Primers and assays for linking regions using polymerases

Abstract

To provide methods and kits for linking separated regions of DNA molecules in a biological sample of a subject.SOLUTION: Particular forward and reverse primers may be used to link distant regions of the same large DNA molecule into a smaller DNA molecule. A reverse primer R1 can have a first portion complementary to an ending sequence of region A and can have a second portion having an overlapping sequence. A forward primer F2 can have a first portion complementary to a starting sequence of region B, where the forward primer includes a complementary overlapping sequence (e.g., the same first portion or a second portion) that is complementary to the overlapping sequence. The first portion of F2 may be the entire primer. The smaller DNA molecules can be used to determine haplotypes of regions. Kits including the particular forward and reverse primers are also described.SELECTED DRAWING: None

Description

[Background technology]

[0001] Humans have two copies of each chromosome, one from each parent. Understanding the combination of variants on the same parental chromosome, i.e., haplotypes, can provide valuable clinical implications. In particular, haplotype information is useful for non-invasive prenatal testing of single-gene diseases and deciphering fetal genomic inheritance (Hui et al. Clin Chem. 2017;63:513-524; Lo et al. Sci Transl Med. 2010;2:61ra91). However, current techniques for determining a particular individual's haplotype can be costly, inaccurate, and low-throughput. Summary of the Invention

[0002] PCR assays using high-throughput polymerases can be used to join distant regions (e.g., 100 bp to 100 kbp apart) of the same large DNA molecule to a smaller DNA molecule, making it easier to measure, for example, the haplotypes of two regions. High-throughput polymerases can add excess nucleotides to only one end of each strand of double-stranded DNA (e.g., an A at the 3' end), which can hinder and cause reduced yields in the production of the smaller joined DNA molecule (also called an extension amplicon).

[0003] To address these issues, specific forward and reverse primers can be used. For example, reverse primer R1 can have a first portion that is complementary to the end sequence of region A and a second portion that has an overlap sequence. Forward primer F2 can have a first portion that is complementary to the start sequence of region B, and the forward primer contains a complementary overlap sequence (e.g., the same first portion or second portion) that is complementary to the overlap sequence. The first portion of F2 can be the entire primer.

[0004] A better understanding of the nature and advantages of the embodiments of the present disclosure may be obtained with reference to the following detailed description and the accompanying drawings.

[0005] term The term "fragment" (e.g., DNA fragment) as used herein may refer to a portion of a polynucleotide or polypeptide sequence comprising at least three consecutive nucleotides. A nucleic acid fragment may retain biological activity and / or some characteristics of the parent polypeptide. A nucleic acid fragment may be double-stranded or single-stranded, methylated or unmethylated, intact or nicked, complexed or uncomplexed with other macromolecules, e.g., lipid particles, proteins. A fragment may be derived from a particular tissue type, e.g., fetus, tumor, transplanted organ, etc.

[0006] The term "assay" generally refers to a technique for determining a characteristic of a nucleic acid. An assay (e.g., a first assay or a second assay) generally refers to a technique for determining the amount of a nucleic acid in a sample, the genomic identity of a nucleic acid in a sample, the copy number variation of a nucleic acid in a sample, the methylation state of a nucleic acid in a sample, the fragment size distribution of a nucleic acid in a sample, the mutation state of a nucleic acid in a sample, or the fragmentation pattern of a nucleic acid in a sample. Any assay known to one of ordinary skill in the art can be used to detect any of the nucleic acid characteristics mentioned herein. An assay can also refer to a technique for combining amplification products from different regions of a DNA molecule to form one or more DNA molecules. The characteristics of a nucleic acid include sequence, amount, genomic identity, copy number, methylation state at one or more nucleotide positions, size of the nucleic acid, mutation of the nucleic acid at one or more nucleotide positions, and the fragmentation pattern of the nucleic acid (e.g., the nucleotide position(s) at which the nucleic acid is fragmented). The term "assay" may be used interchangeably with the term "method". Assays or methods can have a particular sensitivity and / or specificity, and their relative usefulness as diagnostic tools can be measured using the ROC-AUC statistic.

[0007] A "sequence read" refers to a strand of nucleotides sequenced from any portion or all of a nucleic acid molecule. For example, a sequence read can be an entire nucleic acid fragment present in a biological sample. Also by way of example, a sequence read can be a short strand of nucleotides (e.g., about 20-150 bases) sequenced from a nucleic acid fragment, a short strand of nucleotides at one or both ends of a nucleic acid fragment, or the sequencing of the entire nucleic acid fragment present in a biological sample. Paired sequence reads can be aligned to a reference genome, which can provide the length of the fragment. Sequence reads can be obtained, for example, using sequencing techniques, or in a variety of ways using probes, for example, with hybridization arrays or capture probes, or with amplification techniques such as polymerase chain reaction (PCR) or linear amplification using a single primer or isothermal amplification, or based on biophysical measurements such as mass spectrometry. Sequence reads can be obtained from single molecule sequencing. "Single molecule sequencing" refers to sequencing a single template DNA molecule to obtain a sequence read, without the need to interpret base sequence information from a clonal copy of the template DNA molecule. Single molecule sequencing may sequence the entire molecule or only a portion of a DNA molecule. A majority of a DNA molecule may be sequenced, for example, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0008] A "separation value" (or relative abundance) corresponds to a difference or ratio between two values, e.g., involving two read amounts with two different alleles. A separation value can be a simple difference or ratio. As an example, the direct ratio of x / y is a separation value, as is x / (x+y). A separation value can include other factors, e.g., multiplicative factors. As another example, a difference or ratio of a function of the values ​​can be used, e.g., the difference or ratio of the natural logarithms (ln) of two values. A separation value can include a difference and / or a ratio.

[0009] The term "classification" as used herein refers to any number(s) or other feature(s) associated with a particular characteristic of a sample. For example, a "+" symbol (or the word "positive") can mean that the sample is classified as having a deletion or amplification. Classification can be binary (e.g., positive or negative) or can have more levels of classification (e.g., a scale of 1-10 or 0-1).

[0010] The terms "cutoff" and "threshold" refer to a predefined number used in an operation. For example, a cutoff size may refer to a size above which fragments are excluded. A threshold may be a value above or below which a particular classification is applied, for example, a classification of a condition, such as whether a subject has a condition or the severity of the condition. A cutoff or threshold may be a "reference value" or may be derived from a reference value that represents a particular classification or distinguishes between two or more classifications, for example, between which alleles contain a haplotype. Such a reference value may be selected from or determined in various ways, as will be understood by those skilled in the art, for example, from or based on the output of test data. For example, a standard of measurement may be determined for two different cohorts of subjects with different known classifications, and a reference value may be selected as a representative (e.g., average) of one classification or as a value between two clusters of the standard of measurement. Thus, a reference subject with a known classification of haplotypes may be used to determine a reference level for distinguishing between different haplotypes. As another example, a reference value may be determined based on a statistical simulation of the sample. Any of these terms may be used in any of these contexts. As will be appreciated by one of skill in the art, the cutoff can be selected to achieve the desired sensitivity and specificity. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1A shows a four-primer PCR system used to amplify two different target regions, A and B. [Figure 1B]FIG. 1B shows a four-primer PCR system used to amplify two different target regions, A and B. [Figure 1C] FIG. 1C shows a four-primer PCR system used to amplify two different target regions, A and B. [Figure 1D] FIG. 1D shows a four-primer PCR system used to amplify two different target regions, A and B.

[0012] [Figure 2A] FIG. 2A shows a three-primer PCR system used to amplify two different target regions, A and B. [Figure 2B] FIG. 2B shows a three-primer PCR system used to amplify two different target regions, A and B. [Figure 2C] FIG. 2C shows a three-primer PCR system used to amplify two different target regions, A and B.

[0013] [Figure 3A] FIG. 3A illustrates a fusion PCR method using genomic locations with T and A in regions A and B, respectively, according to an embodiment of the present disclosure. [Figure 3B] FIG. 3B illustrates a fusion PCR method using genomic positions with T and A in regions A and B, respectively, according to an embodiment of the present disclosure. [Figure 3C] FIG. 3C illustrates a fusion PCR method using genomic positions with T and A in regions A and B, respectively, according to an embodiment of the present disclosure. [Figure 3D] FIG. 3D illustrates a fusion PCR method using genomic positions with T and A in regions A and B, respectively, according to an embodiment of the present disclosure. [Figure 3E] FIG. 3E illustrates a fusion PCR method using genomic positions with T and A in regions A and B, respectively, according to an embodiment of the present disclosure. [Figure 3F] FIG. 3F illustrates a fusion PCR method using genomic positions with T and A in regions A and B, respectively, according to an embodiment of the present disclosure.

[0014] [Figure 4A] FIG. 4A shows a fusion PCR method using a reverse primer with an A appended thereto, according to an embodiment of the present disclosure. [Figure 4B] FIG. 4B illustrates a fusion PCR method using a reverse primer with an A appended thereto, according to an embodiment of the present disclosure. [Figure 4C] FIG. 4C illustrates a fusion PCR method using a reverse primer with an A appended thereto, according to an embodiment of the present disclosure.

[0015] [Figure 5A] FIG. 5A shows a fusion PCR method using a reverse primer and a forward primer with an A appended thereto according to an embodiment of the present disclosure. [Figure 5B] FIG. 5B illustrates a fusion PCR method using a reverse primer and a forward primer with an A appended thereto according to an embodiment of the present disclosure. [Figure 5C] FIG. 5C illustrates a fusion PCR method using a reverse primer and a forward primer with an A appended thereto according to an embodiment of the present disclosure. [Figure 5D] FIG. 5D illustrates a fusion PCR method using a reverse primer and a forward primer with an A appended thereto according to an embodiment of the present disclosure.

[0016] [Figure 6] FIG. 6 illustrates the principle of fusion and adapter ligation PCR using primers to facilitate amplification of fusion PCR products according to an embodiment of the present disclosure.

[0017] [Figure 7A] FIG. 7A shows an example of using primers to facilitate amplification of a fusion PCR product according to an embodiment of the present disclosure. [Figure 7B] FIG. 7B shows an example of using primers to facilitate amplification of a fusion PCR product according to an embodiment of the present disclosure.

[0018] [Figure 8A] FIG. 8A shows a fusion PCR method for joining more than two regions where only specific regions bind to each other according to an embodiment of the present disclosure. [Figure 8B] FIG. 8B shows a fusion PCR method for joining more than two regions where only specific regions bind to each other according to an embodiment of the present disclosure.

[0019] [Figure 9A] FIG. 9A shows a fusion PCR method for joining more than two regions, where any of the regions can be joined to each other, according to an embodiment of the present disclosure. [Figure 9B] FIG. 9B shows a fusion PCR method for joining more than two regions, where any of the regions can be joined together, according to an embodiment of the present disclosure.

[0020] [Figure 10A] FIG. 10A illustrates how primers can be used to amplify a fusion PCR product having multiple regions, according to an embodiment of the present disclosure. [Figure 10B] FIG. 10B illustrates how primers can be used to amplify a fusion PCR product having multiple regions, according to an embodiment of the present disclosure.

[0021] [Figure 11] FIG. 11 shows six haplotypes containing different combinations of genotypes in exons 1 and 3 of NUDT15.

[0022] [Figure 12A] FIG. 12A shows melting curves distinguishing samples with different NUT15 genotypes in the exon1 (A) and exon3 (B) regions. [Figure 12B] FIG. 12B shows melting curves distinguishing samples with different NUDT15 genotypes in the exon1 (A) and exon3 (B) regions.

[0023] [Figure 13A]FIG. 13A shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. [Figure 13B] FIG. 13B shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. [Figure 13C] FIG. 13C shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. [Figure 13D] FIG. 13D shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. [Figure 13E] FIG. 13E shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. [Figure 13F] FIG. 13F shows the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure.

[0024] [Figure 14A] FIG. 14A shows four regions to be haplotyped divided into several groups of two regions according to an embodiment of the present disclosure. [Figure 14B] FIG. 14B shows four regions to be haplotyped divided into several groups of two regions according to an embodiment of the present disclosure. [Figure 14C] FIG. 14C shows four regions to be haplotyped divided into several groups of two regions according to an embodiment of the present disclosure.

[0025] [Figure 15A] FIG. 15A shows the formation of a three-domain fusion product, with one domain from each of the three groups, according to an embodiment of the present disclosure. [Figure 15B] FIG. 15B shows the formation of a three-domain fusion product, with one domain from each of the three groups, according to an embodiment of the present disclosure. [Figure 15C]FIG. 15C shows the formation of a three-domain fusion product, with one domain from each of the three groups, according to an embodiment of the present disclosure.

[0026] [Figure 16A] FIG. 16A shows the formation of a fusion product without the addition of an adenosine in the primer according to an embodiment of the present disclosure. [Figure 16B] FIG. 16B shows the formation of a fusion product without the addition of an adenosine in the primer according to an embodiment of the present disclosure.

[0027] [Figure 17A] FIG. 17A shows (A) the distance between different SNPs in a DNA molecule and (B) the configuration of primers for the SNPs, according to an embodiment of the present disclosure. [Figure 17B] FIG. 17B shows (A) the distance between different SNPs in a DNA molecule and (B) the configuration of primers for the SNPs, according to an embodiment of the present disclosure.

[0028] [Figure 18] FIG. 18 is a table showing the sequences of primers for amplifying each region according to an embodiment of the present disclosure.

[0029] [Figure 19] FIG. 19 is a table showing phases determined by digital fusion PCR according to an embodiment of the present disclosure.

[0030] [Figure 20-1] FIG. 20 is a table showing primer sequences for SNPs in the HBB gene according to embodiments of the present disclosure. [Figure 20-2] FIG. 20 is a table showing primer sequences for SNPs in the HBB gene according to embodiments of the present disclosure.

[0031] [Figure 21A] FIG. 21A depicts a phase assembly analysis according to an embodiment of the present disclosure. [Figure 21B]FIG. 21B illustrates a phase assembly analysis according to an embodiment of the present disclosure.

[0032] [Figure 22-1] FIG. 22 is a table showing haplotypes derived by fusion PCR for family trios according to an embodiment of the present disclosure. [Figure 22-2] FIG. 22 is a table showing haplotypes derived by fusion PCR for family trios according to an embodiment of the present disclosure.

[0033] [Figure 23] FIG. 23 is a flow chart illustrating a method for joining separate regions of DNA molecules in a biological sample of a subject according to an embodiment of the present disclosure.

[0034] [Figure 24] FIG. 24 illustrates a measurement system according to an embodiment of the present disclosure.

[0035] [Diagram 25] FIG. 25 illustrates a block diagram of an exemplary computer system that can be used with systems and methods according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] It may be beneficial to sequence multiple separate regions of a DNA molecule. Only certain regions of a DNA molecule (e.g., single nucleotide polymorphisms [SNPs]) may be of interest. Certain sequencing techniques may be limited in the length that can be sequenced at one time. Sequencing intermediate regions between regions of interest may unnecessarily consume equipment and operation time. Traditional methods of joining two separate regions together for sequencing suffer from many problems. It may be difficult to control the purity of the amplified DNA molecule. Such traditional methods may be inaccurate. The techniques described herein allow for accurate and efficient methods for joining two or more separate regions of a DNA molecule together. The resulting fused DNA molecule can then be sequenced and / or haplotyped.

[0037] Some embodiments can provide increased yields and corresponding efficiencies by describing the use of high-throughput polymerases with a bias for adding specific nucleotides (e.g., A) at the overhang positions. In one example, such techniques can include designing a reverse primer whose end hybridizes at a position with a complementary nucleotide (e.g., T) to allow the two regions to be joined appropriately. The other region to be joined can have a specific nucleotide after the forward primer, thereby creating a matching overlap region for joining the regions. In another example, the primer itself can include an overlap region with a specific nucleotide (and the complementary nucleotide of the specific primer in certain embodiments) inserted between the two portions. Thus, a forward primer with a portion complementary to a portion of the reverse primer can be used to join the separate regions.

[0038] As another example of using such high throughput polymerases with or without bias, different combinations of regions from the same molecule can be joined together in groups. This combination of regions allows accurate and cost-effective haplotyping of large genomic regions covering multiple regions of interest. Forward and reverse primers can be used to amplify the fusion products. Three or more regions may be joined together. Simultaneous fusion, phasing, and haplotyping of multiple regions would be advantageous when one or more regions are homozygous, since homozygous regions cannot provide haplotype information. Additionally, kits containing such specially designed primers may be provided. Details of these features are described below.

