging the preserved molecular contiguity of proximity ligated DNA molecules is utilized for 3D genome interactions of the genome or portion thereof, genomic rearrangement analysis of the genome or portion thereof, clustering and ordering of contigs of the genome or portion thereof, determining contig orientation of the genome or portion thereof, haplotype phasing of the genome or portion thereof, DNA methylation analysis of the genome or portion thereof, single nucleotide variant (SNV) discovery of the genome or portion thereof, base polishing of long-range sequencing information of the genome or portion thereof, highly sensitive copy number variation (CNV) analysis of the genome or portion thereof or combinations thereof.
Single-Cells
In some embodiments, an optimized 3C protocol is to obtain sequence information from a single cell which provides a single cell profile.
Single-Cell 3C Via Cell/Nuclei Sorting (Either Before or after 3C) (“Plate” Method)
In some embodiments, in situ 3C proximity ligation is carried out “in bulk” (i.e. in a population of cells). Cells/nuclei are sorted using a cell sorting instrument (e.g. FACS and FANS), or manually, into discrete physical compartments such as wells of a microtiter plate. DNA is purified and amplified from each single cell using methods of whole genome amplification known in the art, such as multiple displacement amplification (MDA), or other means. Such an approach is analogous to Flyamer et al. Nature, 544(7648), pp. 110-114 (2017) or Tan et al. Science, 361(6405), pp. 924-928 (2018). Libraries are produced from amplified DNA molecules of each cell/nucleus. Libraries are sequenced and sequence reads are examined to obtain sequence information at single cell resolution.
In some embodiments, more pairwise interactions per cell may be captured by preserving the molecular contiguity of each proximally-ligated DNA molecule from each single cell. In certain embodiments, barcoded transposome linked beads (e.g. TELL-seq beads, Universal Sequencing Technologies, Carlsbad, Calif.) are applied to the purified proximally-ligated DNA in each microwell. Once the transposome-linked beads are applied, libraries are constructed for each individual cell. Reconstruction of the proximally-ligated DNA molecules from each single cell has the potential to dramatically improve the number of pairwise contacts per cell using the concept of “virtual pairs”, which means that 10 restriction fragment ligated together in a ligation product would conventionally be derived from ˜9 ligation junctions and produce 9 pairwise 3D contacts. If the entire 10 fragments on a given ligation product were revealed, this would inform 45 total combinations of pairwise 3D contacts ((10*9)/2), or the equation P=(((n*(n−1))/2), where P is the total number of pairwise 3D contacts obtained per ligation product, and n is the number of restriction fragment concatemerized into the ligation product. If 25 restriction fragments were in a ligation product, this would produce ˜24 pairwise contacts with traditional library prep, or 300 “virtual pairs” if the molecular contiguity of each 3C ligation product was preserved during library prep. This would represent a log-order increase in information content per cell.
Single-Cell 3C Via Droplet Microfluidics Approaches (“Droplet” Method)
In some embodiments, in situ 3C proximity ligation is carried out “in bulk” (i.e. in a population of cells). Cells/nuclei are input into a commercial (e.g. 10× Genomics (Pleasanton, Calif.), Bio-Rad, (Hercules, Calif.), Mission Bio (South San Francisco, Calif.) or homebrew (e.g. Drop-Seq) droplet microfluidics system where reagents are delivered to barcode and amplify proximally-ligated DNA from each single cell/nucleus. Libraries are produced from amplified DNA molecules of each cell/nucleus. Libraries are sequenced and sequence reads are examined to obtain sequence information at single cell resolution.
In some embodiments, 4C is utilized for library preparation (single-cell 4C). For 4C in the plate and droplet single cell methods, targeted amplification with a locus specific primer pair (which is what is done in 4C) comprising cell barcodes rather than whole genome amplification is carried out.
In some embodiments, Capture C is used to enrich for specific targets (templates are enriched by target enrichment and sequenced). Since the templates have the cell barcode(s) based on the protocol used to obtain single cells (see above) the sequence information can be assigned to a single cell.
Spatial Positioning (“Spatial” Method)
In some embodiments, analysis of tissue sections processed using an optimized 3C protocol (or HiC protocol) can provide spatial positioning for sequence information obtained from portions of the tissue section or from single cells. In certain embodiments, in situ 3C (or HiC) proximity ligation is carried out while the tissue is held intact on a surface such as a slide, and then the tissue (now comprised of proximally-ligated nuclei) is micro-dissected into spatially distinct regions. In some embodiments, a spatially distinct region is a grid (e.g. 8×12) sometimes having quadrants, concentric circles (like a bulls eye), peripheral tumor cells that contact non-tumor cells or the tumor microenvironment, cell clusters in sub-regions of a tissue, or a collection of single cells. Each spatially distinct region can be treated as its own “sample” and processed as a distinct physical collection of cells or single-cells can be obtained according to the examples above and processed individually. In certain embodiments, a tissue section is first micro-dissected into spatially distinct regions and each spatially distinct region is treated as its own in situ 3C (or HiC) proximity ligation reaction and processed as a distinct physical collection of cells or single-cells can be obtained according to the examples above and processed individually. During the data analysis phase, tissue 3C (or HiC) profiles of spatially distinct regions or single cell 3C (or HiC) profiles can be attributed to their spatial positioning within a tissue section.
In certain embodiments, each spatially distinct region may not need to be treated as its own separate in situ 3C (or HiC) reaction. In certain embodiments, methods similar to MULTI-seq (McGinnis et al. Nature methods, 16(7), p. 619 (2019)) can be adapted for sample barcoding in the context of single cell 3C (or HiC) analysis. For example, cells/nuclei can be collected from each spatially defined region from a tissue section. The samples would then be reacted with lipid-modified oligonucleotide (LMO) or cholesterol-modified oligonucleotide (CMO), which imbeds into the plasma membrane of a cell membrane or nuclear membrane. The oligonucleotide would comprise a means to be amplified after the proximally-ligated nuclei are partitioned into wells of a plate or droplets. During the data analysis phase, the single cell 3C (or HiC) profiles can be attributed to their spatial positioning within a tissue section, and the co-amplified sample barcode sequence corresponding to each single cell would serve as the sample identifier that was introducing during the sample tagging reaction.
In some embodiments, 4C is utilized in the analysis is of tissue section. Targeted amplification is carried out with a locus specific primer pair using the 3C templates that are produced from each spatially defined region that is micro-dissected from the tissue section.
Library Prep Manipulations
In some embodiments, the above-described 3C methods are combined with target enrichment methods. In certain embodiments, target enrichment is PCR based.
Post Library Prep Manipulations
In some embodiments, the above-described 3C methods are combined with target enrichment methods. In certain embodiments, target enrichment is probe based. In certain embodiments, target enrichment is PCR based.