[0039] I. Haplotype Technology Numerous methods are available for determining an individual's haplotype. These methods are broadly divided into two categories: computational deduction and experimental analysis. However, existing methods for determining an individual's haplotype have their own limitations, such as relatively low accuracy, high cost, low throughput, and inability to target specific regions. In this application, a technique is described that can accurately determine an individual's haplotype in a relatively low-cost and high-throughput manner.

[0040] A. Computational method Computational methods are frequently used to statistically infer an individual's haplotype based on genotypic information of a related population (Browning et al., Nature Reviews Genetics, 2011;12:703-714). Through analysis of the genotypes of many individuals in a population, the common haplotype of the population can be determined. For the tested individuals, the genotype will be experimentally elucidated and compared with the known haplotypes of the population to infer the most likely haplotype of the individual.

[0041] However, the accuracy of such computational methods is affected by the ethnicity and ancestral background of the tested individuals. In areas of mixed ethnicity, computational haplotyping methods may not provide sufficient accuracy for clinical purposes. Furthermore, the accuracy of the technique will decrease when determining haplotypes over long distances and in regions with low linkage disequilibrium. For example, recombination will occur frequently in genomic regions encoding human leukocyte antigens (HLA). Thus, computational inference of haplotypes in these regions with high recombination will be less accurate.

[0042] B. Experimental Analysis Alternatively, the haplotype of an individual can be determined experimentally. The principle of these experimental haplotyping methods is based on the genotyping of different regions of a single long DNA molecule. Three common approaches to achieve this goal include (a) structurally proximal region bridging, (b) compartmentalization, and (c) long-read sequencing.

[0043] 1. Crosslinking Cross-linking takes advantage of the fact that chromosomes fold into a three-dimensional (3-D) structure, making it highly likely that different parts of the same chromosome will be in close proximity. By cross-linking DNA that is close in 3D space, it is possible to connect DNA regions that originate from the same chromosome but are up to several kilobases apart. The sequences of these hybrid molecules can be used to phase single nucleotide polymorphism (SNP) alleles. Examples of this technique include the Hi-C technique (Selvaraj et al. Nat. Biotechnol 2013;31:1111-8) and the modified Hi-C technique "Chicago" (Putnam et al. Genome Res. 2016;26:342-50).

[0044] 2. Compartmentalization Compartmentalization techniques can utilize automated platforms for compartmentalization. The principle of this technique is to separate individual long DNA molecules obtained from a test subject into different compartments. The DNA of a single compartment will then be genotyped. Since the genotype information is obtained from a single DNA molecule, rather than one pair of two parental chromosomes, the obtained allele information will represent a haplotype. Separation of individual long DNA molecules in compartments can be performed manually (Peters et al. Nature 2012;478:190-5) or using a microfluidic system (Zheng et al. Nat. Biotechnol 2016:34:303-11).

[0045] To improve the cost-effectiveness of this approach, long DNA molecules in a single compartment will be fragmented into smaller fragments. Each short fragment resulting from the same long DNA molecule will be coded with the same index. Short DNA fragments from different compartments can then be pooled together and sequenced using massively parallel sequencing. Fragments with the same index can be used to construct haplotypes (Amini et al. Nat. Genet 2014;46:1343-9). Examples of automated platforms that allow fragment indexing include, but are not limited to, 10x genomics' Chromium system and Illumina's phased sequencing solution.

[0046] However, existing methods of haplotyping based on these techniques are primarily designed for haplotyping relatively large genomic regions, and using these methods to haplotype relatively short genomic regions requires, for example, enrichment of associated regions by hybridization capture, which makes it relatively expensive and labor-intensive.

[0047] 3. Long-read Sequencing In another approach, the whole long DNA molecule can be directly sequenced to determine its sequence. This can be performed by using next-generation sequencing platforms, such as, but not limited to, single-molecule real-time sequencing technology (by Pacific Biosystems) and nanopore sequencing technology (by Oxford Nanopore Technologies). However, this system also requires pre-concentration of the region of interest for sequencing.

[0048] II. Compartmentalization using PCR DNA molecules can be compartmentalized into emulsion droplets. Each emulsion droplet can contain a DNA molecule with two regions that can be linked together via amplification. Linking the two regions together allows for more efficient sequencing and haplotyping. Four-primer and three-primer systems have been used for amplification.

[0049] A.4 Primer system Many PCR-based methods have been described to haplotype specific regions. For example, Wetmur et al. developed a coupled emulsion PCR method (Wetmur et al. Nucleic acids research 2005;33:2615-9), in which diluted DNA templates are distributed into emulsion droplets that act as compartments.

[0050] 1A-1D show a four-primer PCR system used to amplify two different target regions A and B. FIG. 1A shows region A and region B as the two targets that require phasing. Forward primer F1 and reverse primer R1 are for amplifying region A. Forward primer F2 and reverse primer R2 are for amplifying region B. The dotted line 105 represents the genomic intermediate region between region A and region B. The two regions may be several kilobases apart and are not drawn to scale.

[0051] In Figure IB, forward primer F2 and reverse primer R1 are partially complementary. In this example, there are 32 complementary nucleotides between F2 and R1, with 10 nt of each primer specific to its respective region. The complementary regions are shown as overlap regions (OLR) 110. Biotin molecules 115 are attached to the 5' ends of F2 and R1, allowing for removal of the unbound strand, e.g., as described below.

[0052] In FIG. 1C, the 3' ends of the PCR amplification products of region A and region B contain OLR110 such that they are complementary to each other. Thus, the 3' end of the amplification product of region A can be used as a primer for the amplification product of region B, and vice versa. This will lead to the joining of the two amplified regions. The joined DNA strands can then be extended to the right on the top strand and to the left on the bottom strand, thereby generating a relatively short molecule containing regions A and B separated.

[0053] Thus, as shown in Figure 1B, there is a 32 nucleotide overlap between the reverse primer of amplicon A and the forward primer of amplicon B. This overlap allows the joining of the two amplicons. When the concentration of DNA template is low and only one DNA template is present in each emulsion droplet, the joined PCR products represent the haplotype of an individual.

[0054] However, in this system, the binding of the two amplicons is not complete (i.e., not all are bound), and there will be unbound DNA molecules from each region. For example, the single-stranded DNA molecules generated by the extension of R1 or F2 only cover region A or region B, respectively. These unbound DNA molecules will affect the subsequent haplotyping analysis. Therefore, these unbound DNA molecules need to be removed by binding of biotinylated PCR primers and capping the unextended single-stranded DNA.

[0055] In Figure ID, unbound PCR products will include biotin-conjugated primers R1 and F2, which can be removed by treatment with strepavidine beads.

[0056] B.3 Primer System Another method described by Tyson et al. combined a three-primer PCR amplification system with emulsion droplet partitioning to haplotype structurally complex regions (Tyson et al. BMC Genomics 2012;13:693). Similar to the method described by Wetmur et al., the PCR system was set up in an emulsion with a very low concentration of target DNA, such that each emulsion droplet contained a single long target DNA molecule. In contrast to the method described by Wetmur et al., the method developed by Tyson et al. used only three primers.

[0057] 2A-2C show a three-primer PCR system used to amplify two different target regions, A and B.

[0058] In FIG. 2A, forward primer F3 and reverse primer R3 are used to amplify region A.

[0059] In Figure 2B, reverse primer R3 consists of two parts: the 3' end 205 contains nucleotides complementary to the sequence of region A, and can therefore function as a primer for amplifying region A. The 5' end 210 of R3 is complementary to the B' region of region B.

[0060] In Figure 2C, after PCR amplification of region A using F3 and R3, the amplification product will contain a 3' end 215 that is complementary to the B' region. This amplification product can serve as a primer to amplify region B with reverse primer R4. As a result, regions A and B can be joined together. Each joined PCR product consists of regions A and B of a single DNA molecule, representing a haplotype.

[0061] Based on the method described by Tyson et al., allele-specific PCR was used as a second round of amplification for the selection of binding molecules with a specific allele in region A. Selection of binding molecules can be performed by amplifying using a forward primer specific for a specific allele in region A and a reverse primer at the end of region B. In this way, only one of the haplotypes is amplified, and the amplified haplotype corresponds to a specific allele in region A. For selected molecules, the molecules can be sequenced using Sanger sequencing to determine the sequence of region B, and the alleles in region A and region B can be phased.

[0062] C.Problem These PCR methods use manual generation of droplet emulsions to distribute long template DNA molecules in very dilute concentrations into individual droplets. The emulsions are typically formed by shaking a mixture of oil and aqueous reagents used in PCR reactions. However, distribution of DNA molecules into individual droplets using this manual method presents numerous problems.

[0063] First, the volume of the droplet cannot be precisely controlled. As a result, there is a relatively high probability that two or more DNA molecules will be split into one large droplet. In such a situation, the phases of alleles at different loci will be determined incorrectly. Furthermore, PCR reactions performed on different droplets do not have uniform efficiency. In these previous methods using emulsion PCR analysis, allele-specific PCR was then used to amplify the combined products to amplify DNA molecules bearing one of the two alleles at a first locus (e.g., region A). Sanger sequencing was then performed to determine the allele at a second locus (e.g., region B) that is combined with a particular allele at the first locus.

[0064] III. Fusion PCR method This patent application describes robust haplotyping methods that utilize microfluidic systems to bind multiple targets on a single DNA molecule and allow the bound molecule to be sequenced by massively parallel sequencing. These methods may be particularly useful for haplotyping regions containing variants that cannot be genotyped by simple allele discrimination techniques, such as allele-specific PCR or allele-specific fluorescent probes. Some embodiments may utilize microfluidic digital PCR systems to generate small droplets, most of which contain one or less long DNA molecules that cover the region of interest. PCR amplification can be performed for two or more regions of interest from one long DNA molecule using highly efficient polymerases, such as but not limited to Taq polymerase, and two sets of primers. Using such polymerases can significantly increase throughput by reducing the time to generate amplification products.

[0065] There are existing commercially available devices that can automatically generate droplets of several thousand nanoliter size in a short time, such as, but not limited to, BioRad QX200 Droplet Generator, Elveflow Droplet Generator Pack, and Micronit Microfluidic Droplet Generator. Other methods for generating individual compartments for PCR reactions can also be used, but are not limited to microfluidic systems. Examples of such systems include the Fluidigm BioMark system and the system provided by Microfluidic Chipshop.

[0066] While the use of such polymerases can improve efficiency and uniformity of droplets, such polymerases can cause problems by adding an additional [A] to the 3' end of the extension sequence. Such additions can cause OLR110 and end 210 to be non-complementary. Embodiments can address such issues while still allowing the use of highly efficient polymerases.

[0067] A. Addition of A to the reverse primer: sequence content of specific genomic locations with regions A, BA, and T The region to be joined can be selected to take advantage of the extra A at the 3' end added by the polymerase, or the region with a T at the 5' end can be selected to complement the extra A added by the polymerase.

[0068] 3A-3F show fusion PCR methods using respective genomic positions with T and A according to embodiments of the present disclosure.

[0069] In FIG. 3A, only the Watson strand of the long DNA molecule is shown. Forward primer F5 and reverse primer R5 are used to amplify region A. Forward primer F6 and reverse primer R6 are used to amplify region B. The 3' end 307 of region A is designed to be a thymine. Region B is specifically designed such that the nucleotide downstream (3') of the 3' end of the F6 primer is an adenosine 308. The dotted line between regions A and B represents an intermediate region 305 between regions A and B, which is a long piece of DNA, for example in the range of 100 bp to 100 kbp.

[0070] In Figure 3B, for reverse primer R5, an additional sequence 310 complementary to F6 is placed at its 5' end. This region of the primer is referred to as the overlap region (OLR). The 3' end of R5 will be used to perform PCR amplification of region A. Adenosine 309 separates OLR 310 from the priming sequence 312 of region A. Adenosine 309 is complementary to thymine 307 of region A.

[0071] In FIG. 3C, after PCR amplification, an additional adenosine [A] (e.g., 319 and 329) will be added by the polymerase to the 3' end of each of the four extended strands (i.e., strands W, X, Y, and Z). Strands X and Y are the two strands of the PCR amplicon of region A, and strands W and Z are the two strands of the PCR amplicon of region B. The additional adenosine 319 at the 3' end of strand X improves the efficiency and improves the specificity of the subsequent joining of the PCR amplicons of region A and region B. Furthermore, most of the existing commercially available microfluidic systems use polymerases that add an additional [A] at the 3' end of the extended sequence.

[0072] Meanwhile, because there is an additional adenosine in each amplified strand of region A, primer F6 is specially designed such that the nucleotide downstream of the 3' end (3') of the F6 primer must be adenosine 308 in Figures 3A and 3C. Similarly, the binding site on region A for R5 to anneal must be thymine [T] (307 as shown in Figure 3A).

[0073] In Figure 3D, strand X and strand Z anneal to each other at the OLR. Thymine [T] 307 at the 5' end of the annealing site of R5 in region A binds to adenosine [A] 329 at the 3' end of strand Z. Thymine [T] 331, created by adenosine 308 adjacent to the 3' end of F6, binds to adenosine [A] 319 at the 3' end of strand X. These two AT binding sites increase the efficiency and specificity of the binding of strand X and strand Z.

[0074] In Figure 3E, after priming, strands X and Z can join together with polymerase extension, i.e., the top strand can be extended to the right (3' direction) and the bottom strand can be extended to the left (5' direction) using polymerase.

[0075] In FIG. 3F, in the reaction compartment, the concentration of R5 and F6 may be lower than that of F5 and R6, for example, after binding, the extended amplicon is amplified. Examples of the ratio between these primer groups (R5&F6 vs. F5&R6) include, but are not limited to, 1:100000, 1:50000, 1:10000, 1:5000, 1:1000, 1:500, 1:200, 1:150, 1:100, 1:80, 1:60, 1:40, 1:30, 1:20, 1:10 and 1:5. The reason for the lower concentration of R5 and F6 is likely because they are exhausted after forming sufficient strands X and Z, and there is no excess amount of strands Y and W. The separated strands Y and W do not carry haplotype information with respect to regions A and B. Furthermore, strands Y and W may interact or fuse with the amplification products of region A and region B from another compartment of the downstream process. This may affect the accuracy of the haplotyping method. Higher concentrations of F5 and R6 allow to perform the final PCR amplification of the sequence bounded by these two primers. In other embodiments, the extension amplicon may be amplified using a separate amplification procedure, or may not be amplified at all, for example, when analyzing the extension amplicon using single molecule sequencing.

[0076] After the extended amplicons are generated, they can be analyzed using, for example, allele-specific PCR or sequencing.

[0077] In FIG. 3A, the 3' end 307 of region A is designed to be a thymine, and the complementary primer portion of R5 contains A309 at the end. This particular arrangement of adenosine [A] 309 may be useful for fusing region A and region B. In PCR amplification of region B using F6 and R6, an additional adenosine [A] 329 was added to the 3' end of strand Z (FIG. 3C). OLR 310 shares the same sequence as OLR on strand Z (FIG. 3D). To generate a fusion product containing region A and region B, the sequences of the 3' ends of strand X and strand Z need to be completely complementary (FIG. 3D). The design of adenosine [A] 309 in R5 is to ensure that the additional adenosine [A] 329 added to the 3' end of strand Z is complementary to thymine [T] 307 of strand X. Similarly, an additional adenosine [A] 319 is added by the polymerase to the 3' end of strand X. The adenosine [A] 308 on strand W is to ensure that adenosine [A] 319 is complementary to thymine [T] 331 on strand Z (Figure 3D). This arrangement allows for more complete amplification and more efficiently yields extended amplicons that cover both regions.

[0078] B. Addition of A in the reverse primer: non-specific genomic location As described above in Figure 3A, the 3' end 307 of region A is designed to be a thymine, and the complementary primer portion of R5 includes A309 at the end. However, in some embodiments, the complementary primer portion can end before the A, with A being an additional base (third portion) between the complementary portion and the OLR portion. In this way, the end of region A does not need to be a T. Instead, an additional A can allow a T to be added during amplification.

[0079] 4A-4C show a fusion PCR method using a reverse primer with an added A according to an embodiment of the present disclosure. FIG. 4A shows a DNA molecule having a region A and a region B. In contrast to region A in FIG. 3A, region A does not have to be terminated with thymine [T] at its 3' end. Thus, in this alternative design of FIG. 4A, the annealing site at the 5' end of R5 does not have to be thymine [T].

[0080] 4B shows the reverse primer R5. An additional adenosine [A] 409 can be placed between the priming sequence 403 for region A and the OLR 410.

[0081] Figure 4C shows the PCR amplicon formed by the primers. An additional [A] 409 can generate [T] 413 on strand X to bind to [A] 415 at the 3' end of strand Z. [A] 415 is an additional adenosine that is added to the 3' end of the amplicon by the polymerase.

[0082] The additional adenosine [A] in the reverse primer allows the region to be designed to terminate at any nucleotide at the 3' end. The region can be designed based on factors other than terminating at a thymine [T]. For example, the region can be designed for a particular length or to cover a particular SNP, without regard to whether a thymine is present at the end of the region.

[0083] C. Add A to the reverse and forward primers In the scenario of Figure 4A, region B still has a need for A404 after the OLR region. In some embodiments, an additional A can be added to both the reverse primer R5 and the forward primer F6.

[0084] 5A-5D show a fusion PCR method using a reverse primer and a forward primer with an added A according to an embodiment of the present disclosure. In another alternative design, the 5' ends of R5 and F6 can be designed to be artificial sequences.

[0085] In Figure 5B, the two artificial sequences are complementary to each other. An additional adenosine [A] 509 can be placed between the priming sequence 503 and the OLR 510, where the A is still part of the artificial sequence but is not part of the OLR.

[0086] In Figure 5C, adenosine [A] 509 on R5 generates [T] 513 on strand X, located between the OLR and the PCR amplicon. This [T] 513 binds to [A] 523 at the 3' end of strand Z. Adenosine 559 on F6 generates [T] 563 between the OLR on strand Z and the PCR amplicon. This [T] binds to [A] at the 3' end of strand X.

[0087] In this example, the OLR of the reverse primer is complementary to the OLR of the forward primer, and there are no sequence requirements for the region since the OLR is fully processed by the primer.

[0088] In Figure 5D, the binding of strand X and strand Z in the OLR is shown. Each strand can be extended to form a double-stranded molecule with both strands of region A and both strands of region B.

[0089] D. Primers for amplifying the fusion PCR product Specific primers can be used to promote the amplification of fusion PCR products with two regions rather than the amplification products that contain only a single region.Specific primers can amplify fusion PCR products without amplifying the regions that are not yet amplified themselves.These primers can be included at high concentrations compared to other primers to form large amounts of fusion PCR products.

[0090] 1. A forward primer with no separate overlap region Figure 6 shows the principle of fusion and adapter ligation PCR method using primers to promote the amplification of fusion PCR products. A DNA template and six primers (F1, R1, F2, R2, F3, and R3) were added in the reaction compartment. Primers F1, R1, F2, and R2 can be similar to primers F5, R5, F6, and R6 in Figures 3A-3F, respectively. Region 1 and Region 2 are two targets that are targets for haplotype phasing analysis. Region 1 can be similar to Region A in Figure 3, and Region 2 can be similar to Region B in Figure 3.

[0091] The dotted line represents the genomic region between Region 1 and Region 2. The two regions are several kilobases apart and the length of the dotted line is not drawn to scale. F1 and R1 are primers for amplifying Region 1. F2 and R2 are primers used to amplify Region 2. The 5' end of R1 contains an overlap region (OLR) that is complementary to F2. This overlap allows for the joining of Region 1 and Region 2 (as shown in Figures 3E and 3F).

[0092] One important difference in Figure 6 compared to Figures 3A-3F and 4A-4C is the addition of an additional pair of primers F3 and R3. This primer pair is used to amplify a fusion PCR product that includes both Region 1 and Region 2. The inclusion of this primer pair (F3 and R3) is particularly useful when fusing more than two regions. Its use for fusing more than two regions is described in more detail below. The 5' end 604 of R1 is complementary to F2, so that a PCR product of Region 1 using F1 and R1 can be fused to a PCR product of Region 2. The 5' ends of F1 and R2 (608 and 612) are designed to be the same sequence as the 3' ends of F3 and R3 (616 and 620), respectively. The overlap region between F1 and F3 is designated as Sequencing Adapter Region 1 (SAR1), and the overlap region of R2 and R3 is designated as Sequencing Adapter Region 2 (SAR2). As a result of SAR1 and SAR2, F3 and R3 can be used to amplify the fusion product of region 1 and region 2.

[0093] The extension amplicon formed by F1, R1, F2, and R2 may resemble the strand in FIG. 3F. However, since F1 contains SAR1 and R2 contains SAR2, the double strand of DNA at one end of the extension amplicon will have a sequence complementary to SAR1 and SAR1. The double strand of DNA at the other end will have a sequence complementary to SAR2 and SAR2. F3 and R3 primers can amplify this extension amplicon. The amplified extension amplicon then contains sections 624 of F3 and 628 of R3 designed for sequencing, and their complements, such as adapters attached to a flow cell. Thus, the amplified extension amplicon can be further amplified using F3 and R3 primers, which can then be perfectly complementary to the start or end sequence of the amplified extension amplicon.

[0094] In order to reduce the interference of non-fusion PCR products of region 1 and region 2, the concentration of F3 and R3 can be set higher than that of other primers F1, F2, R1, and R2. Examples of ratios of the concentration of F3 and / or R3 to the concentration of any one or more of F1, F2, R1, and R2 include, but are not limited to, 5:1, 10:1, 20:1, 50:1, 75:1, 100:1, 200:1, 500:1, 1000:1, 2000:1, 5000:1, 10000:1, 20000:1, 50000:1, or 100000:1 or more. The lower the concentration of F1, R1, F2, and R2, the more likely they will be exhausted after forming fusion products, and there will not be a large amount of non-fusion products. The 5' end of F3 and R3 is designed to facilitate sequencing of the fusion products. The unfused product may fuse with an unfused product from another compartment in downstream analytical processes, affecting the accuracy of the haplotyping analysis.

[0095] In embodiments, adapter sequences for massively parallel sequencing platforms, e.g., Illumina sequencing adapter sequences, can be placed at the 5' end, 624 in F3 and 628 in R3, to allow sequencing of the fusion product. In some embodiments, a sequence indicating the identity of the sample, i.e., a sample index, can be included so that samples from different samples or experiments can be pooled for sequencing or other subsequent analysis. After sequencing, the sequenced reads can be attributed to their respective original samples based on the sample index sequencing. These adapter sequences or sample indexes are examples of the portions of F3 and R3 labeled as "designed for sequencing" in FIG. 6.

[0096] 2. Forward primers with separate overlap regions FIG. 7A shows another example of using primers to facilitate amplification of a fusion PCR product. The 5' ends 704 and 708 of R1 and F2, respectively, can be engineered to be artificial sequences. The two artificial sequences are complementary to each other, similar to R5 and F6 in FIG. 5. As shown in FIG. 6, primers F3 and R3 can be added to facilitate amplification of a fusion PCR product. The 5' ends 712 and 716 of F1 and R2 are designed to be the same sequence as the 3' ends 720 and 724 of F3 and R3, respectively. The overlap region between F1 and F3 is shown as Sequencing Adapter Region 1 (SAR1), and the overlap region of R2 and R3 is shown as Sequencing Adapter Region 2 (SAR2). F3 and R3 can be used to amplify the fusion product of Region 1 and Region 2. In order to reduce the interference of non-fusion PCR products of region 1 and region 2, the concentration of F3 and R3 is set higher than that of other primers F1, F2, R1, and R2. Examples of the ratio of the concentration of F3 and R3 to the concentration of F1, F2, R1, and R2 include, but are not limited to, 5:1, 10:1, 20:1, 50:1, 75:1, 100:1, 200:1, 500:1, 1000:1, 2000:1, 5000:1, 10000:1, 20000:1, 50000:1, or 100000:1 or more. The lower the concentration of F1, R1, F2, and R2, the more likely they are used up after forming fusion products, and there will not be a large amount of non-fusion products.

[0097] Figure 7B shows the fusion PCR product, which contains the sequence of the OLR between the two target regions.

[0098] IV.2 Combination of domains beyond In some embodiments, multiple regions can be linked together in an extended amplicon. Linking multiple regions allows for more regions to be sequenced than if the regions were not linked together. The 5' end of the reverse primer of the first region can be designed to be complementary to a portion of the forward primer of the downstream region (3'). The pattern of reverse primer and forward primer can continue to additional regions downstream of the first region.

[0099] A. Specific regions that bind to each other Figures 8A-8B show fusion PCR methods for linking more than two regions, where only specific regions link to each other, according to an embodiment of the present disclosure. In Figures 8A and 8B, the OLR of the reverse primer of one region is specifically complementary to the end of another region, similar to Figures 4A-4C, where the OLR is complementary to the next forward priming sequence. Thus, using the various strategies described above, the OLR can be designed to link more than two regions to each other.

[0100] In FIG. 8A, the OLR at the 5' end of the reverse primer is complementary to the forward primer of the next region. For example, OLR804 of added R1 is complementary to F2, OLR808 of added R2 is complementary to F3, etc. The resulting extended amplicon will contain regions 1-3 in that order, or will contain an extended amplicon of only regions 1 and 2 and regions 2 and 3. By using higher concentrations of primers F1 and R3, the fully extended amplicon can be favored. The outermost primers can be used at similar concentrations to just the two regions. This design allows multiple regions to be amplified from a single long piece of DNA and joined together. The phase of the alleles in the different regions can be determined using downstream analysis, such as, but not limited to, massively parallel sequencing.

[0101] In Figure 8B, both the reverse and forward primers contain an additional OLR in addition to the priming sequence. The sequence of the OLR is a specific binding of the region, for example, the sequence of the OLR between R1 and F2 (812 and 816) is different from the sequence of the OLR between R2 and F3. The resulting extended amplicon contains regions 1-3 in that order, as in Figure 8A, but the technique of Figures 5A-5D is used. Thus, in this alternative design, the OLR is engineered in the reverse and forward primers. The OLR of the reverse primer of the previous region is complementary to the OLR of the forward primer of the next region.

[0102] B. Any region that can be bonded to each other In some embodiments, the OLR region can be the same for all regions. In this way, the combination does not need to be continuous, since there may be pairwise combinations of regions. The paired extension amplicons can be analyzed (e.g., sequenced) to provide haplotype information. For example, the pair information of regions 1 and 2 can provide the haplotype information of 1, 2, and 3, together with the pair information of 2 and 3 (or 1 and 3). Thus, a pair can be sufficient. This allows essentially random combination between any two pairs of regions.

[0103] Figures 9A-9B show fusion PCR methods for combining more than two regions, where any region can be combined with each other, according to an embodiment of the present disclosure. In another variation, the OLRs located in the reverse primer and forward primer can be inclusive. Figure 9A shows a molecule with three regions (regions 1, 2, and 3) and the associated forward and reverse primers.

[0104] Figure 9B shows the details of the primers. For example, OLR904 located at the 5' end of the reverse primer R1 for region 1 is complementary to the forward primer located at the 5' end of the forward primer for regions 2 and 3 (as well as the forward primers for regions 4, 5, 6, etc., if present). Thus, all primers have an OLR. That is, any F and R primers are complementary to each other. In this design, the alleles of region 1 can be phased with other regions (regions 2, 3, 4, etc.). The phases of all regions can then be derived.

[0105] As an example, suppose there are three regions with the SNP A / T. If A in region 1 is determined to be linked to A in region 2, then the two As are on the same haplotype. And if A in region 1 is linked to A in region 3, then the haplotype across all regions can be determined to be AAA.

[0106] C. Primers for amplifying fusion PCR products with multiple regions Forward and reverse primers designed to amplify the fusion product can be used in multiple regions. Forward and reverse primers can be designed similarly to the primers for joining the two regions in Figures 6, 7A, and 7B.

[0107] FIG. 10A shows how primers can be used to amplify fusion PCR products with multiple regions. Using the various strategies described above, OLRs can be designed to link three or more regions together. The OLR at the 5' end of the reverse primer is complementary to the forward primer of the next region. This design allows multiple regions to be amplified and linked together from a single long piece of DNA. This method is similar to the method described in FIGS. 8A-8B, except for the addition of a pair of primers (F4 and R4) whose 3' ends have the same sequence as the 5' ends of F1 and R3, respectively. The addition of this primer pair is to amplify a fully fused product that contains all the regions that are fused. Preferential amplification of the fully fused product can be achieved by a higher concentration of F4 and R4 compared to the other primers, including F1, F2, F3, R1, R2, and R3. Only fully fused products that link all three regions can be amplified with F4 and R4. Partially fused products that only join two regions, or non-fused PCR products of any of the three regions cannot be amplified by F4 and R4. Because of the high concentration of F4 and R4, the amount of fully fused products may be much greater than non-fused PCR products and partially fused products. Examples of ratios of concentrations of F4 and R3 to those of F1, F2, R1, and R2 include, but are not limited to, 5:1, 10:1, 20:1, 50:1, 75:1, 100:1, 200:1, 500:1, 1000:1, 2000:1, 5000:1, 10000:1, 20000:1, 50000:1, or 100000:1 or more.

[0108] Figure 10B shows the amplification of a fusion PCR product with three regions by a forward primer with an OLR where the portion is separated from the portion complementary to the region, as opposed to a forward primer with an OLR equal to the portion complementary to the region. The fusion of PCR products is similar to the scheme shown in Figure 7A. In Figure 10B, the use of higher concentrations of F4 and R4 compared to F1, F2, F3, R1, R2, and R3 can enrich for fully fused products, similar to the use of F4 and R4 in 10A.

[0109] Example of haplotyping using the V.NUDT15 gene Haplotyping of variants of the NUDT15 gene is used as an example to illustrate this haplotyping method.

[0110] A.NUDT15 The NUDT15 gene encodes the enzyme Nudix hydrolase 15, which metabolizes the thiopurine drug 6-mercaptopurine (6-MP), a major component of maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Many variants in the human NUDT15 gene as important determinants of thiopurine intolerance (Moriyama et al., Nature Genetics, 2016;48:367-373). Variants located in exon 1 and exon 3 of the NUDT15 gene cause loss of function of the Nudix hydrolase 15 enzyme, which can lead to severe immunosuppression when these patients are prescribed regular doses of 6-MP. The combination of genotypes in these variants gives rise to many haplotypes.

[0111] FIG. 11 shows six haplotypes containing different combinations of genotypes in exons 1 and 3 of NUDT15.

[0112] For patients with genetic variants in both exons 1 and 3, it is important to determine the phase of the variants in the two loci, as this will affect the metabolic rate of the immunosuppressant and therefore the optimal dose administered to the patient. For example, if a heterozygous p.Val18_Val19insGlyVal variant is detected in exon 1 and a heterozygous p.Arg139Cys variant is detected in exon 3, the two variants can be located on the same or different chromosomes. If the two variants are present on the same chromosome, the patient will have haplotypes *1 and *2. The patient will still be able to produce the functional enzyme Nudix hydrolase 15, since they have a normal copy of the NUDT15 gene. The dose of the immunosuppressant will be adjusted to a moderate dose, as if there was only one variant.

[0113] On the other hand, if the two variants are present on two different chromosomes, the patient will have haplotypes *3 and *6. In this situation, the patient will not be able to produce functional Nudix hydrolase 15 and will need to significantly reduce the dose of immunosuppressants. In a meta-analysis by Yin et al (Yin et al., Oncotarget, 2017;8:13575-85), patients with one or two copies of the NUDT15 risk allele required a 23% and 65% reduction in the average daily thiopurine dose, respectively, compared to patients with wild-type NUDT15.

[0114] Haplotyping of NUDT15 variants is challenging for two reasons. First, the distance between the mutation hotspots in exon1 and exon3 is approximately 8 kb. As the size of the amplicon increases, the efficiency and robustness of PCR decreases, limiting long-range PCR approaches that would lead to artificial recombination. Another problem is that V18V19insGV is a 6 bp tandem repeat variant. Therefore, haplotype phasing methods that rely on allele discrimination by primers or probes, such as multiplex duplex PCR, cannot be used (Regan et al., PLoS One, 2015;10:e0118270). Technologies that provide long-range information with short-read sequencing (i.e., 10X Genomics) or direct sequencing of single molecules at long ranges (i.e., PacBio) are relatively costly and have low throughput. Therefore, it is a suitable candidate locus to demonstrate the power of this new haplotyping method.