In some embodiments, Capture C is used to enrich for specific targets (templates are enriched by target enrichment and sequenced).
Kits
In some embodiments, provided are kits for carrying out methods described herein. Kits often comprise one or more containers that contain one or more components described herein. A kit comprises one or more components in any number of separate containers, packets, tubes, vials, multiwell plates and the like, or components may be combined in various combinations in such containers. Kit components and reagents are as described herein.
HiC Kits
In some embodiments, a kit comprises one or more of (a) three or more restriction endonucleases; (b) a restriction endonuclease buffer; and (c) one or more of a biotinylated nucleotide, unlabeled nucleotides, a DNA polymerase, ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking.
In some embodiments, a kit comprises one or more of (a) four restriction endonucleases; (b) a restriction endonuclease buffer; and (c) one or more of a biotinylated nucleotide, unlabeled nucleotides, a DNA polymerase, ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking. In certain embodiments, the four restriction endonucleases are: MboI, HinfI, MseI and DdeI. In certain embodiments, the four restriction endonucleases are: HpyCH4IV, HinfI, HinP1I and MseI.
In some embodiments, a kit comprises one or more of: four restriction endonucleases; (b) two or more restriction endonuclease buffers; and (c) one or more of a biotinylated nucleotide, unlabeled nucleotides, a DNA polymerase, ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking. In some embodiments, the two or more restriction endonuclease buffers are in separate containers from the four restriction endonucleases. In some embodiments, each restriction endonuclease has a theoretical digestion frequency of at least 1 in 256. In some embodiments, at least two of the restriction endonucleases require unique buffers for high level activity.
In some embodiments, the restriction endonucleases are in separate containers. In some embodiments, the restriction endonucleases are in a single container. In some embodiments, each restriction endonuclease has a high activity level in a common restriction endonuclease buffer and each restriction endonuclease has a theoretical digestion frequency of at least 1 in 256. In some embodiments, the restriction endonuclease buffer is in a separate container from the restriction endonucleases.
3C Kits
In some embodiments, a kit comprises one or more of (a) two or more restriction endonucleases; (b) a restriction endonuclease buffer; and (c) one or more of ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking, one or more additional buffers and reagents for size selection, a bead-linked transposome, primers with barcode oligonucleotides, one or more reagents to create a sequencing library and does not include a biotinylated nucleotide or a labelled nucleotide.
In some embodiments, a kit comprises one or more of (a) two restriction endonucleases; (b) a restriction endonuclease buffer; and (c) one or more of ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking, one or more additional buffers and reagents for size selection, a bead-linked transposome, primers with barcode oligonucleotides, one or more reagents to create a sequencing library and does not include a biotinylated nucleotide or a labelled nucleotide. In certain embodiments, one of the restriction endonucleases is NlaIII. In certain embodiments, one of the restriction endonucleases is NlaIII and the other restriction endonuclease is MboI or MseI.
In some embodiments, a kit comprises one or more of (a) three restriction endonucleases; (b) one or more of restriction endonuclease buffers; and (c) one or more of ligase, ligase buffer, one or more additional buffers and reagents for reversing cross-linking, one or more additional buffers and reagents for size selection, a bead-linked transposome, primers with barcode oligonucleotides, one or more reagents to create a sequencing library and does not include a biotinylated nucleotide or a labelled nucleotide. In certain embodiments, one of the restriction endonucleases is NlaIII. In certain embodiments, one of the restriction endonucleases is NlaIII and one of the other restriction endonucleases is MboI or MseI. In certain embodiments, the restriction endonucleases are: NlaIII, MboI and MseI.
In some embodiments, the restriction endonucleases of a kit produce the same overhanging sequence. In some embodiments, the restriction endonucleases of a kit produce different overhanging sequences. In some embodiments, digestion with the two or more restriction endonucleases of a kit can be carried out at the same time. In some embodiments, digestion with two or more restriction endonucleases of a kit cannot be carried out at the same time.
In some embodiments, the restriction endonucleases of a kit are in separate containers. In some embodiments, the restriction endonucleases of a kit are in a single container. In some embodiments, the restriction endonucleases of a kit are in more than one container and at least one container contains more than one restriction endonuclease. In some embodiments, each restriction endonuclease of a kit has a high activity level in a common restriction endonuclease buffer and the buffer is in one container. In some embodiments, more than one buffer is in a kit and the buffers are in separate containers. In some embodiments, a restriction endonuclease buffer is in a separate container from a restriction endonuclease.
In certain embodiments, the kit comprises instructions. In some embodiments, the instructions recite the order that the restriction enzymes of a kit are to be used.
A kit sometimes is utilized in conjunction with a process, and can include instructions for performing one or more processes and/or a description of one or more compositions. A kit may be utilized to carry out a process described herein. Instructions and/or descriptions may be in tangible form (e.g., paper and the like) or electronic form (e.g., computer readable file on a tangle medium (e.g., compact disc) and the like) and may be included in a kit insert. A kit also may include a written description of an internet location that provides such instructions or descriptions.
Libraries
In some embodiments, libraries are constructed as described herein based on the use of HiC or optimized 3C methods.
The examples set forth below illustrate certain embodiments and do not limit the technology.
Crosslinked GM19240 cells were digested with increasing amounts of HinfI for 30 min, in replicate. After digestion, crosslinks were reversed, DNA was purified, and gel electrophoresis was performed. At least 100 U of HinfI were required for efficient chromatin digestion, evidenced by the smaller molecular weight of the digested DNA sample. Because HinfI can reach efficiency levels of crosslinked chromatin digestion with a reasonable amount of RE units (e.g., 100 units), and is compatible with the same buffer as MboI, HinfI can be used in conjunction with MboI (see
To select additional RE to further increase coverage uniformity, 4 additional 4-cutters (BfaI, DdeI, MseI, and MspI) with 100% reported activity levels in a RE buffer that is also compatible with MboI and HinfI (CutSmart™ Buffer) were identified. Crosslinked GM12878 cells were digested with a maximum practical amount of each enzyme, in replicate. After digestion, crosslinks were reversed, DNA was purified, and gel electrophoresis was performed. Surprising, despite the reasonable RE concentrations, buffer compatibility, and in silico cut site frequency (1 in 256), only 2/4 of the RE showed efficient RE digestion during HiC (see
Twenty vertebrate genome assemblies, two plant genome assemblies, two insect genome assemblies, and two parasite genome assemblies were downloaded from various sources such as GenomeArk (https://vgp.github.io/genomeark/) for vertebrates and NCBI (https://www.ncbi.nlm.nih.gov/genome/) for other genomes. Genomes were then digested in silico using either the four restriction enzymes cut site motifs for MboI, MseI, DdeI, and HinfI, or, for just the single restriction enzyme MboI to mimic a relatively low density restriction enzyme method. To estimate the expected coverage, or what fraction of the genomic bases would be “visible” to HiC, the fraction of genomic bases that are within 250 bp from a restriction enzyme cut site was calculated. These fractions are plotted on the y-axis for each genome (x-axis labels) (
The results indicate that HiCoverage using a combination of restriction enzymes enables near complete genomic coverage across representative plant and animal species, and therefore various plant and animal species should be robust to the unique benefits of HiCoverage data described herein.