[0115] Sixty-three children with ALL were recruited from the Department of Chemical Pathology and Pediatrics at the Prince of Wales Hospital (PWH), Hong Kong. These patients were initially referred for TPMT genotyping after myelosuppression occurred during thiopurine treatment. NUDT15 genotyping was performed for all patients by Sanger sequencing. The parents or grandparents of four patients who were heterozygous for both the V18V19insGV and R139C variants were also genotyped to determine the true haplotype of each patient. The phase of the two variants in these patients was determined by family analysis. In addition, 506 healthy subjects were also analyzed for potential mutations at the two NUDT15 loci.

[0116] B. Genotyping of the NUDT15 gene by high-resolution melting curve analysis As these subjects would benefit from haplotyping analysis to select the appropriate dose of thiopurine treatment, we first identified subjects who were heterozygous at both loci of interest before proceeding with the haplotype analysis. Two high-resolution melting curve analysis (HRM) assays were designed. One assay targeted the mutation hotspot in exon1 of the NUDT15 gene, including the V18V19insGV and V18I variants. Another assay amplifies a fragment covering the R139C and R139H variants in exon3. The primer sequences are listed in Table 1.

[0117] [Table 1]

[0118] PCR reactions were performed in duplicate for each sample. A 20 μL reaction mix was prepared containing 10 μL of 2× LightCycler® 480 High Resolution Melting Master, both forward and reverse primers at a final concentration of 300 nmol / L (exon1 assay) or 250 nmol / L (exon3 assay), MgCl2 at a final concentration of 3.5 mmol / L (exon1 assay) or 2.5 mmol / L (exon3 assay), and 20 ng of DNA. For the exon1 assay, 10% dimethyl sulfoxide was added to the PCR reaction to improve amplification.

[0119] PCR reactions were performed using a LightCycler® 96 Instrument. The PCR profile consisted of a preincubation at 95°C for 10 min, followed by an amplification program. For the exon1 assay, the amplification program consisted of 50 cycles of 95°C for 15 s, 59°C for 20 s with a ramp rate of 1.5°C / s, and 72°C for 20 s. For the exon3 assay, the amplification program consisted of 95°C for 10 s, 59°C for 15 s with a ramp rate of 2.2°C / s, and 72°C for 15 s. After PCR amplification, melting cycles were performed according to the protocol: 95°C for 1 min, 40°C for 1 min, followed by continuous fluorescence collection while heating from 60°C to 90°C at a rate of 0.04°C / s. High-resolution melting curve analysis data were analyzed using the LightCycler® 96 application software. A wild-type sample was run on each plate as a negative control.

[0120] For 73 ALL-related subjects (i.e., 63 ALL patients and 10 family members), the mutation status in exon1 and exon3 of the NUDT15 gene was identified by Sanger sequencing. Sixteen of these subjects had mutations in exon1, of which 15 were heterozygous V18V19insGV mutants and one was V18I mutant. For exon3, 48 subjects were homozygous for the wild-type sequence, 21 subjects were heterozygous for R139C and wild-type, and 4 subjects were homozygous for R139C. High-resolution melting curve analysis was performed on the genotypes of exon1 and exon3 of the NUDT15 gene for all subjects.

[0121] Figures 12A and 12B show melting curves distinguishing samples with different NUDT15 genotypes in the exon1 (A) and exon3 (B) regions. In Figure 12A, HRM analysis targeting exon1 of the NUDT15 gene distinguished the heterozygous V18V19insGV and heterozygous V18I mutants from the wild-type sequence based on the shape of the normalized melting peaks. In Figure 12B, HRM analysis distinguished two genotypes in exon3 of the NUDT15 gene (i.e., heterozygous R139C and homozygous R139C) from the wild-type sequence.

[0122] Droplet digital haplotype fusion PCR analysis of C.NUDT15 gene To phase the variants at the two NUDT15 loci, we developed a droplet digital PCR-based fusion PCR haplotyping method. High molecular weight DNA extracted from buffy coats was distributed into nanoliter droplets and fusion PCR was performed using a BioRad Droplet Digital PCR system. The concentration of DNA template was controlled to fill only about 10% of the droplets, and more than 99% of the droplets contained only one or no template DNA covering the two regions of interest. Fusion PCR was performed to amplify the two regions harboring the variants from a single long DNA molecule and fused together.

[0123] Figures 13A-13F show the application of fusion PCR to exons 1 and 3 of the NUDT15 gene according to an embodiment of the present disclosure. Figures 13A-13F mimic 3A-3F, although other method techniques can be used, such as Figures 4A-4C and 5A-5D.

[0124] In Figure 13A, the fusion PCR assay used for haplotyping the NUDT15 gene contains two pairs of primers, one pair (F1 and R1) amplifies the mutation region on exon 1, and the other pair (F2 and R2) amplifies the mutation region on exon 3.

[0125] In FIG. 13B, the reverse primer for exon 1 (R1) has a linker sequence at its 5′ end that is complementary to the sequence of the forward primer for exon 3 (F2).

[0126] In Figure 13C, in the first round of PCR reactions, the two regions are amplified separately. The two PCRs were run simultaneously in a single droplet. As explained before, an additional adenosine [A] is added by Taq polymerase at the 3' end of the growing strand of DNA.

[0127] In FIG. 13D, an OLR designed at the 5′ end of R1 makes the 3′ end of strand X complementary to the 3′ end of strand Z.

[0128] In FIG. 13E, the two strands can be ligated to one another, following extension with Taq polymerase or other polymerase.

[0129] In Figure 13F, because the concentrations of F1 and R2 were higher than those of R1 and F2, further PCR amplification will be performed on the fusion product using primers F1 and R2. The size of the fusion PCR product should be 190 bp for haplotypes carrying the p.V18V19insGV mutation, or 184 bp otherwise.

[0130] [Table 2]

[0131] PCR reactions were prepared in quadruplicate for each sample in a volume of 20 μL, consisting of 2.5 ng template DNA, 10 μL of 2× ddPCR Supermix for Probes (No dUTP) (Bio-Rad), both F1 and R2 at a final concentration of 1 μmol / L, and both R1 and F2 at a final concentration of 30 nmol / L. The PCR reaction mix was then split using a QX100 / QX200 Droplet Digital PCR Generator (Bio-Rad). The thermal profile of the assay was initiated at 95°C for 10 min, followed by 45 cycles of 94°C for 30 s, 61°C for 1 min, and a final incubation at 98°C for 10 min.

[0132] After the PCR reaction, droplets from duplicate wells from one sample were pooled and PCR products were recovered according to the manufacturer's protocol with the minor adjustment of using 40 μL of TE buffer per well instead of 20 μL, which improved droplet disruption.

[0133] To enrich for the desired fusion PCR products of 184 or 190 bp in length, the recovered PCR products were further purified using AMPure XP beads according to the manufacturer's dual bead-based size selection protocol. The bead / DNA ratios (i.e., the ratio of the cumulative volume of added beads to the volume of the original sample) for the first and second bead selections were 0.8 and 1.6, respectively. The size profiles of the purified fusion PCR products were then analyzed using Reagents on a D1000SreenTape and 4200TapeStation instrument (Agilent Technologies).

[0134] D. Sequencing of NUDT15 gene fusion PCR products Fusion PCR products were analyzed by next-generation sequencing, although other measurement techniques can be used if suitable for sequencing of the joined regions. Indexed libraries were constructed for the fusion PCR products from each sample using the KAPA Library Preparation Kit (Kapa Biosystems) according to the manufacturer's recommendations. Up to 48 indexed libraries were multiplexed and sequenced using the Illumina MiSeq sequencing platform (76x2 cycles). Six possible sequences of the fusion PCR products corresponding to the six haplotypes were used as reference sequences for mapping the sequencing data (sequence reads). Paired-end reads were aligned to the six reference sequences using the Bowtie2 program (http: / / bowtie-bio.sourceforge.net / bowtie2 / ). Paired-end reads that aligned at both ends to the reference sequences without mismatches were used for further analysis.

[0135] The reads mapped to each haplotype sequence in the sample were counted using a Perl script. The percentage of reads mapped to each haplotype in the sample (denoted as Hap%) was then calculated by dividing the number of reads mapped to each haplotype by the total number of reads mapped to all haplotypes. Hap%<10%, 40%-60%, and >90% were set as cutoffs for carrying 0, 1, and 2 copies of a haplotype in the sample, respectively. In other embodiments, other percentages can be used for the cutoff for carrying 0 haplotypes in the sample, for example, but not limited to, 1%, 2%, 5%, 15%, 20%. Similarly, examples of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% can be used as cutoffs for carrying 1 haplotype. Similarly, examples of 75%, 80%, 85%, 92%, 94%, 98%, 99% can be used as cutoffs for carrying 2 haplotypes.

[0136] Most haplotype (i.e., diplotype) combinations of the 73 subjects could be determined from the results of genotyping by HRM analysis, except for 9 subjects with double heterozygous mutations of V18V19insGV and R139C. Eight of these 9 subjects were identified as *1*2 by family analysis. To directly determine the diplotypes, droplet digital fusion PCR was performed, followed by next-generation sequencing for all subjects. The copy numbers of each of the six potential haplotypes for all subjects were calculated according to the above criteria. The results are summarized in Table 3. The diplotypes of the NUDT15 gene were accurately identified in all subjects.

[0137] [Table 3]

[0138] E. Genotyping and haplotyping of the NUDT15 gene in 506 healthy subjects To evaluate the power and applicability of using HMR and digital fusion PCR haplotyping methods for NUDT15 gene testing, we further analyzed 506 healthy subjects.

[0139] Five hundred and six subjects were initially screened for NUDT15 variants using two HMR assays. The results are listed in Table 4. Six different diplotypes, including *1*1, *1*3, *1*5, *1*6, *2*3, and *3*3, could be determined directly from the genotyping results. In addition, 22 samples harbored double heterozygous variants of V18V19insGV and R139C, and one subject had an unknown genotype in exon 1. For these 23 samples, digital fusion PCR and subsequent sequencing were used to determine their diplotypes.

[0140] All 22 compound heterozygous subjects were determined as *1*2 diplotype from the sequencing results. The subject with unknown exon1 genotype was identified as carrying both V18V19insGV and V18I mutations. Thus, this subject's diplotype is *5*6, which is a previously unknown diplotype. The NUDT15 diplotype frequencies in our cohort are also listed in Table 4. These results are comparable to those determined based on phased data from the 1000 Genomes Project in East Asia (Moriyama et al. Nat Genet. 2016;48:367-373).

[0141] [Table 4]

[0142] VI. Haplotyping by Phase Assembly Analysis Haplotyping may involve determining alleles at more than two heterozygous loci, and therefore more than two regions with SNPs. In such cases, these regions can be assembled into a fusion product of only two or three regions. Haplotypes can then be determined through the analysis of the resulting two or three region products. The products of different regions can be linked together by a common region. This analysis of haplotyping, which uses a common region to link different fusion products, is described in more detail below.

[0143] A. Region grouping Figures 14A, 14B, and 14C show that the four regions are divided into several groups of two regions. In Figure 14A, four regions are shown in this illustrative example. However, the number of regions is not limited and can be equal to any number of regions to be phased. Each of the four regions has a corresponding forward primer and reverse primer, and may have a primer portion and an overlap portion.

[0144] FIG. 14B shows the configuration of the regions and primers for the two groups. For illustration, the four regions are divided into two groups (group A and group B). The number of groups depends on the number of regions to be phased or haplotyped. The length of the DNA can also be determined by downstream analysis, for example, massively parallel sequencing. For example, using the Illumina sequencing platform, paired-end sequencing of 150 bp at both ends can be performed. In this arrangement, for any fusion DNA molecule, a total length of 300 bp is sequenced. Assuming that the amplification product of each region is 75 bp and the junction region is 25 bp, sequencing of 300 bp can reveal the sequence of up to three regions in the fusion molecule. In such an arrangement, up to three groups can be used to group the regions. If 30 regions need to be haplotyped, each group consists of 10 regions. The number of regions in each group may or may not be equal. More groups can be used if downstream analysis can reveal the sequence of a longer DNA molecule.

[0145] FIG. 14B also shows the configuration of primers based on two groups. The primers in each group have the same overlap region that is complementary to the overlap region of the other group. Here, overlap region 1 (OLR1) located at the 5' end of the group A reverse primer is complementary to overlap region 2 (OLR2) located at the 5' end of the group B forward primer. This design of primers is similar to the method described in 7A above. F5 and R5 are a pair of primers that can specifically amplify the fusion product containing the region of group A and the region of group B. The concentration of F5 and R5 may be higher than the concentration of other primers (F1-F4 and R1-R4). With these higher concentrations of F5 and R5, after fusion PCR, the concentration of the fusion product may be much higher than the concentration of the non-fusion PCR product.

[0146] FIG. 14C shows a fusion product containing group A and group B regions. After fusion PCR, any member of group A can be fused to a member of group B. In this example, there are two members in group A (region 1 and region 3) and two members in group B (region 2 and region 4). Thus, there are four types of fusion products: region 1 fused to region 2, region 1 fused to region 4, region 3 fused to region 2, and region 3 fused to region 4. Using this design, the size of the fusion product can be easily controlled. In this example, all fusion products contain only two regions. Examples of three regions are shown below.

[0147] The advantage of fusing smaller groups of regions than the sequentially linked method described in Figure 10A or Figure 10B is the ability to phase more regions. For example, the Illumina sequencing platform is one of the commonly used platforms for sequencing individual molecules. The number of regions that can be phased using the sequentially fused method described in Figure 10A or Figure 10B is limited by the length of the sequenced read of the readout. For example, using paired-end sequencing of 300 nucleotides at each end, molecules of up to 600 nucleotides can be sequenced. If each region to be phased is 50 nucleotides, up to 12 regions can be phased using the sequential fused method.

[0148] However, using the grouping method described here, there is no theoretical upper limit to the number of regions to be phased. In this arrangement, a multiplex PCR can be performed in a single compartment that simultaneously amplifies the target regions (regions to be haplotyped). The amplification products of these regions are then fused with the method described in this application. Methods have been described to improve the number of multiplexes in a single reaction (Wei et al. Journal of Virological Methods 2008;151:132-139; Mo et al. Clin Chem Lab Med;50:649-654). For example, using four groups with 20 regions in each group, a total of 80 regions can be phased. Each fusion product can contain four regions. If each region is 50 nucleotides, the fusion product has 200 nucleotides, which is within the capabilities of the Illumina sequencing platform.

[0149] Haplotyping also includes bioinformatics analysis that assigns different alleles to the same haplotype based on pairwise relationships. Thus, the number of regions to be haplotyped may not be limited by the length of the template DNA used for fusion PCR. The common region between two sets of fusion PCR products can be used to link the haplotypes of other regions of the fusion products to each other. For example, the haplotypes of three regions (e.g., SNP1, SNP2, and SNP3) can be inferred when the phases of SNP1 and SNP2 and the phases of SNP2 and SNP3 are determined. This analysis can be repeated to haplotype multiple regions, as described below.

[0150] Figures 15A, 15B, and 15C show the use of one region from each of the three groups to form a three-region fusion product. Figure 15A shows, as an example, randomly dividing the regions into three groups. However, more than three groups (e.g., 4, 5, 6, 7, or more than 7) can be used in this method. For illustration purposes, the regions are assigned to the three groups in sequence. The order in which the regions are assigned to the groups may not affect the results. In some embodiments, the assignment of the regions to the groups may be random rather than sequential.

[0151] Figure 15B shows the primer configuration based on groups. The OLR located at the 5' end of each reverse primer of one group is complementary to the OLR located at the 5' end of each forward primer of the next group. In this illustrative example, the OLR of the group A reverse primer is complementary to the OLR of the group B forward primer, and the OLR is represented by a dotted line. The OLR of the group B reverse primer is complementary to the OLR of the group C forward primer, and the OLR is represented by a dashed line.

[0152] A pair of outer primers Fz and Rz are used to amplify the complete fusion product. Fz has the same OLR as the 5' end of F1 and F4, and the OLR is shown by a double solid line. Rz has the same OLR as the 5' end of R3 and R6, and the OLR is shown by a double dashed line. The concentration of Fz and Rz can be higher than the concentration of all other primers used to amplify a specific region to facilitate the amplification of the fusion product of the three regions. Examples of the ratio of the concentration of Fz and Rz to the concentration of other outer primers include, but are not limited to, 5:1, 10:1, 20:1, 50:1, 75:1, 100:1, 200:1, 500:1, 1000:1, 2000:1, 5000:1, 10000:1, 20000:1, 50000:1, or 100000:1 or more.

[0153] Figure 15C shows the fusion PCR products. In this design, each complete fusion product contains three regions: a group A region, a group B region, and a group C region.

[0154] B. Fusion PCR using a polymerase without adding an adenosine at the 3' end In an embodiment, fusion PCR can be performed using a polymerase that does not add an adenosine to the 3' end of the extended strand of DNA. Figure 16A shows the design of the primers. The illustrated design is similar to that described in Figure 7A. However, primer R1 in Figure 16A is modified to remove the adenosine adjacent to the priming sequence for region 1 and the OLR. Similarly, primer F2 is modified to remove the adenosine adjacent to the priming sequence for region 2 and the OLR. Primer F3 is modified to remove the thymine adjacent to SAR1 and the sequence located at the 5' end that is designed for sequencing. Primer R3 is modified to remove the thymine adjacent to SAR2 and the sequence located at the 5' end that is designed for sequencing. Figure 16B shows a fusion PCR product with two regions. The methods described in Figures 14A-14C and Figures 15A-15C can be used with this modification in primer design and polymerase use without adding excess adenosine to the 3' end of the extended DNA strand.

[0155] C. Effect of length between regions Regions spaced at various distances from each other are joined together. The fusion regions were then analyzed to determine how length affects the accuracy of haplotype analysis.

[0156] 1.10 SNP composition example Figure 17A shows the lengths between different SNPs in a DNA molecule. These different lengths were used to determine the effect of length on the fusion PCR method. Each fusion PCR aims to fuse two regions; one is SNP1, and the other is one member of SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, or SNP10. The distance between the two regions to be fused ranged from 2.5 kb to 100 kb.

[0157] FIG. 17B shows the configuration of the primers for the SNPs. The two regions were fused using the method described in FIG. 7A and 7B. The 5' end of the reverse primer (primer R1) for amplifying SNP1 contains overlap region 1 (OLR1). The 5' end of the forward primer for amplifying SNP2-SNP10 contains overlap region 2 (OLR2). OLR1 and OLR2 are complementary. This design allows the PCR product of SNP1 to be fused with each PCR product of SNP2-SNP10. However, the ability to correctly phase SNP1 and one of SNP2-SNP10 may depend on whether an intact long DNA molecule covering the two SNPs to be phased is present in a single compartment, which may be, for example, a droplet in droplet digital PCR.

[0158] 2.Results Pregnant women were recruited from the Department of Obstetrics and Gynecology at Prince of Wales Hospital (PWH), Hong Kong. Peripheral blood samples were collected and maternal buffy coats were retrieved. Fetal samples were also obtained by sampling the placenta after delivery. Genotyping of maternal buffy coats and fetal placental tissues was performed using an Illumina microarray platform (HumanOmni2.5). Ten SNPs located in the hemoglobin gamma 2 and hemoglobin epsilon 1 genes from chromosome 11 were selected. The distances of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth SNPs from the first SNP were approximately 2.5, 5, 10, 20, 30, 40, 50, 75, and 100 kb, respectively. The SNPs were homozygous in the fetus and heterozygous in the mother. In this case, the fetus inherited two identical haplotypes, one from each parent. Then, based on the fetal haplotype information and the maternal genotype information, two maternal haplotypes can be derived, which can serve as a gold standard for determining the accuracy of digital fusion PCR.

[0159] FIG. 18 shows the sequences of primers for amplifying each region. The first column shows the SNP ID for SNP 10. The second column shows the coordinates of the SNP on chromosome 11. The third column shows the distance in kilobases from the first SNP. The fourth column lists the genotypes of the SNPs. The fifth column lists the sequences of the forward primers. The sixth column lists the sequences of the reverse primers. The reverse primer for SNP1 contains OLR1 (highlighted in yellow and underlined). OLR1 is complementary to each of the OLR2s (highlighted in green and underlined) in the forward primers for SNP2 through SNP10. The base "A", highlighted in bold red, represents an additional base between the OLR and the priming sequence.

[0160] High molecular weight DNA was extracted from maternal buffy coats using the MagAttract HMW DNA Kit (Qiagen). For each assay, PCR reactions were prepared in a volume of 20 μL consisting of 3.2 ng of template DNA, 10 μL of 2x ddPCR Supermix for Probes (No dUTP) (Bio-Rad), inner adapter primers of each pair at a final concentration of 1 μmol / L, reverse primers of SNP1 and forward primers of another SNP at a final concentration of 40 nmol / L, and forward primers of SNP1 and reverse primers of another SNP at a final concentration of 50 nmol / L. The PCR reaction mix was then split using a QX100 / QX200 Droplet Digital PCR Generator (Bio-Rad). The thermal profile of the assay was as follows: start at 95°C for 10 min, followed by 50 cycles of 94°C for 45 s, 59°C for 1 min, and 72°C for 3 min, and a final incubation at 98°C for 10 min.

[0161] After the PCR reaction, droplets were disrupted and PCR products were recovered according to the manufacturer's protocol with a slight adjustment to use 60 μL of TE buffer per well instead of 20 μL, which improved droplet disruption.

[0162] The PCR products were further purified using AMPure XP beads to remove residual primers and other undesired short fragments (size <100 bp) according to the manufacturer's protocol. The bead / DNA ratio (i.e., the ratio of the volume of beads added to the volume of sample) was 0.9.

[0163] To prepare libraries for sequencing, outer adapter sequences were ligated to the fusion PCR products by PCR. The outer adapter primers included a forward primer with the sequence 5'-AATGATACGGCGACCACCGAGATCTACACATACGAGATCCGT-3' and an index reverse primer with the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTC-3'. PCR reactions were prepared for each sample in a volume of 50 μL consisting of the fusion PCR product, 25 μL of KAPA HiFi HotStart ReadyMix (Roche), and both outer adapter primers at a final concentration of 0.5 μmol / L. The thermal profile of the assay was as follows: start at 98°C for 45 s, followed by 10 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s, and a final incubation at 72°C for 1 min.

[0164] To enrich for desired library sequences of 200-300 bp in length, the recovered PCR products were further purified using AMPure XP beads according to the manufacturer's dual bead-based size selection protocol. The bead / DNA ratios (i.e., the ratio of the cumulative volume of added beads to the volume of the original sample) for the first and second bead selections were 0.6 and 1.4, respectively. The size profile of the purified libraries was then analyzed using Reagents with D1000SreenTape and 4200TapeStation instruments (Agilent Technologies).

[0165] Indexed libraries were multiplexed and sequenced using an Illumina MiSeq sequencing platform (76x2 cycles). PhiX Control v2 Library (Illumina) was employed as a quality control. Spike-in PhiX may also improve sequencing quality due to increased library complexity, especially for low diversity and highly repetitive libraries such as fusion DNA libraries. Examples of spike-in PhiX percentages include, but are not limited to, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%. In one embodiment, 33% of PhiX was spiked. Sequencing primers used for read 1 were customized to run on the Illumina system. 2 / 3 of the custom read 1 primer was mixed with 1 / 3 of the Illumina read 1 primer. The custom read 1 primer allows for sequencing of the fusion PCR product, while the Illumina read 1 primer allows for sequencing of the PhiX library. The sequence of the custom read 1 primer is 5'-CATACGAGATCCGTAATCGGGAAGCTGAAG-3'.

[0166] Sequencing reads were processed using Cutadapt (Martin et al. EMBnet. journal. 2011;17:10-12) to remove adapter and OLR sequences. Chromosome 11 sequence was generated to use as a reference. Sequencing reads were aligned to the reference using BWA software (Li et al. Bioinformatics. 2009;25:1754-1760). Sequencing reads mapped correctly to the fusion sequence; i.e., one part mapped to the SNP1 region and another part mapped to one of the other nine SNP regions.

[0167] The two haplotypes of the mother were then determined as described above. The haplotypes with C and T alleles at SNP1 were designated as HapI and HapII, respectively. For HapI, a Python script was used to count fragments with various combinations of two alleles from two regions, designated as allele contigs. For each pair of regions (SNPs), the top two counted allele contigs were retrieved and their relative frequency was calculated, which summed to 100%. The frequency of the top allele contig was set as cutoff at more than 60% to identify it as a haplotype. If the frequency of the top allele contig was less than 60%, the haplotype could not be determined. In other embodiments, other percentages, for example but not limited to, >65%, 70%, 80%, 90%, can be used as cutoffs to identify the top allele contig as a haplotype in the sample. Similarly, the sequence reads of HapII were analyzed.

[0168] FIG. 19 shows the phases determined by digital fusion PCR. The first column lists the SNP pairings of SNP1 with another SNP. The alleles of the 10 SNPs of the haplotype are listed in order. The second and third columns are HapI for SNP1. The second column lists the frequency of the allele combinations of SNP1 with another SNP. The third column lists the determined alleles of HapI. For each allele site, it may include a base (allele) value, a dash for sites not covered by pairing reads, or a cross for undetermined sites. The fourth and fifth columns are HapII for SNP1. The fourth column lists the frequency of the allele combinations of SNP1 with another SNP. The fifth column lists the determined alleles of HapII.

[0169] As shown in Figure 19, the fusion PCR method can accurately physically phase two regions that are up to 50 kb apart (the distance between SNP8 and SNP1) based on a cutoff frequency of 60%. Measurement of HapI alone successfully phased up to 75 kb (SNP1). The accuracy of this method is determined by the quality and intactness of the DNA. Methods that extract high quality and longer size DNA can improve the accuracy and increase the distance that can be phased by this fusion PCR method.

[0170] D. An example of haplotyping using the hemoglobin subunit beta gene We used the haplotyping of variants in the hemoglobin subunit beta (HBB) gene as an example to illustrate this haplotyping method for phasing multiple regions. The HBB gene encodes the hemoglobin beta subunit. Mutations in the HBB gene can cause beta thalassemia, an autosomal recessive genetic disease characterized by severe anemia. Inheritance of the disease requires having two mutations from both parents. Lo et al. developed a method to non-invasively identify whether a fetus inherits two beta-thalassemia mutations from both parents (Lo et al. Sci Transl Med. 2010;2:61ra91). The authors obtained paternal genotypes and maternal haplotypes. From maternal plasma DNA sequence data, the paternal mutations inherited by the fetus can be found. To assess fetal inheritance of the maternal mutation, the authors used relative haplotype dosage (RHDO) analysis to confirm whether the fetus inherited the maternal haplotype containing the mutation. Maternal haplotype information is required for RHDO analysis. In this proof-of-principle study, Lo et al. derived maternal haplotypes from chorionic villus samples. The authors then used the 10X Genomics platform to directly phase the maternal haplotypes (Hui et al. Clin Chem. 2017;63:513-524). These haplotyping methods can be complicated or costly. Here, we demonstrated the potential application of digital fusion PCR in non-invasive prenatal single-gene disease detection by phasing haplotypes of the HBB gene.

[0171] To verify the accuracy of the digital fusion PCR method, genomic DNA of trios including the father, mother, and fetus of a family was used. Four trios were recruited from the Department of Chemical Pathology and Pediatrics at Prince of Wales Hospital (PWH) in Hong Kong. Then, paternal buffy coat, maternal buffy coat, and fetal placenta tissues were obtained. An assay for phasing 20 SNPs was designed using the method shown in Figures 15A, 15B, and 15C. The 20 SNPs were divided into two groups. Group A included SNPs 1, 4, 6, 8, 10, 11, 13, 15, 17, and 20, and Group B included SNPs 2, 3, 5, 7, 9, 12, 14, 16, 18, and 19. In other embodiments, other grouping configurations can be applied, for example, but not limited to, SNPs 1-10 as Group A and SNPs 11-20 as Group B, and SNPs 1 as Group A and SNPs 2-20 as Group B. The internal primer sequences used are similar to those shown in Figure 15B: the 5' end of the reverse primer for the group A region is complementary to the 5' end of the group B forward primer.

[0172] FIG. 20 shows primer sequences for SNPs in the HBB gene. The first column shows the region numbers of the 20 SNPs in the HBB gene. The second column shows the SNP ID. The third column shows the 5' to 3' forward primer sequences. The fourth column shows the 5' to 3' reverse primer sequences. The reverse primer for each SNP in group A contains OLR1 (highlighted in yellow and underlined). OLR1 is complementary to OLR2 (highlighted in green and underlined) of the forward primer for each SNP in group B. The base "A" is highlighted in red and shown in bold, representing an additional base between the OLR and the priming sequence.

[0173] DNA extracted from the paternal buffy coat, maternal buffy coat, or fetal placenta was distributed into droplets and linking PCR was performed using a BioRad Droplet Digital PCR system.

[0174] PCR reactions are prepared for each sample in a volume of 20 μL consisting of 3.2 ng of template DNA, 10 μL of 2x ddPCR Supermix for Probes (No dUTP) (Bio-Rad), each pair of internal adapter primers at a final concentration of 1 μmol / L, each group A reverse primer and group B forward primer at a final concentration of 40 nmol / L, and each group A forward primer and group B reverse primer at a final concentration of 50 nmol / L. The PCR reaction mix was then split using a QX100 / QX200 Droplet Digital PCR Generator (Bio-Rad). The thermal profile of the assay is as follows: start at 95°C for 10 min, followed by 50 cycles of 94°C for 45 s, 59°C for 1 min, and 72°C for 3 min, and a final incubation at 98°C for 10 min. The fusion PCR products were recovered from the droplets and ligated to the outer sequencing adapters using the protocol described herein. Indexed libraries were multiplexed and sequenced on an Illumina MiSeq sequencing platform. Sequencing reads were processed and aligned to chromosome 11. Haplotypes for the 20 SNPs were assembled using a Python script.

[0175] Figures 21A and 21B show phase assembly analysis. In this example, the principle of phase assembly analysis is demonstrated by phasing four SNPs. Figure 21A shows the four SNPs for phasing divided into two groups, group A with SNPs 1 and 3 (gray triangles) and SNPs 2 and 4 in group B (white triangles).

[0176] Figure 21B shows the determination of haplotypes from the fusion PCR products of the two regions. From the sequencing data, any allele of group A (gray box) can be bound to any allele of group B (white box). The two phased alleles of the fusion product reveal part of the haplotype. Then, the two haplotypes can be inferred using all the phased alleles.

[0177] For example, in the fusion PCR product of SNP1 and SNP4, region X802 may be determined to have the A allele of SNP1, and region X804 may be determined to have the T allele of SNP4. In the fusion PCR product of SNP1 and SNP2, region X806 may be determined to have the A allele of SNP1, and region X808 may be determined to have the G allele of SNP2. SNP1, which has the same allele A, helps to combine SNP2 (allele G) and SNP4 (allele T). A similar process can be used to determine SNP3.

[0178] The fusion product of one haplotype can be distinguished from the fusion product of another haplotype by the mismatch of alleles. For example, the region X806 represents the allele A of SNP1, and the region X810 represents the allele T of SNP1. Furthermore, the region X808 represents the allele G of SNP2, and the region X812 represents the allele A of SNP2. As a result, the fusion product of the regions X806 and X808 is determined to be a different haplotype from the fusion product of the regions X810 and X812.

[0179] Figure 22 shows a table of haplotypes derived by fusion PCR for family trios. The first column of the table shows the individuals for which haplotypes are derived by fusion PCR. The table shows four families with a father, mother, and fetus. The second column shows the haplotype labels. The remaining columns show the alleles of the 20 SNPs.

[0180] The haplotype of each individual was determined by fusion PCR. The haplotypes determined by this method were perfectly consistent with the haplotypes derived from the genotypes of the family trio consisting of the father, mother, and child. The agreement between the haplotypes derived from the genotype analysis and those determined by fusion PCR validates the accuracy of the fusion PCR method. For each family, the haplotypes highlighted in blue represent the haplotypes transmitted by the father to his children, and the haplotypes highlighted in red represent the haplotypes transmitted by the mother to her children. The parental haplotypes inherited by the children are also indicated by an asterisk in the second column.

[0181] VII. Method FIG. 23 is a flow chart illustrating a method for joining separate regions of DNA molecules in a biological sample of a subject according to an embodiment of the present disclosure.