Crosslinked GM12878 cells were subject to HiCoverage experiment using MboI, MseI, DdeI, and HinfI and sequenced to approximately 37× raw depth. Depth-matched low density HiC data using MboI in GM12878 cells were downloaded from Rao, Cell, 2014. Each dataset was mapped to the hg19 reference genome using bwa mem −SP5M and deduplicated using PicardTools. The genome coverage histograms were then generated using DeepTools. As illustrated in
Crosslinked GM12878 cells were digested with either one, two, or three restriction enzymes (denoted across categorical axis labels of
The sequencing results shown in
Crosslinked GM12878 cells were digested with either one, two, or three restriction enzymes sequentially, in duplicate, using either MboI, NlaIII, or MseI. The order of restriction enzyme digestion is denoted as categorical axis labels (see
The sequencing results indicate that sample digestion with >1 restriction enzyme improve the preservation of spatial-proximal contiguity in the nucleic acid templates relative to digestion with a single enzyme. This result is surprising given that digestion with multiple restriction enzymes creates incompatible ends for proximity ligation, yet proximity ligation is still evidence by the increase in fraction of long-range cis read-outs. For example, sequential digestion with NlaIII and MseI, in either order, furthest improve the preservation of spatial-proximal contiguity in the nucleic acid templates. The sequencing results also indicate the order of sequential digestion appears to impact the sequencing results, (e.g., the condition starting with MseI and followed by NlaIII have the greatest preservation of spatial-proximal contiguity in the nucleic acid templates). However, similar to co-digestion results (
Crosslinked GM12878 cells were digested with NlaIII. After digestion, proximity ligation was performed using a ligase. Then, crosslinks were reversed and proximally-ligated DNA was purified. Proximally-ligated DNA was then sheared and split into 3 groups of DNA and subject to DNA size selection using either a 0.7×, 0.6×, or 0.5× ratio of Ampure Beads to sample volume, in quadruplicate. Illumina sequencing libraries were constructed using the 12 DNA samples and PCR amplified. After PCR amplification, 2 libraries from each group were purified using a 0.6× ratio of Ampure Beads to sample volume, with the other 2 libraries from each group were purified (and size selected) using a 0.8× ratio of Ampure Beads to sample volume. 3C libraries were sequenced on a MiniSeq yielding ˜1M raw PE reads per sample. After mapping and deduplication, the fraction of read-pairs that represent long-range (>15 kb insert size) intra-chromosomal interactions were enumerated and plotted along the y-axis for each permutation of post-shearing and post-PCR size selection conditions.
The sequencing results shown in
Crosslinked GM12878 cells were subject to two consecutive rounds of digestion and proximity ligation reactions. In the first round, GM12878 nuclei were digested with MboI and then proximity ligation was performed using ligase. Then nuclei were pelleted and resuspended in 1× restriction digestion buffer (CutSmart). Nuclei were then subject to a second round of restriction digestion using NlaIII, and then subject to a second round of proximity ligation using a ligase. As a control, some nuclei were set aside after the first round of digestion and proximity ligation. Then, crosslinks were reversed in all nuclei samples and proximally-ligated DNA was purified. Proximally-ligated DNA was then sheared and size selected using a 0.7× ratio of Ampure Beads to sample volume. Lastly, Illumina sequencing libraries were constructed, PCR amplified, and purified using a 0.8× ratio of Ampure Beads to sample volume. 3C libraries were sequenced on a MiniSeq yielding ˜1M raw PE reads per sample. After mapping and deduplication, the fraction of read-pairs that represent long-range (>15 kb inserts) intra-chromosomal interactions were enumerated and plotted along the y-axis for each condition. Throughout the experiment, a small aliquot of nuclei was taken after each digestion and ligation reaction (4 aliquots total) in order to obtain the molecular size of DNA after each step. DNA is these aliquots of nuclei were obtained by crosslink reversal and DNA purification. DNA was then analyzed by gel electrophoresis using a FlashGel (Lonza) with a molecular weight ladder as indicated.
A1. A method for preparing DNA molecules from a sample comprising:
A2. The method of embodiment A1, wherein the fragments spanning the ligation junctions comprise fragments up 750 base pairs.
A3. The method of embodiment A1 or A2, wherein each restriction endonuclease of the set has a high activity level in a common buffer and each restriction endonuclease of the set has a theoretical digestion frequency of at least 1 in 256.
A4. The method of any one of embodiments A1 to A3, wherein the set of restriction endonucleases consists of four restriction endonucleases.
A5. The method of embodiment A4, wherein the restriction endonucleases are: MboI, HinfI, MseI and DdeI.
A5.1. The method of embodiment A4, wherein the restriction endonucleases are: HpyCH4IV, HinfI, HinP1I and MseI.
A6. The method of anyone of embodiments A1 to A5.1, wherein the DNA molecules are obtained from a sample selected from nuclei, cells, tissues, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples or cell-free DNA.
A7. The method of anyone of embodiments A1 to A5.1, wherein the DNA molecules are obtained from a single cell.
A7.1. The method of anyone of embodiments A1 to A5.1, wherein the DNA molecules are obtained from two or more cells.
A8. The method of any one of embodiments A1 to A5.1, wherein the cross-linked DNA molecules of a sample comprise two or more genomes or portions thereof.
A9. The method of anyone of embodiments A1 to A8, wherein the proximity-ligated DNA molecules are analyzed in a chromatin conformation assay.
A10. The method of embodiment A9, wherein the chromatin conformation assay is Capture-C, 3C, 4C, 5C, HiC, Capture-HiC, HiChIP, PLAC-seq, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC, HiChIRP or combinations thereof.
A11. The method of embodiment A9, wherein the assay is genome-wide.
A11.1. The method of embodiment A11, wherein the assay is 3C, HiC, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC or combinations thereof.
A12. The method of embodiment A9, wherein the assay is directed to one or more target regions in the genome.
A12.1. The method of embodiment A12, wherein the assays is Capture-C, 4C, 5C, Capture-HiC, HiChIP, PLAC-seq, HiChIRP or combinations thereof.
A13. The method of embodiment A12, wherein the targets are single nucleotide variations, insertions, deletions, copy number variations, genomic rearrangements or targets for phasing.
A14. The method embodiment A12 or A13, wherein the sample comprises a cancer genome and the target region is associated with a phenotype.
A15. The method of any one of embodiments A1 to A14, wherein the fragments of the proximity-ligated DNA molecules comprising fragments spanning the ligation junctions are used to prepare a library of template molecules for DNA sequencing.
A15.1. The method of embodiment A15, wherein the ligation junctions are marked with an affinity purification marker.
A15.2 The method of embodiment A15.1, wherein the affinity purification marker is biotin conjugated to a nucleotide.
A15.3. The method of embodiment A15.2, whereby enrichment is by affinity purification of the affinity purification marker with an affinity purification molecule.
A16. The method of embodiment A15.3, wherein fragments spanning the ligation junctions are enriched to prepare a library of template molecules for DNA sequencing.
A17. The method of any one of embodiments A15 to A16 that are used is in a HiC, Capture-HiC, HiSCIP, PLAC-seq, HiCulfite or Methyl-HiC method.
A17.1. The method of embodiment A15.3, wherein the affinity purification molecule is streptavidin.
A17.2. The method of embodiment A16, where enrichment for fragmented proximity-ligated DNA molecules comprising ligation junctions is by size selection.
A18. The method of any one of embodiments A15 to A17.2, wherein the library of template molecules provides uniform genome-wide coverage of a genome or portion thereof.
A18.1. The method of any one of embodiments A15 to A18, wherein the library of template molecules is sequenced to generate sequence reads comprising sequence information.
A19. The method of embodiment A18.1, wherein the sequencing is short read sequencing.
A20. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for genomic rearrangement analysis of the genome or portion thereof.
A21. The method of embodiment A20, wherein the genomic rearrangement analysis comprises identification of a breakpoint.
A22. The method of embodiment A21, wherein sequence information of a given sequence read is located upstream and downstream of the breakpoint.
A23. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for clustering and ordering of contigs of the genome or portion thereof.
A24. The method of embodiment A23, wherein sequence information includes sequence information for each contig that is clustered and ordered.
A25. The method of embodiment A18.1 or A19, wherein the sequence information is utilized to determine contig orientation of the genome or portion thereof.
A26. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for clustering, ordering and orientating contigs of the genome or portion thereof.
A27. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for detection of pairwise 3D genome interactions of the genome or portion thereof.
A28. The method of embodiment A27, wherein the 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences.
A29. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for protein factor location analysis and 3D conformation analysis of the genome or portion thereof.
A30. The method of embodiment A29, wherein the protein factor location analysis and 3D conformation analysis comprises PLAC-seq or HiChIP.
A31. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for haplotype phasing of the genome or portion thereof.
A32. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for genome assembly and 3D conformation analysis of the genome or portion thereof.
A33. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for DNA methylation analysis of the genome or portion thereof.
A33.1. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for DNA methylation analysis and detection of 3D genome interactions of the genome or portion thereof.
A34. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for single nucleotide variant (SNV) discovery of the genome or portion thereof.
A35. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for base polishing of long-range sequencing information of the genome or portion thereof.
A36. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for highly sensitive copy number variation (CNV) analysis of the genome or portion thereof.
A37. The method of embodiment A36, wherein the copy number variation (CNV) is an amplification.
A38. The method of embodiment A36, wherein the copy number variation (CNV) is a heterozygous or homozygous deletion.
A39. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for variant discovery, haplotype phasing and genome assembly of the genome or portion thereof.
A39.1 The method of embodiment A18.1 or A19, wherein the sequence information is utilized for variant discovery and haplotype phasing in a first sample comprising a paternal genome and a second sample comprising a maternal genome and the phased variants of the paternal genome and the maternal genome are used to analyze sequence data of a fetal genome obtained from cfDNA of the mother.
A40. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for haplotype phasing and genome assembly of the genome or portion thereof.
A41. The method of embodiment A18.1 or A19, wherein the sequence information is utilized for genome assembly and detection of 3D genome interaction of the genome or portion thereof.
B1. A method for preparing DNA molecules from a sample comprising:
B2. The method of embodiment B1, wherein the fragments spanning the first, second, third and fourth ligation junctions and of lengths that can be templates for short range sequencing comprise up 750 base pairs.
B3. The method of embodiments B1 or B2, wherein the first, second, third and fourth restriction endonucleases are selected from enzymes that generate molecules with ends having 5′ overhangs, 3′ overhangs or that are blunt and combinations thereof.
B4. The method of embodiment B3, wherein the first, second, third and fourth restriction endonucleases generate molecules with the same type of end.
B5. The method of embodiment B3, wherein two or more of the first, second, third and fourth restriction endonucleases generate molecules with different types of ends.
B5.1. The method of any one of embodiments B1 to B5, wherein one or more of the first, second, third and fourth restriction endonucleases require a specific buffer for high activity level different from a buffer required for high activity level by another of the first, second, third or fourth restriction endonucleases.
B5.2. The method of any one of embodiments B1 to B4, wherein the product of one or more of the first, second, third and fourth restriction endonucleases can incorporate a different label from the label incorporated by another of the first, second, third or fourth restriction endonucleases.
B6. The method of anyone of embodiments B1 to B5.2, wherein the DNA molecules are obtained from a sample selected from nuclei, cells, tissues, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples or cell-free DNA.
B7. The method of anyone of embodiments B1 to B5.4, wherein the DNA molecules are obtained from a single cell.
B7.1. The method of anyone of embodiments B1 to B5.4, wherein the DNA molecules are obtained from two or more cells.
B8. The method of any one of embodiments B1 to A5.4, wherein the cross-linked DNA molecules of a sample comprise two or more genomes or portions thereof.
B9. The method of anyone of embodiments B1 to B8, wherein the proximity-ligated DNA molecules are analyzed in a chromatin conformation assay.
B10. The method of embodiment B9, wherein the chromatin conformation assay is Capture-C, 3C, 4C, 5C, HiC, Capture-HiC, HiChIP, PLAC-seq, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC, HiChIRP or combinations thereof.
B11. The method of embodiment B9, wherein the assay is genome-wide.
B11.1. The method of embodiment B11, wherein the assay is 3C, HiC, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC or combinations thereof.
B12. The method of embodiment B9, wherein the assay is directed to one or more target regions in the genome.
B12.1. The method of embodiment B12, wherein the assays is Capture-C, 4C, 5C, Capture-HiC, HiChIP, PLAC-seq, HiChIRP or combinations thereof.
B13. The method of embodiment B12, wherein the targets are single nucleotide variations, insertions, deletions, copy number variations, genomic rearrangements or targets for phasing.
B14. The method embodiment B12 or B13, wherein the sample comprises a cancer genome and the target region is associated with a phenotype.
B15. The method of any one of embodiments B1 to B14, wherein the fragmented proximity-ligated DNA molecules are used to prepare a library of template molecules for DNA sequencing.