[0182] In block 2310, DNA molecules from a biological sample are divided into multiple compartments (e.g., droplets). The division can be such that a compartment generally contains at most only one DNA molecule. Some compartments may contain less than one DNA molecule. As an example, a compartment can be a droplet. As examples, there are existing commercially available devices that can automatically generate droplets of thousands of nanoliters size in a short time, such as, but not limited to, BioRad QX200 Droplet Generator, Elveflow Droplet Generator Pack, and Micronit Microfluidic Droplet Generator. Various reagents (e.g., primers) can be added to the compartments. Other compartments include Eppendorf tubes, wells of a PCR plate, or any other container.

[0183] Of the set of compartments containing DNA molecules, each compartment can contain a specific reagent. For example, a first compartment of the plurality of compartments can contain (1) a first DNA molecule having a region A and a region B separated by a middle region, (2) a polymerase with a bias for adding a specific nucleotide at an overhang position, (3) a reverse primer R1 having a first portion complementary to a termination sequence of region A, the reverse primer R1 including a second portion having an overlap sequence, and (4) a forward primer F2 having a first portion complementary to a start sequence of region B, the forward primer F2 including a complementary overlap sequence that is complementary to the overlap sequence. The compartment can also contain a forward primer F1 at the start of region A and a reverse primer R2 at the end of region B.

[0184] The primer hybridizes to the 3' end of one strand and extends to the 3' end of the other strand. The forward primer hybridizes to one of the Crick or Watson strands at the 3' end and extends to the 3' end of the other strand. When the forward primer hybridizes to the Crick strand, the reverse primer hybridizes to the 3' end of the Watson strand and extends to the 3' end of the Crick strand. The start sequence of the region is at the 5' end of the Watson strand. The end sequence of the region is at the 3' end of the Watson strand. Similarly, when the forward primer hybridizes to the Watson strand, the reverse primer hybridizes to the 3' end of the Crick strand and extends to the 3' end of the Watson strand. The start sequence of the region is at the 5' end of the Crick strand. The end sequence of the region is at the 3' end of the Crick strand.

[0185] By way of example, the intermediate region may have a length of 100 kb or less, including 2.5 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 75 kb, or any range between these lengths.

[0186] The first DNA molecule can include regions as shown in Figure 3A or other similar figures. Figure 13A shows that region A is exon 1 of the NUDT15 gene and region B is exon 3 of the NUDT15 gene.

[0187] As explained above, the polymerase can have a bias for adding a particular nucleotide (e.g., A) at an overhang position. A sticky end is an end of a double-stranded DNA that has at least one outermost nucleotide that is not hybridized to the other strand. A sticky end is also called an overhang or a jagged end. An overhang position is a position that is not hybridized to the other strand and may correspond to the first such position. A319 or 329 in FIG. 3C is an example. Other polymerases may have a bias for other nucleotides other than A, and the embodiments can be used with such polymerases as well. In some embodiments, including those described below, the polymerase does not need to have a bias for adding a particular nucleotide.

[0188] As an example, reverse primer R1 may correspond to that labeled as R5 in Figures 3A, 4A, and 5A, or may correspond to that labeled as R1 in Figures 8A, 9A, and 13A. As an example, the first portion may correspond to a priming sequence, e.g., 403 in Figure 4B. As an example, the second portion with the overlap sequence may correspond to OLR310, 410, 510, and other similar portions described herein. Reverse primer R1 may include a specific nucleotide (e.g., A409 or 509) between the first portion and the second portion. In some embodiments, reverse primer R1 may not include a specific nucleotide if the method uses a polymerase that does not add an additional adenosine to the 3' end of the DNA strand it extends.

[0189] As an example, forward primer F2 can correspond to that labeled as F6 in Figures 3A, 4A, and 5A, or can correspond to that labeled as F2 in Figures 8A, 9A, and 13A. As an example, the first portion can correspond to a priming sequence, e.g., 553 in Figure 5B. As an example, the complementary overlap sequence can be the first portion (e.g., corresponding to the start of region B), e.g., as shown by F6 in Figures 3A and 4A or F2 in Figure 8A. Thus, the first portion of the forward primer can include a complementary overlap sequence.

[0190] As another example, the complementary overlap sequence may correspond to the second portion of the forward primer F2, such as OLR550 in FIG. 5B and other similar portions described herein. Thus, the forward primer F2 can include a second portion with a complementary overlap sequence. The forward primer F2 can include a specific nucleotide (e.g., A) between the first portion and the second portion, for example, as shown in FIG. 5B. In some embodiments, the forward primer F2 can be free of the specific nucleotide if the method uses a polymerase that does not add additional adenosines to the DNA strand it extends.

[0191] In block 2320, a polymerase and reverse primer R1 are used to amplify region A to obtain a first set of amplicons having a specific nucleotide (e.g., A319 of strand X) at the first 3' end and a complementary nucleotide (e.g., T513 of strand X) between sequence strings corresponding to the first and second parts of reverse primer R1. The complementary nucleotide is complementary to the specific nucleotide. One of the sequence strings can be a part of a complementary overlap sequence, e.g., the OLR of FIG. 5C. In some embodiments, if the polymerase does not add additional bases (e.g., adenosine) to the DNA strand it extends, the additional nucleotides located at the 3' and 5' ends of the OLR are not present or are not needed.

[0192] The complementary nucleotides may or may not be part of the first DNA molecule of the DNA molecule, for example as shown in Figures 3A-3F (included) and 4A-4C (not included). Thus, the complementary nucleotides of the first set of the amplicon may be present in the first DNA molecule (e.g., at the outermost terminal position).

[0193] At block 2330, region B is amplified using a polymerase and forward primer F2 to obtain a second set of amplicons having a particular nucleotide at the second 3' end (e.g., A329 of strand Z in FIG. 3C) and a complementary nucleotide at the opposite end of the overlap sequence (e.g., T563 of strand Z). In some embodiments, the second set of amplicons may not include the particular nucleotide and the complementary nucleotide.

[0194] In block 2340, a polymerase is used to extend a first set of amplicons to include region B and a second set of amplicons to include region A. In this manner, extended amplicons can be obtained that include region A and region B, and exclude the middle region. The extended amplicons can be amplified using forward primer F1 and reverse primer R2.

[0195] Extension can be performed in the same compartment or as part of a separate assay. If performed in the same compartment as above, F1 and R2 will be at higher concentrations than F2 and R1 and ultimately most of the product will be the extension amplicon.

[0196] In block 2350, the extension amplicon is analyzed to determine the haplotype. Any number of suitable assays can be performed to analyze the extension amplicon. For example, sequencing can be performed, including but not limited to, Illumina sequencing systems (e.g., HiSeq2500, NextSeq550 and MiSeq), Pacific Biosystems sequencer and Ion Proton sequencing system. As another example, allele-specific PCR can be performed to detect at least one allele in region A and at least one other allele in region B. Despite being a specific technique, a first allele can be detected in region A and a second allele can be detected in region B, thereby detecting that the first allele and the second allele are on the same haplotype.

[0197] An allele (e.g., a first allele in region A or a second allele in region B) can be detected by measuring the amount of the allele in the respective region. The amount can be a concentration, percentage, fraction, or count. The amount can be compared to a cutoff value. If the amount is greater than the cutoff value, the allele can be determined to be detected. The cutoff value can include 50%, 60%, 70%, 80%, 90%, 95%, or equivalent fractions.

[0198] In some embodiments, the extension amplicons can be analyzed to determine the presence of amplifications or deletions. The extension amplicons can be sequenced to obtain sequencing reads. The sequencing reads can be aligned to a reference genome. Amplifications and deletions can be identified from misalignments between the sequencing reads and the reference genome.

[0199] In some embodiments, molecules can be added to the forward primer F2 and reverse primer R1 to capture and remove unbound (unextended) amplicons.

[0200] A. Primers for amplifying the fusion product The method may include using a forward primer Fz and a reverse primer Rz to amplify the fusion product. Examples of forward primer Fz and reverse primer Rz include F3 and R3 in FIG. 6, R4 and F4 in FIG. 10A, or Fz or Rz in FIG. 15B. Forward primer F1 may include a second portion that is not the first portion. The first compartment may further include a forward primer Fz having a first portion identical to the second portion of forward primer F1. Forward primer Fz may also have a second portion. Forward primer Fz may include complementary nucleotides. Reverse primer R2 may include a second portion that is not the first portion. The first compartment may also include a reverse primer Rz having a first portion identical to the second portion of reverse primer R2. Reverse primer Rz may have a second portion. Reverse primer Rz may have complementary nucleotides.

[0201] The fusion product formed using forward primer F1 and reverse primer R2 can be amplified using forward primer Fz and reverse primer Rz. The extension amplicon formed using forward primer F1 and reverse primer R2 can be a first extension amplicon. The first extension amplicon can include a second portion of forward primer F1 and a second portion of reverse primer R2. The method can include amplifying the first extension amplicon using a polymerase, forward primer Fz and reverse primer Rz to obtain a second extension amplicon. Each second extension amplicon can include a second portion of forward primer Fz and a second portion of reverse primer Rz.

[0202] In embodiments using forward primer Fz and reverse primer Rz to amplify the fusion product, the polymerase may not have a bias for adding a specific nucleotide, and therefore these forward and reverse primers (Fz and Rz) may not contain a nucleotide complementary to a specific nucleotide at the end of the overlap sequence.

[0203] B. Integration of the three fields In some embodiments, the method may be used to fuse three regions of a first DNA molecule to each other. For example, the three regions may be fused as in Figures 8A, 8B, 10A, or 10B. Although the figures label the regions as Region 1, Region 2, and Region 3, embodiments are described that refer to them as Region A, Region B, and Region C. The naming of the regions by numbers or letters does not affect the method. The regions may be Region A (e.g., labeled as Region 1 in some figures), Region B (e.g., labeled as Region 2 in some figures), and Region C (e.g., labeled as Region 3 in some figures). The intermediate region between Region A and Region B may be a first intermediate region. Region C may be separated from Region B by a second intermediate region. The overlap sequence of the second portion of the reverse primer R1 may be a first overlap sequence. The complementary overlap sequence of the forward primer F2 may be a first complementary overlap sequence.

[0204] The first compartment may also include a reverse primer R2 having a first portion complementary to the end sequence of region B. The reverse primer R2 may include a second portion having a second overlap sequence. The first compartment may also include a forward primer F3 having a first portion complementary to the start sequence of region C. The forward primer F3 may include a second complementary overlap sequence complementary to the second overlap sequence. The second complementary overlap sequence may be present in the first portion of the forward primer F3 or in the second portion of the forward primer F3.

[0205] The method may include amplifying region C using a polymerase and forward primer F3 to obtain a third set of amplicons having a particular nucleotide at a 3' end of the third and a complementary nucleotide at an opposite end of the second overlapping sequence. Amplifying region B to obtain the second set of amplicons may include using reverse primer R2.

[0206] The method may further include extending the three sets of amplicons using a polymerase. The first extended amplicon (having region A and region B) may be extended to include region C. The third set of amplicons may be extended to include region A and region B. The result of the extension may be a second extended amplicon that includes region A, region B, and region C, and excludes the first intermediate region and the second intermediate region.

[0207] Extending the first extension amplicon to include region C can include hybridizing (at the end sequence of region C) to a second overlap sequence toward the 3' end of the fourth. Extending the third set of amplicons to include region A and region B can include hybridizing (at the start sequence of region B) to a second complementary overlap sequence toward the 3' end of the second.

[0208] In embodiments fusing three or more regions, the polymerase may not have a bias for adding a particular nucleotide. Similarly, the reverse primer may not contain a particular nucleotide at the end of the overlap sequence, and the forward primer may not contain a nucleotide complementary to a particular nucleotide at the end of the complementary overlap sequence.

[0209] C. Fusion of different groups' territories The method may also include forming a fusion PCR product that does not include all regions in one fusion product. For example, three regions may be fused, but the regions may be fused into two fusion products, each with two regions (e.g., FIG. 14). For example, the first DNA molecule may have a region D (e.g., region 4 in FIG. 14). Region D may be separated from region A (e.g., region 1 in FIG. 14) by a second intermediate region. The first compartment may further include a forward primer F4 having a first portion complementary to the start sequence of region D. The forward primer F4 may include a second portion having a complementary overlap sequence also in the forward primer F2. The first compartment may also include a reverse primer R4 having a first portion complementary to the end sequence of region D.

[0210] The method may include amplifying region D using a polymerase and forward primer F4 to obtain a third set of amplicons. The third set of amplicons may have a specific nucleotide at the third 3' end and a complementary nucleotide at the opposite end of the overlapping sequence. Using a polymerase, the first set of amplicons may be extended to include region D. Using a polymerase, the third set of amplicons may be extended to include region A. A second extended amplicon may be obtained that includes region A and region D. The second extended amplicon may exclude the second intermediate region. The first extended amplicon is an extended amplicon that includes region A and region B (e.g., region 2 in FIG. 14).

[0211] In some embodiments, the method may include combining regions from three different groups, as in FIG. 15A. Although the regions are described by letters, in an example of combining four regions, region A is a region of the first group (e.g., region 1 in FIG. 15A). Region B (e.g., region 2 in the figure) and region D (e.g., region 5 in the figure) are regions of the second group. Region C (e.g., region 3 in the figure) is a region of the third group. Region A (region 1) can combine with either region B (region 2) or region D (region 5). Region B (region 2) or region D (region 5) can combine with region C (region 3). The complementary sequence of forward primer F2 can be a first complementary overlap sequence. The overlap sequence of reverse primer R1 can be a first overlap sequence. The first compartment may further comprise a reverse primer R2 having a first portion complementary to the termination sequence of region B (region 2). The reverse primer R2 may comprise a second portion having a second overlap sequence. The first compartment may also comprise a reverse primer R4 having a first portion complementary to the end sequence of region D (region 5). The reverse primer R4 may comprise a second portion having a second overlap sequence. The first compartment may further comprise a forward primer F3 having a first portion complementary to the start sequence of region C (region 3) of the first DNA molecule. Region C (region 3) may be separated from region B (region 2) by a third intermediate region. The first intermediate region or the second intermediate region may comprise region C (region 3). The forward primer F3 may comprise a second portion having a second complementary overlap sequence.

[0212] Amplification of region B (region 2) may include use of reverse primer R2. Amplification of region D (region 5) may include use of reverse primer R4. The method may further include amplifying region C (region 3) using a polymerase and forward primer F3 to obtain a fourth set of amplicons. The method may further include extending the second extension amplicon to include region C (region 3) using a polymerase, thereby obtaining a third extension amplicon that includes region A (region 1), region D (region 5), and region C (region 3). The method also includes extending the first extension amplicon to include region C (region 3) using a polymerase, thereby obtaining a fourth extension amplicon that includes region A (region 1), region B (region 2), and region C (region 3).

[0213] In some embodiments, two regions can be combined to select a first region from the first group and a second region from the second group, as in FIG. 14A. For example, the first group can include region A (i.e., region 1 in the figure) and region C (i.e., region 3 in the figure). The second group can include region B (i.e., region 2 in the figure) and region D (i.e., region 4 in the figure). As explained above, region A (region 1) and region B (region 2) can be fused, and region A (region 1) and region D (region 4) can be fused. Furthermore, region C (region 3) and region B (region 2) can be fused, and region C (region 3) and region D (region 4) can be fused. The first compartment can include a reverse primer R3 having a first portion complementary to the termination sequence of region C (region 3) of the first DNA molecule. Region C (region 3) can be separated from region B (region 2) by a third intermediate region. Region C (Region 3) may be separated from Region D (Region 4) by a fourth intermediate region. Reverse primer R3 may include a second portion having an overlap sequence that is the same overlap sequence of reverse primer R1.

[0214] The method may include using a polymerase and reverse primer R3 to amplify region C (region 3) to obtain a fourth set of amplicons having a specific nucleotide at the fourth 3' end and a complementary nucleotide at the opposite end of the overlap sequence. The method may also include using a polymerase to extend the fourth set of amplicons to include region B (region 2) and extend the second set of amplicons to include region C (region 3), thereby obtaining a third extended amplicon that includes region C (region 3) and region B (region 2) and excludes the third intermediate region. The method may also further include using a polymerase to extend the fourth set of amplicons to include region D (region 4) and extend the third set of amplicons to include region C (region 3), thereby obtaining a third extended amplicon that includes region C (region 3) and region D (region 4) and excludes the fourth intermediate region.