B16. The method of embodiment B15, wherein the fragmented proximity-ligated molecules are enriched for fragmented proximity-ligated DNA molecules comprising ligation junctions and the fragmented proximity-ligated DNA molecules comprising ligation junctions are used to prepare a library of template molecules for DNA sequencing.
B17. The method of embodiment B16, wherein the assay is HiC, Capture-HiC, HiSCIP, PLAC-seq, HiCulfite or Methyl-HiC and the ligation junctions are marked with an affinity purification marker.
B17.1. The method of embodiment B17, whereby enrichment is by affinity purification of the affinity purification marker with an affinity purification molecule.
B17.2. The method of embodiment B17.1, wherein the affinity purification molecule is streptavidin.
B17.3. The method of embodiment B16, where enrichment for fragmented proximity-ligated DNA molecules comprising ligation junctions is by size selection.
B18. The method of any one of embodiments B15 to B17.3, wherein the library of template molecules provides uniform genome-wide coverage of a genome or portion thereof.
B18.1. The method of any one of embodiments B15 to A18, wherein the library of template molecules is sequenced to generate sequence reads comprising sequence information.
B19. The method of embodiment B18.1, wherein the sequencing is short read sequencing.
B20. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for genomic rearrangement analysis of the genome or portion thereof.
B21. The method of embodiment B20, wherein the genomic rearrangement analysis comprises identification of a breakpoint.
B22. The method of embodiment B21, wherein sequence information of a given sequence read is located upstream and downstream of the breakpoint.
B23. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for clustering and ordering of contigs of the genome or portion thereof.
B24. The method of embodiment B23, wherein sequence information includes sequence information for each contig that is clustered and ordered.
B25. The method of embodiment B18.1 or B19, wherein the sequence information is utilized to determine contig orientation of the genome or portion thereof.
B26. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for clustering, ordering and orientating contigs of the genome or portion thereof.
B27. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for detection of pairwise 3D genome interactions of the genome or portion thereof.
B28. The method of embodiment B27, wherein the 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences.
B29. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for protein factor location analysis and 3D conformation analysis of the genome or portion thereof.
B30. The method of embodiment B29, wherein the protein factor location analysis and 3D conformation analysis comprises PLAC-seq or HiChIP.
B31. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for haplotype phasing of the genome or portion thereof.
B32. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for genome assembly and 3D conformation analysis of the genome or portion thereof.
B33. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for DNA methylation analysis of the genome or portion thereof.
B33.1. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for DNA methylation analysis and detection of 3D genome interactions of the genome or portion thereof.
B34. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for single nucleotide variant (SNV) discovery of the genome or portion thereof.
B35. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for base polishing of long-range sequencing information of the genome or portion thereof.
B36. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for highly sensitive copy number variation (CNV) analysis of the genome or portion thereof.
B37. The method of embodiment B36, wherein the copy number variation (CNV) is an amplification.
B38. The method of embodiment B36, wherein the copy number variation (CNV) is a heterozygous or homozygous deletion.
B39. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for variant discovery, haplotype phasing and genome assembly of the genome or portion thereof.
B40. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for haplotype phasing and genome assembly of the genome or portion thereof.
B41. The method of embodiment B18.1 or B19, wherein the sequence information is utilized for genome assembly and detection of 3D genome interaction of the genome or portion thereof.
C1. A method for preparing DNA molecules from a sample comprising:
C2. The method of embodiment C1, wherein the fragments spanning the ligation junctions comprise fragments up 750 base pairs.
C3. The method of embodiment C1 or C2, where the streptavidin comprises streptavidin coated beads.
C4. The method of any one of embodiments C1 to C3, wherein each restriction endonuclease of the set has a high activity level in a common buffer and each restriction endonuclease of the set has a theoretical digestion frequency of at least 1 in 256.
C5. The method of any one of embodiments C1 to C4, wherein the restriction endonucleases are: MboI, HinfI, MseI and DdeI.
C5.1. The method of any one of embodiments C1 to C4, wherein the restriction endonucleases are: HpyCH4IV, HinfI, HinP1I and MseI.
C6. The method of anyone of embodiments C1 to C5.1, wherein the DNA molecules are obtained from a sample selected from nuclei, cells, tissues, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples or cell-free DNA.
C7. The method of anyone of embodiments C1 to C5.1, wherein the DNA molecules are obtained from a single cell.
C7.1. The method of anyone of embodiments C1 to C5.1, wherein the DNA molecules are obtained from two or more cells.
C8. The method of any one of embodiments C1 to C5.1, wherein the cross-linked DNA molecules of a sample comprise two or more genomes or portions thereof.
C9. The method of anyone of embodiments C1 to C8, wherein the proximity-ligated DNA molecules are analyzed in a chromatin conformation assay.
C10. The method of embodiment C9, wherein the chromatin conformation assay is Capture-C, 3C, 4C, 5C, HiC, Capture-HiC, HiChIP, PLAC-seq, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC, HiChIRP or combinations thereof.
C11. The method of embodiment C9, wherein the assay is genome-wide.
C11.1. The method of embodiment C11, wherein the assay is 3C, HiC, tethered chromosome capture (TCC), HiCulfite, Methyl-HiC or combinations thereof.
C12. The method of embodiment C9, wherein the assay is directed to one or more target regions in the genome.
C12.1. The method of embodiment C12, wherein the assays is Capture-C, 4C, 5C, Capture-HiC, HiChIP, PLAC-seq, HiChIRP or combinations thereof.
C13. The method of embodiment C12, wherein the targets are single nucleotide variations, insertions, deletions, copy number variations, genomic rearrangements or targets for phasing.
C14. The method embodiment C12 or C13, wherein the sample comprises a cancer genome and the target region is associated with a phenotype.
C15. The method of any one of embodiments C1 to C14, wherein the fragmented proximity-ligated DNA molecules are used to prepare a library of template molecules for DNA sequencing.
C16. The method of embodiment C15, wherein the fragmented proximity-ligated molecules are enriched for fragmented proximity-ligated DNA molecules comprising ligation junctions and the fragmented proximity-ligated DNA molecules comprising ligation junctions are used to prepare a library of template molecules for DNA sequencing.
C17. The method of embodiment C16, wherein the assay is HiC, Capture-HiC, HiSCIP, PLAC-seq, HiCulfite or Methyl-HiC and the ligation junctions are marked with an affinity purification marker.
C17.1. The method of embodiment C17, whereby enrichment is by affinity purification of the affinity purification marker with an affinity purification molecule.
C17.2. The method of embodiment C17.1, wherein the affinity purification molecule is streptavidin.
C17.3. The method of embodiment C16, where enrichment for fragmented proximity-ligated DNA molecules comprising ligation junctions is by size selection.