[0215] In the embodiment of fusing various combinations of at least two regions in the same compartment, the polymerase may not have a bias for adding a specific nucleotide. Thus, the reverse primer may not include a specific nucleotide at the end of the overlap sequence, and the forward primer may not include a nucleotide that is complementary to a specific nucleotide at the end of the complementary overlap sequence. The forward and reverse primers Fz and Rz may not include a specific nucleotide at the end of the overlap sequence.

[0216] A phase assembly analysis such as that described in FIG. 21B may be performed on the first extension amplicon and the second extension amplicon. The first allele may be detected in region A of the first extension amplicon (e.g., region 1 in FIG. 21B). The second allele may be detected in region B of the first extension amplicon (e.g., region 2 in FIG. 21B). The first allele may also be detected in region A of the second extension amplicon. The third allele may be detected in region D of the second extension amplicon (e.g., region 4 in FIG. 21B). As a result of the first allele being detected in region A of both extension amplicons, the first allele in region A, the second allele in region B, and the third allele in region D may be detected in the same haplotype.

[0217] In some embodiments, the first allele may not be detected in region A of the second extension amplicon. Alternatively, the fourth allele may be detected in region A of the second extension amplicon. In these cases, the third allele of region D may be determined to not be on the same haplotype as the first allele of region A.

[0218] The above process can be repeated for each of a plurality of compartments to determine the haplotype in each of the compartments that contain DNA molecules.

[0219] VIII. Kit The embodiments may include a kit for joining separate regions of DNA molecules in a biological sample of a subject. The kit may include a polymerase with a bias for adding a specific nucleotide at an overhang position. In some embodiments, the polymerase may not include a bias for adding a specific nucleotide at an overhang position. The embodiments include a kit for amplifying a fusion product using a forward primer Fz and a reverse primer Rz, a kit for fusing three or more regions, and a kit for fusing various combinations of at least two regions in the same compartment, similar to the above method.

[0220] The kit may also include a reverse primer R1 having a first portion that is complementary to the termination sequence of region A of the first DNA molecule. The reverse primer R1 may include a second portion that has an overlap sequence. A specific nucleotide, for which the polymerase has a bias to add, may be present between the first portion and the second portion.

[0221] The kit may further comprise a forward primer F2 having a first portion complementary to the start sequence of region B of the first DNA molecule. Region B may be separated from region A by an intermediate region. The forward primer F2 may comprise a complementary overlap sequence complementary to the overlap sequence of the reverse primer R1. The complementary overlap sequence may be present in a second portion of the forward primer F2, and the second portion is not the first portion. In some embodiments, the first portion of the forward primer F2 may comprise a complementary overlap sequence.

[0222] The kit may also include a forward primer F1 having a first portion that is complementary to the initiation sequence of region A. The forward primer F1 may include a second portion that is not the first portion.

[0223] The kit may further comprise a reverse primer R2 having a first portion that is complementary to a termination sequence of region B. The reverse primer R may comprise a second portion that is not the first portion.

[0224] The kit may include a forward primer Fz and a reverse primer Rz, both of which can be used to amplify the fusion product. The forward primer Fz may have a first portion identical to the second portion of the forward primer F1. The reverse primer Rz may have a first portion identical to the second portion of the reverse primer R2. The forward primer Fz may include a nucleotide complementary to a specific nucleotide. The reverse primer Rz may include a nucleotide complementary to a specific nucleotide.

[0225] The ratio of forward primer Fz to any primer other than forward primer F1 or reverse primer Rz can be at least 5: 1. In some embodiments, the ratio can be at least 10: 1, 20: 1, 50: 1, 75: 1, 100: 1, 200: 1, 500: 1, 1000: 1, 2000: 1, 5000: 1, 10000: 1, 20000: 1, 50000: 1, or 100000: 1. The ratio of reverse primer Rz to reverse primer R2 or any primer other than forward primer Fz can be at least 5:1, 10:1, 20:1, 50:1, 75:1, 100:1, 200:1, 500:1, 1000:1, 2000:1, 5000:1, 10000:1, 20000:1, 50000:1, or 100000:1.

[0226] The forward primer Fz and the reverse primer Rz may each include a second portion. The second portion may be designed for sequencing. For example, the second portion may be a sample index or an adapter sequence for a particular sequencing platform.

[0227] In some embodiments, the kit may further include a reverse primer R2 having a first portion complementary to the end sequence of region B. The reverse primer R2 may include a second portion having a second overlap sequence. The kit may also include a forward primer F3 having a first portion complementary to the start sequence of region C of the first DNA molecule. Region C may be separated from region B by a second intermediate region. The forward primer F3 may include a second complementary overlap sequence complementary to the second overlap sequence of the reverse primer R2. The second overlap sequence may not be the overlap sequence of the reverse primer R1. In some embodiments, the second overlap sequence may be the overlap sequence of the reverse primer R1.

[0228] In some embodiments, the kit may include a reverse primer R3. The reverse primer R3 may have a first portion that is complementary to the termination sequence of region C. The reverse primer R3 may have a second portion that is not the first portion. In region C, the reverse primer Rz may have a first portion that is identical to the second portion of the reverse primer R3.

[0229] The overlap sequence in the primer can be at least 12 bases, including 12-15, 15-20, 20-25, or more than 25 bases. The portion of the primer that is complementary to the sequence of the region can be at least 12 bases, including 12-15, 15-20, 20-25, or more than 25 bases.

[0230] The portion of the primer that is complementary to the sequence of the region may be at the 3' end of the primer. The portion of the primer that is the overlap sequence or is complementary to the overlap sequence may be at the 5' end of the primer. The forward primer Fz and the reverse primer Rz may have the same portion as the forward primer F1, and the reverse primer R2 may be at the 3' end of the forward primer Fz and the reverse primer Rz.

[0231] The kit may include a first DNA molecule. The kit may include reagents for PCR, a microfluidic cartilage tissue for generating individual compartments for PCR, and primers for the fusion reaction.

[0232] IX. Exemplary Systems FIG. 24 illustrates a measurement system 2400 according to an embodiment of the invention. The system shown includes a sample 2405, such as cell-free DNA molecules in a sample holder 2410, which may contact an assay 2408 to provide a signal of a physical property 2415. An example of a sample holder may be a flow cell, which includes probes and / or primers of an assay, or a tube through which a droplet travels (along with the droplet containing the assay). The physical property 2415 (e.g., fluorescence intensity, voltage, or current) from the sample is detected by a detector 2420. The detector 2420 may measure at intervals (e.g., periodic intervals) to obtain data points that constitute a data signal. In one embodiment, an analog-to-digital converter converts the analog signal from the detector to digital form multiple times. The sample holder 2410 and the detector 2420 may form an assay device, such as a sequencing device that performs sequencing according to embodiments described herein. The data signal 2425 is transmitted from the detector 2420 to a logic system 2430. The data signal 2425 may be stored in a local memory 2435 , an external memory 2440 , or a storage device 2445 .

[0233] Logic system 2430 may be or include a computer system, ASIC, microprocessor, etc. Logic system 2430 may also include or be coupled to a display (e.g., monitor, LED display, etc.) and user input devices (e.g., mouse, keyboard, buttons, etc.). Logic system 2430 and other components may be part of a stand-alone or networked computer system, or may be directly attached to or incorporated into an apparatus (e.g., a sequencing apparatus) that includes detector 2420 and / or sample holder 2410. Logic system 2430 may also include software that executes in processor 2450. Logic system 2430 may include a computer readable medium that stores instructions for controlling system 2400 to perform any of the methods described herein. For example, logic system 2430 may provide commands to a system that includes sample holder 2410 such that sequencing or other physical operations are performed. Such physical operations may be performed in a particular order, such as, for example, reagents are added and removed in a particular order. Such physical manipulations can be performed by a robotic system, including, for example, a robotic arm, such that the system can be used to obtain samples and perform assays.

[0234] Any of the computer systems referred to herein may utilize any suitable number of subsystems. An example of such a subsystem is shown in computer system 2510 in FIG. 25. In some embodiments, a computer system includes a single computing device, and the subsystems may be components of the computing device. In other embodiments, a computer system may include multiple computing devices, each of which is a subsystem and has internal components. Computer systems may include desktop and laptop computers, tablets, mobile phones, and other portable devices.

[0235] The subsystems shown in FIG. 25 are interconnected via a system bus 2575. Additional subsystems are shown such as a printer 2574, a keyboard 2578, storage device(s) 2579, a monitor 2576 (e.g., a display screen such as LED) connected to a display adapter 2582, and others. Peripherals and input / output (I / O) devices coupled to the I / O controller 71 may be connected to the computer system by any number of means known in the art, such as input / output (I / O) ports 2577 (e.g., USB, FireWire). For example, the I / O ports 2577 or an external interface 2581 (e.g., Ethernet, Wi-Fi, etc.) may be used to connect the computer system 2510 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via a system bus 2575 allows a central processor 2573 to communicate with each subsystem and control the execution of instructions from a system memory 2572 or storage device(s) 2579 (e.g., a fixed disk such as a hard drive or optical disk) and the exchange of information between the subsystems. The system memory 2572 and / or the storage device(s) 2579 may embody a computer-readable medium. Another subsystem is a data collection device 2585 such as a camera, microphone, and accelerometer, and the like. Any of the data mentioned herein may be output from one component to another or to a user.

[0236] A computer system may include multiple identical components or subsystems connected to each other, for example, by an external interface 2581, by an internal interface, or through a removable storage device that can be connected from one component to another. In some embodiments, computer systems, subsystems, or devices may communicate over a network. In such an example, one computer may be considered a client and another computer a server, each of which may be part of the same computer system. The client and server may each include multiple systems, subsystems, or components.

[0237] Aspects of the embodiments can be implemented in the form of control logic using hardware circuitry (e.g., application specific integrated circuits or field programmable gate arrays) and / or using computer software in conjunction with a processor that is generally programmable in a modular or integrated manner. As used herein, a processor may include a single-core processor, a multi-core processor on the same integrated chip, or multiple processing units on a single circuit board or networked, as well as dedicated hardware. Based on this disclosure and the teachings provided herein, those skilled in the art will recognize and understand other ways and / or methods for implementing embodiments of the present invention using hardware and combinations of hardware and software.

[0238] Any of the software components or functions described in this application may be implemented as software code executed by a processor using any suitable computer language, such as, for example, Java, C, C++, C#, Objective-C, Swift, or a scripting language, such as, for example, Perl or Python, using conventional or object-oriented techniques. The software code may be stored as a series of instructions or commands on a computer-readable medium for storage and / or transmission. Suitable non-transitory computer-readable media may include random access memory (RAM), read-only memory (ROM), magnetic media (such as a hard drive or floppy disk), or optical media (such as a compact disk (CD) or DVD (digital versatile disk)), or Blu-ray disks and flash memories, etc. The computer-readable medium may be any combination of such storage or transmission devices.

[0239] Such programs may also be coded and transmitted using carrier signals adapted for transmission over wired, optical, and / or wireless networks following various protocols, including the Internet. Thus, computer-readable media may be created using data signals coded with such programs. Computer-readable media coded with program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer-readable medium may be present on or within a single computer product (e.g., a hard drive, CD, or an entire computer system), or may be present on or within different computer products in a system or network. A computer system may include a monitor, printer, or other suitable display for providing a user with any of the results described herein.

[0240] Any of the methods described herein may be implemented, in whole or in part, using a computer system including one or more processors that may be configured to perform the steps. Thus, embodiments may be directed to a computer system configured to perform the steps of any of the methods described herein, with potentially different components performing each step or each group of steps. Although presented as numbered steps, steps of the methods herein may be performed simultaneously or at different times, or in different orders. In addition, parts of these steps may be combined with parts of other steps from other methods. Also, all or parts of a step may be optional. In addition, any steps of any of the methods may be performed by modules, units, circuits, or other means of a system for performing these steps.

[0241] The specific details of the particular embodiments may be combined in any suitable manner without departing from the spirit and scope of the embodiments of the invention, however, other embodiments of the invention may be directed to particular embodiments relating to each individual aspect, or particular combinations of these individual aspects.

[0242] The above description of exemplary embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form described, and many modifications and variations are possible in light of the above teachings.

[0243] References to "a," "an," or "the" are intended to mean "one or more," unless specifically indicated to the contrary. The use of "or" is intended to mean "inclusive or" rather than "exclusive or," unless specifically indicated to the contrary. A reference to a "first" element does not necessarily require that a second element be provided. Furthermore, a reference to a "first" or "second" element does not limit the referenced element to a particular location unless expressly stated. The term "based on" is intended to mean "based at least in part on."

[0244] All patents, patent applications, publications, and descriptions referred to herein are incorporated by reference in their entirety for all purposes. Nothing is admitted to be prior art.

Claims

1. 1. A method for joining separated regions of DNA molecules in a biological sample of a subject, comprising: partitioning a plurality of DNA molecules from the biological sample into a plurality of compartments, a first compartment of the plurality of compartments comprising: a first DNA molecule having a region A and a region B separated by an intermediate region; a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to the termination sequence of the region A and a second portion having an overlap sequence; a forward primer F2 having a first portion complementary to the initiation sequence of region B and comprising a complementary overlap sequence complementary to the overlap sequence; amplifying the region A using the polymerase and the reverse primer R1 to obtain a first set of amplicons having a specific nucleotide at a first 3' end and a complementary nucleotide between sequences corresponding to the first portion and the second portion of the reverse primer R1, wherein the complementary nucleotide is complementary to the specific nucleotide; amplifying the region A to obtain a first set of amplicons; amplifying said region B using said polymerase and said forward primer F2 to obtain a second set of amplicons having a particular nucleotide at a second 3' end and said complementary nucleotide at an opposite end of said overlapping sequence; using said polymerase to extend a first set of said amplicons to include said region B and extend a second set of said amplicons to include said region A, thereby obtaining extended amplicons that include said region A and said region B, and exclude the intermediate region.

2. 2. The method of claim 1, wherein the first DNA molecule comprises the same complementary nucleotides as the complementary nucleotides of the first set of the amplicon.

3. The method of claim 1 , wherein the reverse primer R1 comprises the specific nucleotide between the first portion and the second portion.

4. The method of claim 1 , wherein the compartment is a droplet.

5. 2. The method of claim 1, further comprising amplifying the extended amplicon using forward primer F1 and reverse primer R2.

6. 2. The method of claim 1, further comprising detecting a first allele at region A and a second allele at region B, thereby detecting that the first allele and the second allele are on the same haplotype.

7. detecting the first allele in region A; determining the amount of said first allele in said region A; and comparing the amount to a cutoff value.

8. 2. The method of claim 1, wherein the biological sample is diluted so that on average only one DNA molecule is present in a compartment.

9. 10. The method of claim 1, further comprising repeating the method for other compartments to determine the haplotypes of each of the other DNA molecules in the other compartments.

10. The first compartment comprises: a forward primer F1 having a first portion complementary to the initiation sequence of region A and a second portion; a reverse primer R2 having a first portion complementary to the termination sequence of region B and a second portion; a forward primer Fz having a first portion identical to the second portion of the forward primer F1 and having a second portion; a reverse primer Rz having a first portion identical to the second portion of the reverse primer R2 and a second portion; the extension amplicon is a first extension amplicon, each first extension amplicon comprises the second portion of the forward primer F1 and the second portion of the reverse primer R2; The method further comprising:

2. The method of claim 1, further comprising amplifying the first extension amplicon using the polymerase, the forward primer Fz, and the reverse primer Rz to obtain second extension amplicons, each second extension amplicon comprising the second portion of the forward primer Fz and the second portion of the reverse primer Rz.

11. the forward primer Fz comprises a complementary nucleotide, the complementary nucleotide being complementary to the specific nucleotide; The method of claim 10, wherein the reverse primer Rz comprises the complementary nucleotide.