C18. The method of any one of embodiments C15 to C17.3, wherein the library of template molecules provides uniform genome-wide coverage of a genome or portion thereof.
C18.1. The method of any one of embodiments C15 to C18, wherein the library of template molecules is sequenced to generate sequence reads comprising sequence information.
C19. The method of embodiment C18.1, wherein the sequencing is short read sequencing.
C20. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for genomic rearrangement analysis of the genome or portion thereof.
C21. The method of embodiment C20, wherein the genomic rearrangement analysis comprises identification of a breakpoint.
C22. The method of embodiment C21, wherein sequence information of a given sequence read is located upstream and downstream of the breakpoint.
C23. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for clustering and ordering of contigs of the genome or portion thereof.
C24. The method of embodiment C23, wherein sequence information includes sequence information for each contig that is clustered and ordered.
C25. The method of embodiment C18.1 or C19, wherein the sequence information is utilized to determine contig orientation of the genome or portion thereof.
C26. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for clustering, ordering and orientating contigs of the genome or portion thereof.
C27. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for detection of pairwise 3D genome interactions of the genome or portion thereof.
C28. The method of embodiment C27, wherein the 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences.
C29. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for protein factor location analysis and 3D conformation analysis of the genome or portion thereof.
C30. The method of embodiment C29, wherein the protein factor location analysis and 3D conformation analysis comprises PLAC-seq or HiChIP.
C31. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for haplotype phasing of the genome or portion thereof.
C32. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for genome assembly and 3D conformation analysis of the genome or portion thereof.
C33. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for DNA methylation analysis of the genome or portion thereof.
C33.1. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for DNA methylation analysis and detection of 3D genome interactions of the genome or portion thereof.
C34. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for single nucleotide variant (SNV) discovery of the genome or portion thereof.
C35. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for base polishing of long-range sequencing information of the genome or portion thereof.
C36. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for highly sensitive copy number variation (CNV) analysis of the genome or portion thereof.
C37. The method of embodiment C36, wherein the copy number variation (CNV) is an amplification.
C38. The method of embodiment C36, wherein the copy number variation (CNV) is a heterozygous or homozygous deletion.
C39. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for variant discovery, haplotype phasing and genome assembly of the genome or portion thereof.
C40. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for haplotype phasing and genome assembly of the genome or portion thereof.
C41. The method of embodiment C18.1 or C19, wherein the sequence information is utilized for genome assembly and detection of 3D genome interaction of the genome or portion thereof.
D1. A kit comprising:
D2. The kit of embodiment D1, wherein the restriction endonucleases are in separate containers.
D3. The kit of embodiment D1, wherein the restriction endonucleases are in a single container.
D4. The kit of any one of embodiments D1 to D3, wherein each restriction endonuclease has a high activity level in a common restriction endonuclease buffer and each restriction endonuclease has a theoretical digestion frequency of at least 1 in 256.
D5. The kit of any one of embodiments D1 to D4, wherein the restriction endonuclease buffer is in a separate container from the restriction endonucleases.
D6. The kit of any one of embodiments D1 to D5, further comprising instructions.
E1. A kit comprising:
E2. The kit of embodiment E1, wherein the four restriction endonucleases are in separate containers.
E3. The kit of embodiment E1, wherein the four restriction endonucleases are in a single container.
E4. The kit of any one of embodiments E1 to E3, wherein the restriction endonuclease buffer is in a separate container from the four restriction endonucleases.
E5. The kit of any one of embodiments E1 to E4, wherein each restriction endonuclease has a high activity level in a common restriction endonuclease buffer and each restriction endonuclease has a theoretical digestion frequency of at least 1 in 256.
E6. The kit of any one of embodiments E1 to E5, wherein the four restriction endonucleases are: MboI, HinfI, MseI and DdeI.
E7. The kit of any one of embodiments E1 to E5, wherein the four restriction endonucleases are: HpyCH4IV, HinfI, HinP1I and MseI.
E8. The kit of any one of embodiments E1 to E7, further comprising instructions.
F1. A kit comprising:
F2. The kit of embodiment F1, wherein the four restriction endonucleases are in separate containers.
F3. The kit of any one of embodiments F1 to F3, wherein the two or more restriction endonuclease buffers are in separate containers from the four restriction endonucleases.
F4. The kit of any one of embodiments F1 to F3, wherein each restriction endonuclease has a theoretical digestion frequency of at least 1 in 256.
F5. The kit of any one of embodiments F1 to F4, wherein at least two of the restriction endonucleases require unique buffers for high level activity.
F6. The kit of any one of embodiments F1 to F5, further comprising instructions.
G1. A method for preparing DNA molecules from a sample comprising:
G2. The method of embodiment G1, wherein the spatially-proximal DNA molecules comprise crosslinked DNA molecules.
G2.1. The method of embodiment G1 or G2, wherein the spatially-proximal DNA molecules with stable spatial interactions of a sample are within cells/nuclei and the contacting steps are in situ.
G2.2. The method of embodiment G1 or G2, wherein the spatially-proximal DNA molecules comprise a genome or portion thereof.
G3. The method of any one of embodiments G1 to G2.2, wherein there are two restriction endonucleases.
G4. The method of any one of embodiments G1 to G2.2, wherein there are at least three restriction endonucleases.
G4.1. The method of embodiment G4, wherein there are three restriction endonucleases.
G5. The method of any one of embodiments G1 to G4.1, wherein one of the restriction endonucleases is NlaIII.
G6. The method of any one of embodiments G1 to G5, wherein one of the restriction endonucleases is NlaIII and the other restriction endonuclease is MboI or MseI.
G7. The method of any one of embodiments G1 to G4.1, wherein one of the restriction endonucleases is NlaIII and another other restriction endonuclease is MboI or MseI.
G8. The method of embodiment G4 or G4.1, wherein the restriction endonucleases are: NlaIII, MboI and MseI.
G9. The method of any one of embodiments G1 to G5, wherein the restriction endonucleases produce the same overhanging sequence.
G10. The method of any one of embodiments G1 to G8, wherein the restriction endonucleases produce different overhanging sequences.
G11. The method of anyone of embodiments G1 to G10, wherein contact and digestion with all of the restriction endonucleases is at one time.
G12. The method of anyone of embodiments G1 to G10, wherein contact and digestion with each restriction endonucleases is sequential.
G12.1. The method of embodiment G12, wherein the digestion with a prior endonuclease or endonucleases has essentially completed.
G12.2. The method of embodiment G12, wherein the digestion with a prior endonuclease or endonucleases has not completed.
G13. The method of any one of embodiments G4 to G10, wherein contact and digestion with restriction endonucleases is sequential and at least one contact and digestion is with at least two restriction endonucleases.
G14. The method of any one of embodiments G12 to G13, wherein the sequential contact and digestion has a determined order for the restriction endonucleases.
G14.1. The method of embodiment G11, wherein contact with ligase is after completion of the digestion by the restriction endonucleases.
G14.2. The method of any one of embodiments G12 to G14, wherein contact with ligase is after completion of the sequential contact and digestion with all the restriction endonucleases.
G15. The method of any one of embodiments G12 to G14, wherein each contact and digestion with one or more restriction endonucleases is followed by contact with ligase.
G16. The method of anyone of embodiments G1 to G15, wherein the DNA molecules are obtained from a sample selected from nuclei, cells, tissues, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples or cell-free DNA.
G16.1 The method of embodiment G16, wherein the sample is in an aqueous solution or affixed to a solid surface.
G17. The method of anyone of embodiments G1 to G16.1, wherein the DNA molecules are obtained from a single cell.
G18. The method of anyone of embodiments G1 to G16.1, wherein the DNA molecules are obtained from two or more cells.
G19. The method of any one of embodiments G1 to G18, wherein the DNA molecules of a sample comprise two or more genomes or portions thereof.
G20. The method of any one of embodiments G1 to G19, wherein the method comprises one or more steps specific to a 4C, 5C, Capture-C, 3C-ChIP or Methyl-3C method.
G21. The method of any one of embodiments G1 to G20, wherein the proximity-ligated DNA molecules comprising ligation junctions are derived from sequences representing essentially an entire genome.
G22. The method of any one of embodiments G1 to G21, wherein the proximity-ligated DNA molecules comprising ligation junctions are purified.
G23. The method of any one of embodiments G2 to G22, wherein the crosslinked proximity-ligated DNA molecules comprising ligation junctions are contacted with a reagent that reverses crosslinking.
G24. The method of any one of embodiments, G1 to G23, wherein proximity-ligated DNA molecules comprising ligation junctions are enriched for DNA molecules with ligation junctions.
G24.1. The method of embodiment G24, wherein enrichment for DNA molecules with ligation junctions is by size selection.
G24.2. The method of embodiment G24.1, wherein size selection comprises the use of beads.
G24.3. The method of embodiment G24.1, wherein size selection comprises gel extraction or size selective DNA precipitation.
G25. The method of any one of embodiments G1 to G24.3, wherein a library of template molecules for DNA sequencing is prepared from the proximity-ligated DNA molecules.
G25.1. The method of embodiment G25, wherein size selection to enrich for DNA molecules with ligation junctions is performed before or after an amplification step when constructing the library.
G26. The method of embodiment G25 or G25.1, wherein the library of template molecules is sequenced to generate sequence reads comprising sequence information.
G27. The method of embodiment G26, wherein the sequencing is short-read sequencing.
G27.1. The method of any one of embodiments G1 to G27, wherein at least 30% of the nucleic acid templates are long-range cis molecules.
G27.2. The method of any one of embodiments G1 to G27, wherein at least 40% of the nucleic acid templates are long-range cis molecules.
G27.3. The method of any one of embodiments G1 to G27, wherein at least 50% of the nucleic acid templates are long-range cis molecules.
G27.4. The method of any one of embodiments G1 to G27, wherein at least 60% of the nucleic acid templates are long-range cis molecules.
G27.5. The method of embodiment G27, wherein the proximity-ligated DNA molecules are fragmented to generate fragments of proximity-ligated DNA molecules comprising fragments spanning the ligation junctions prior to the preparation of a library.
G27.6. The method of embodiment G26, wherein the sequencing is long-read sequencing.
G28. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for detection of pairwise 3D genome interactions of the genome or portion thereof.
G29. The method of embodiment G28, wherein the 3D genome interaction is between promoters, enhancers, gene regulatory elements, GWAS loci, chromatin loop and topological domain anchors, repetitive elements, polycomb regions, gene bodies, exons or integrated viral sequences.
G30. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for protein factor location analysis and 3D conformation analysis of the genome or portion thereof.
G31. The method of embodiment G30, wherein the protein factor location analysis and 3D conformation analysis comprises 3C-ChIP.
G32. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for genomic rearrangement analysis of the genome or portion thereof.
G33. The method of embodiment G32, wherein the genomic rearrangement analysis comprises identification of a breakpoint.
G34. The method of embodiment G33, wherein sequence information of a given sequence read is located upstream and downstream of the breakpoint.
G35. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for clustering and ordering of contigs of the genome or portion thereof.
G36. The method of embodiment G35, wherein sequence information includes sequence information for each contig that is clustered and ordered.
G37. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized to determine contig orientation of the genome or portion thereof.
G38. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for clustering, ordering and orientating contigs of the genome or portion thereof.
G39. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for haplotype phasing of the genome or portion thereof.
G40. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for DNA methylation analysis of the genome or portion thereof.
G41. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for single nucleotide variant (SNV) discovery of the genome or portion thereof.
G42. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for base polishing of long-range sequencing information of the genome or portion thereof.
G43. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for highly sensitive copy number variation (CNV) analysis of the genome or portion thereof.
G44. The method of embodiment G43, wherein the copy number variation (CNV) is an amplification.
G45. The method of embodiment G43, wherein the copy number variation (CNV) is a heterozygous or homozygous deletion.
G46. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for variant discovery, haplotype phasing and genome assembly of the genome or portion thereof.
G47. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for variant discovery and haplotype phasing in a first sample comprising a paternal genome and a second sample comprising a maternal genome and the phased variants of the paternal genome and the maternal genome are used to analyze sequence data of a fetal genome obtained from cfDNA of the mother.
G48. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for haplotype phasing and genome assembly of the genome or portion thereof.
G49. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for genome assembly and 3D conformation analysis of the genome or portion thereof.
G50. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for DNA methylation analysis and detection of 3D genome interactions of the genome or portion thereof.
G51. The method of any one of embodiments G26 to G27.6, wherein the sequence information is utilized for genome assembly and detection of 3D genome interaction of the genome or portion thereof.
G52. The method of any one of embodiments G1 to G51, wherein molecular contiguity of proximity-ligated DNA molecules is preserved in barcodes.
G53. The method of embodiment G52, wherein barcodes are introduced into the proximity-ligated DNA molecules by contacting proximally-ligated DNA with a barcoded transposome linked bead prior to library preparation.
G54. The method of embodiment G52 to G53, wherein the sequence information is utilized for detection of higher-order 3D genome interactions of a genome or portion thereof, by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules.
G55. The method of any one of embodiments G52 to G54, wherein the sequence information is utilized for detection of three or more concurrent 3D genome interactions of the genome or portion thereof, by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules.
G56. The method of any one of embodiments G52 to G55, wherein the sequence information is utilized for detection of virtual pairwise 3D genome interactions by leveraging the preserved molecular contiguity of proximity-ligated DNA molecules.