12. the overlap sequence is a first overlap sequence, the complementary overlap sequence is a first complementary overlap sequence, the intermediate region is a first intermediate region, the extension amplicon is a first extension amplicon, The first compartment comprises: a reverse primer R2 having a first portion complementary to a termination sequence of region B, the reverse primer R2 comprising a second portion having a second overlap sequence; and a forward primer F3 having a first portion complementary to a start sequence of region C of the first DNA molecule, the region C being separated from region B by a second intermediate region, the forward primer F3 comprising a second complementary overlap sequence complementary to the second overlap sequence; amplifying the region B to obtain the second set of amplicons comprises using the reverse primer R2; The method further comprising: amplifying region C using said polymerase and said forward primer F3 to obtain a third set of amplicons having said particular nucleotide at a third 3' end and said complementary nucleotide at the opposite end of said second overlapping sequence; Using the polymerase, extending said first extension amplicon to include said region C; extending the third set of amplicons to include region A and region B; 2. The method of claim 1 , further comprising: thereby obtaining a second extension amplicon that includes region A, region B, and region C, and excludes the first intermediate region and the second intermediate region.

13. 13. The method of claim 12, wherein the forward primer F3 comprises a second portion having the second complementary overlap sequence.

14. 13. The method of claim 12, wherein the first portion of the forward primer F3 comprises a second complementary overlap sequence.

15. extending the first extension amplicon to include region C; 13. The method of claim 12, comprising hybridizing nucleotides toward the fourth 3' end of region C to the second overlapping sequence and to strands of a third set of amplicons.

16. extending the third set of amplicons to include region A and region B; 13. The method of claim 12, comprising hybridizing a nucleotide towards the second 3' end to the second complementary overlap sequence and to the first extended amplicon strand.

17. The method of claim 1 , wherein the first portion of the forward primer F2 comprises the complementary overlap sequence.

18. The method of claim 1 , wherein the forward primer F2 comprises a second portion having the complementary overlap sequence.

19. 19. The method of claim 18, wherein the forward primer F2 comprises the specific nucleotide between the first portion and the second portion.

20. the intermediate region is a first intermediate region, the extension amplicon is a first extension amplicon, The first compartment comprises: a forward primer F4 having a first portion complementary to a start sequence of a region D of the first DNA molecule, said region D being separated from said region A by a second intermediate region, said forward primer F4 comprising a second portion having said complementary overlap sequence; The method further comprising: amplifying the region D using the polymerase and the forward primer F4 to obtain a third set of amplicons having the specific nucleotide at a third 3' end and the complementary nucleotide at the opposite end of the overlapping sequence; 20. The method of claim 18, further comprising using the polymerase to extend the first set of amplicons to include region D and extend the third set of amplicons to include region A, thereby obtaining a second extended amplicon that includes region A and region D and excludes the second intermediate region.

21. The first compartment comprises: a reverse primer R3 having a first portion complementary to a termination sequence of region C of the first DNA molecule, said region C being separated from region B by a third intermediate region, said region C being separated from region D by a fourth intermediate region, said reverse primer R3 comprising a second portion having said overlap sequence; The method further comprising: amplifying the region C using the polymerase and the reverse primer R3 to obtain a fourth set of amplicons having the specific nucleotide at a fourth 3' end and the complementary nucleotide at the opposite end of the overlapping sequence; using said polymerase to extend said fourth set of amplicons to include said region B and extend said second set of amplicons to include said region C, thereby obtaining a third extended amplicon that includes said region C and said region B and excludes said third intermediate region; 21. The method of claim 20, further comprising using the polymerase to extend the fourth set of amplicons to include region D and extend the third set of amplicons to include region C, thereby obtaining a fourth extended amplicon that includes region C and region D and excludes the fourth intermediate region.

22. detecting a first allele in said region A of said first extension amplicon; detecting a second allele in said region B of said first extension amplicon; detecting the first allele in region A of the second extension amplicon; detecting a third allele in said region D of said second extension amplicon; 21. The method of claim 20, further comprising detecting the first allele of region A, wherein the second allele of region B and the third allele of region D are present on the same haplotype.

23. the complementary overlap sequence is a first complementary overlap sequence, the overlap sequence is a first overlap sequence, The first compartment comprises: a reverse primer R2 having a first portion complementary to a termination sequence of the region B, the second portion having a second overlap sequence; a reverse primer R4 having a first portion complementary to the termination sequence of the region D, the second portion having the second overlap sequence; a forward primer F3 having a first portion complementary to a start sequence of region C of the first DNA molecule, said region C being separated from said region B by a third intermediate region, said forward primer F3 comprising a second portion having a second complementary overlap sequence, said second complementary overlap sequence being complementary to said second overlap sequence; amplifying said region B comprises using said reverse primer R2; amplifying said region D comprises using said reverse primer R4; The method further comprising: amplifying said region C using said polymerase and said forward primer F3 to obtain a fourth set of amplicons; extending said second extension amplicon using said polymerase to include said region C, thereby obtaining a third extension amplicon comprising said region A, said region D, and said region C; 21. The method of claim 20, further comprising using the polymerase to extend the first extension amplicon to include region C, thereby obtaining a fourth extension amplicon that includes region A, region B, and region C.

24. 1. A method for joining separated regions of DNA molecules in a biological sample of a subject, comprising: partitioning a plurality of DNA molecules from the biological sample into a plurality of compartments, a first compartment of the plurality of compartments comprising: a first DNA molecule having a region A and a region B separated by an intermediate region; a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of the region A, the reverse primer R1 including a second portion having an overlap sequence; a forward primer F2 having a first portion complementary to the initiation sequence of the region B, the forward primer F2 comprising a complementary overlap sequence complementary to the overlap sequence; a forward primer F1 having a first portion complementary to the initiation sequence of region A and a second portion; a reverse primer R2 having a first portion complementary to the termination sequence of region B and a second portion; a forward primer Fz having a first portion identical to the second portion of the forward primer F1 and a second portion; a reverse primer Rz having a first portion identical to the second portion of the reverse primer R2 and a second portion; amplifying the region A using the polymerase and the reverse primer R1 to obtain a first set of amplicons having a specific nucleotide at a first 3' end and a complementary nucleotide between sequences corresponding to the first portion and the second portion of the reverse primer R1, wherein the complementary nucleotide is complementary to the specific nucleotide; amplifying the region A to obtain a first set of amplicons; amplifying the region B using the polymerase and the forward primer F2 to obtain a second set of amplicons having a particular nucleotide at a second 3' end and the complementary nucleotide at an opposite end of the overlapping sequence; extending the first set of amplicons using the polymerase to include the region B and extending the second set of amplicons to include the region A, thereby obtaining first extended amplicons that include the region A and the region B and exclude the middle region, wherein each first extended amplicon includes the second portion of the forward primer F1 and the second portion of the reverse primer R2, extending the first set and extending the second set to obtain first extended amplicons; amplifying the first extension amplicon to obtain second extension amplicons using the polymerase, the forward primer Fz, and the reverse primer Rz, each second extension amplicon comprising the second portion of the forward primer Fz and the second portion of the reverse primer Rz.

25. 25. The method of claim 24, wherein the forward primer F2 comprises a second portion having the complementary overlap sequence.

26. 25. The method of claim 24, wherein the first portion of the forward primer F2 comprises the complementary overlap sequence.

27. 1. A method for joining separated regions of DNA molecules in a biological sample of a subject, comprising: partitioning a plurality of DNA molecules from the biological sample into a plurality of compartments, a first compartment of the plurality of compartments comprising: a first DNA molecule having a region A and a region B separated by a first intermediate region; a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of the region A, the reverse primer R1 comprising a second portion having a first overlap sequence; a forward primer F2 having a first portion complementary to the initiation sequence of the region B, the forward primer F2 comprising a first complementary overlap sequence complementary to the first overlap sequence; a reverse primer R2 having a first portion complementary to a termination sequence of the region B, the second portion having a second overlap sequence; a forward primer F3 having a first portion complementary to an initiation sequence of region C of the first DNA molecule, said region C being separated from region B by a second intermediate region, said forward primer F3 comprising a second complementary overlap sequence complementary to said second overlap sequence; amplifying the region A using the polymerase and the reverse primer R1 to obtain a first set of amplicons having a specific nucleotide at a first 3' end and a complementary nucleotide between sequences corresponding to the first portion and the second portion of the reverse primer R1, wherein the complementary nucleotide is complementary to the specific nucleotide; amplifying the region A to obtain a first set of amplicons; amplifying the region B using the polymerase, the forward primer F2, and the reverse primer R2 to obtain a second set of amplicons having a particular nucleotide at a second 3' end and the complementary nucleotide at an opposite end to the first overlap sequence; amplifying region C using said polymerase and said forward primer F3 to obtain a third set of amplicons; Using the polymerase, extending the first set of amplicons to include region B and region C; extending a third set of amplicons to include region A and region B, thereby obtaining extended amplicons that include region A, region B, and region C, and exclude the first intermediate region and the second intermediate region.

28. 1. A method for joining separated regions of DNA molecules in a biological sample of a subject, comprising: partitioning a plurality of DNA molecules from the biological sample into a plurality of compartments, a first compartment of the plurality of compartments comprising: a first DNA molecule having a region A and a region B separated by a first intermediate region; a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of the region A, the reverse primer R1 including a second portion having an overlap sequence; a forward primer F2 having a first portion complementary to the initiation sequence of region B, the second portion having a complementary overlap sequence complementary to the overlap sequence; a forward primer F4 having a first portion complementary to a start sequence of region D of the first DNA molecule, said region D being separated from said region A by a second intermediate region, said forward primer F4 comprising a second portion having said complementary overlap sequence; amplifying the region A using the polymerase and the reverse primer R1 to obtain a first set of amplicons having a specific nucleotide at a first 3' end and a complementary nucleotide between sequences corresponding to the first portion and the second portion of the reverse primer R1, wherein the complementary nucleotide is complementary to the specific nucleotide; amplifying the region A to obtain a first set of amplicons; amplifying said region B using said polymerase and said forward primer F2 to obtain a second set of amplicons having a particular nucleotide at a second 3' end and said complementary nucleotide at an opposite end of said overlapping sequence; amplifying said region D using said polymerase and said forward primer F4 to obtain a third set of amplicons; using said polymerase to extend said first set of amplicons to include said region B and extend said second set of amplicons to include said region A, thereby obtaining a first extended amplicon that includes said region A and said region B and excludes said first intermediate region; using the polymerase to extend the first set of amplicons to include region D and extend the third set of amplicons to include region A, thereby obtaining a second extended amplicon that includes region A and region D and excludes the second intermediate region.

29. detecting a first allele in said region A of said first extension amplicon; detecting a second allele in said region B of said first extension amplicon; detecting the first allele in region A of the second extension amplicon; detecting a third allele in said region D of said second extension amplicon; 29. The method of claim 28, further comprising detecting the first allele of region A, wherein the second allele of region B and the third allele of region D are present on the same haplotype.

30. the complementary overlap sequence is a first complementary overlap sequence, the overlap sequence is a first overlap sequence, The first compartment comprises: a reverse primer R2 having a first portion complementary to a termination sequence of the region B, the second portion having a second overlap sequence; a reverse primer R4 having a first portion complementary to the termination sequence of the region D, the second portion having the second overlap sequence; a forward primer F3 having a first portion complementary to a start sequence of region C of the first DNA molecule, said region C being separated from said region B by a third intermediate region, said forward primer F3 comprising a second portion having a second complementary overlap sequence, said second complementary overlap sequence being complementary to said second overlap sequence; amplifying said region B comprises using said reverse primer R2; amplifying said region D comprises using said reverse primer R4; The method further comprising: amplifying said region C using said polymerase and said forward primer F3 to obtain a fourth set of amplicons; extending said second extension amplicon using said polymerase to include said region C, thereby obtaining a third extension amplicon comprising said region A, said region D, and said region C; 29. The method of claim 28, further comprising using the polymerase to extend the first extension amplicon to include region C, thereby obtaining a fourth extension amplicon that includes region A, region B, and region C.

31. 1. A kit for joining separated regions of DNA molecules in a biological sample of a subject, comprising: a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of region A of the first DNA molecule, the reverse primer R1 including a second portion having an overlap sequence; a forward primer F2 having a first portion complementary to the initiation sequence of region B of the first DNA molecule; Including, the region B is separated from the region A by an intermediate region, the forward primer F2 comprises a second portion having a complementary overlap sequence that is complementary to the overlap sequence, and the first portion of the forward primer F2 does not overlap with the second portion of the forward primer F2; A kit for joining separated regions of DNA molecules in a biological sample of a subject.

32. 32. The kit of claim 31 , wherein the reverse primer R1 comprises the specific nucleotide between the first portion and the second portion.

33. a forward primer F1 having a first portion complementary to the initiation sequence of region A; and a reverse primer R2 having a first portion complementary to a termination sequence of region B.

34. the forward primer F1 comprises a second portion that is not the first portion, the reverse primer R2 comprises a second portion that is not the first portion; The kit comprises: a forward primer Fz having a first portion identical to the second portion of the forward primer F1; 34. The kit of claim 33, further comprising a reverse primer Rz having a first portion identical to the second portion of the reverse primer R2.

35. the forward primer Fz comprises a nucleotide complementary to the specific nucleotide, The kit of claim 34, wherein the reverse primer Rz comprises a nucleotide complementary to the specific nucleotide.

36. the ratio of the forward primer Fz to the forward primer F1 is at least 5:1; 35. The kit of claim 34, wherein the ratio of reverse primer Rz to reverse primer R2 is at least 5:

1.

37. the overlap sequence is a first overlap sequence, the complementary overlap sequence is a first complementary overlap sequence, the intermediate region is a first intermediate region, The kit comprises: a reverse primer R2 having a first portion complementary to a termination sequence of the region B, the second portion having a second overlap sequence; 32. The kit of claim 31, further comprising a forward primer F3 having a first portion complementary to a start sequence of region C of the first DNA molecule, said region C being separated from region B by a second intermediate region, said forward primer F3 comprising a second complementary overlap sequence complementary to the second overlap sequence.

38. 38. The kit of claim 37, wherein the first overlapping sequence is not the second overlapping sequence.

39. 1. A kit for joining separated regions of DNA molecules in a biological sample of a subject, comprising: a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of region A of the first DNA molecule, the reverse primer R1 including a second portion having an overlap sequence; a forward primer F2 having a first portion complementary to a start sequence of region B of the first DNA molecule, said region B being separated from region A by an intermediate region, said forward primer F2 comprising a second portion having a complementary overlap sequence complementary to said overlap sequence, said first portion of said forward primer F2 not overlapping with said second portion of said forward primer F2; a forward primer F1 having a first portion complementary to the initiation sequence of region A and a second portion; a reverse primer R2 having a first portion complementary to the termination sequence of region B and a second portion; a forward primer Fz having a first portion identical to the second portion of the forward primer F1 and having a second portion; a reverse primer Rz having a first portion identical to the second portion of the reverse primer R2 and a second portion; Including, A kit for joining separated regions of DNA molecules in a biological sample of a subject.

40. 1. A kit for joining separated regions of DNA molecules in a biological sample of a subject, comprising: a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of region A of the first DNA molecule, the reverse primer R1 comprising a second portion having a first overlap sequence; a forward primer F2 having a first portion complementary to a start sequence of region B of the first DNA molecule, said region B being separated from region A by an intermediate region, said forward primer F2 comprising a second portion having a first complementary overlap sequence complementary to said first overlap sequence, said first portion of said forward primer F2 not overlapping with said second portion of said forward primer F2; a reverse primer R2 having a first portion complementary to a stop sequence of region B, said reverse primer R2 comprising a second portion having a second overlap sequence; 1. A kit for binding separated regions of DNA molecules in a biological sample of a subject comprising: a forward primer F3 having a first portion complementary to a start sequence of region C of the first DNA molecule, said region C being separated from region B by a second intermediate region, said forward primer F3 comprising a second complementary overlap sequence complementary to the second overlap sequence.

41. 1. A kit for joining separated regions of DNA molecules in a biological sample of a subject, comprising: a polymerase having a bias for adding specific nucleotides at the overhang positions; a reverse primer R1 having a first portion complementary to a termination sequence of region A of the first DNA molecule, the reverse primer R1 including a second portion having an overlap sequence; 1. A kit for binding separated regions of DNA molecules in a biological sample of a subject comprising: a forward primer F2 having a first portion complementary to a start sequence of region B of the first DNA molecule, wherein region B is separated from region A by an intermediate region, and wherein the forward primer F2 comprises a second portion having a complementary overlap sequence that is complementary to the overlap sequence, and wherein the first portion of the forward primer F2 does not overlap with the second portion of the forward primer F2; and a forward primer F4 having a first portion complementary to a start sequence of region D of the first DNA molecule, wherein region D is separated from region A by a second intermediate region, and wherein the forward primer F4 comprises the complementary overlap sequence.

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