G57. The method of embodiment G56, wherein a virtual pairwise 3D genome interaction is between restriction fragments that are not directly ligated to one another within a given proximity-ligated DNA molecule of the genome or portion thereof.
G58. The method of any one of embodiments G52 to G57, wherein the pairwise interactions, virtual pairwise interactions, and/or higher order interactions obtained by leveraging the preserved molecular contiguity of proximity ligated DNA molecules is utilized for 3D genome interactions of the genome or portion thereof, genomic rearrangement analysis of the genome or portion thereof, clustering and ordering of contigs of the genome or portion thereof, determining contig orientation of the genome or portion thereof, haplotype phasing of the genome or portion thereof, DNA methylation analysis of the genome or portion thereof, single nucleotide variant (SNV) discovery of the genome or portion thereof, base polishing of long-range sequencing information of the genome or portion thereof, highly sensitive copy number variation (CNV) analysis of the genome or portion thereof or combinations thereof.
H1. A method for preparing DNA molecules from a sample comprising:
H2. The method of embodiment H1, comprising:
H3. A method for preparing DNA molecules from a sample comprising:
H4. The method of embodiment H3, comprising:
H5. The method of any one of embodiments H1 to H4, wherein the restriction endonucleases produce the same overhanging sequence.
H6. The method of any one of embodiments H1 to H4, wherein the restriction endonucleases produce different overhanging sequences.
11. A method of obtaining the spatial positioning of sequence information obtained from a proximity-ligated tissue section comprising:
12. A method of obtaining the spatial positioning of sequence information obtained from a proximity-ligated tissue section comprising:
13. A method of obtaining the spatial positioning of sequence information obtained from a proximity-ligated tissue section comprising:
14. A method of obtaining the spatial positioning of sequence information obtained from a proximity-ligated tissue section comprising:
J1. A library of DNA template molecules for sequencing prepared by a method comprising any of the methods of embodiments A1 to A18.
J2. A library of DNA template molecules for sequencing prepared by a method comprising any of the methods of embodiments B1 to B14.
J3. A library of DNA template molecules for sequencing prepared by a method comprising any of the methods of embodiments C1 to C14.
J4. A library of DNA template molecules for sequencing prepared by a method comprising any of the methods of embodiments G1 to G27.5.
J5. A library of DNA template molecules for sequencing prepared by a method comprising any of the methods of embodiments H1 to H16.
K1. A kit comprising one or more of:
K2. A kit comprising one or more of:
K2.1. The kit of embodiment K2, wherein one of the restriction endonucleases is NlaIII.
K2.2. The kit of embodiment K2.1, wherein the other restriction endonuclease is MboI or MseI.
K3. A kit comprising one or more of:
K3.1. The kit of embodiment K3, wherein one of the restriction endonucleases is NlaIII.
K3.2. The kit of embodiment K3.1, wherein one of the endonucleases is MboI or MseI.
K3.3. The kit of embodiment K3, wherein the restriction endonucleases are: NlaIII, MboI and MseI.
K4. The kit of any one of embodiments K1 to K3.3, wherein the restriction endonucleases of the kit produce the same overhanging sequence.
K5. The kit of any one of embodiments K1 to K3.3, wherein the restriction endonucleases of the kit produce different overhanging sequences.
K6. The kit of any one of embodiments K1 to K5, wherein digestion with the two or more restriction endonucleases of the kit can be carried out at the same time.
K7. The kit of any one of embodiments K1 to K5, wherein digestion with one or more restriction endonucleases of the kit cannot can be carried out at the same time.
K8. The kit of any one of embodiments K1 to K7, wherein the restriction endonucleases of the kit are in separate containers.
K9. The kit of embodiment K6, wherein the restriction endonucleases of the kit are in a single container.
K10. The kit of any one of embodiments K1 to K7, wherein the restriction endonucleases of the kit are in more than one container.
K10.1. The kit of embodiment K10, wherein at least one container contains more than one restriction endonuclease.
K11. The kit of any one of embodiments K1 to K6, wherein each restriction endonuclease of the kit has a high activity level in a common restriction endonuclease buffer and the buffer is in one container.
K12. The kit of any one of embodiments K1 to K10.1, wherein more than one restriction endonuclease buffer is in the kit and the buffers are in separate containers.
K13. The kit of any one of embodiments K1 to K12, wherein a restriction endonuclease buffer is in a separate container from a restriction endonuclease.
K14. The kit of any one of embodiments K1 to K13, wherein the kit comprises instructions.
K14.1. The kit of embodiment K14, wherein the instructions recite the order that the restriction enzymes of a kit are to be used.
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.
Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.
The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.
Certain embodiments of the technology are set forth in the claim(s) that follow(s).
This application is a 35 U.S.C. 371 national phase application of International Patent Cooperation Treaty (PCT) Application No. PCT/US2020/033666, filed on May 19, 2020, entitled METHODS AND COMPOSITIONS FOR ENHANCED GENOME COVERAGE AND PRESERVATION OF SPATIAL PROXIMAL CONTIGUITY, naming Anthony SCHMITT et al. as inventors, and designated by attorney docket no. AMG-1004-PC, which claims the benefit of U.S. Provisional Patent Application No. 62/850,449 filed May 20, 2019 entitled METHODS AND COMPOSITIONS FOR ENHANCED GENOME COVERAGE, naming Anthony Schmitt, Bret Reid, Stephen Mac, Xiang Zhou and Siddarth Selvaraj as inventors and assigned attorney docket no. AMG-1004-PV. This application is related to U.S. application Ser. No. 16/689,002 filed Nov. 19, 2019 entitled METHODS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITY INFORMATION, naming Anthony Schmitt, Catherine Tan, Derek Reid, Chris De La Torre and Siddarth Selvaraj as inventors and assigned attorney docket no. AMG-1003-UT. This application is also related to U.S. application Ser. No. 16/764,787 filed May 15, 2020 entitled PRESERVING SPATIAL-PROXIMAL CONTIGUITY AND MOLECULAR CONTIGUITY IN NUCLEIC ACID TEMPLATES, naming Siddarth Selvaraj, Anthony Schmitt and Bret Reid as inventors and assigned attorney docket no. AMG-1002-US. This application is also related to U.S. application Ser. No. 15/738,871 filed Dec. 21, 2017, entitled ACCURATE MOLECULAR DECONVOLUTION OF MIXTURE SAMPLES, naming Siddarth Selvaraj, Nathaniel Heintzman and Christian Edgar Laing as inventors and assigned attorney docket no. AMG-1001-US. The entire content of the foregoing patent applications are incorporated herein by reference, including all text, tables and drawings.
This invention was made with government support under Contract Nos. 1R44HG009584-01 and 2R44HG008118-04A1 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/033666 | 5/19/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62850449 | May 2019 | US |
