Description:FIELD OF INVENTION
The current invention relates to compositions and methods for enriching and purifying large molecular weight genomic DNA from genomic DNA sample using a two-step magnetic nanoparticle based method.
BACKGROUND
Isolating and purifying nucleic acids is a basic requirement for biotechnology research and applications. Several methods for isolation, purification of nucleic acids have been developed. Such methods of isolating and purifying nucleic acids involve separating nucleic acids of interest from other unwanted sample components or impurities. Isolation and purification of nucleic acids is many times followed by enrichment of target nucleic acids based on certain criteria / distinguishing factors. For many applications requiring isolation and use of genomic DNA, difference in the size is an important criterion to distinguish target nucleic acids from non-target nucleic acids. Size selective fractionation of DNA e.g. by mechanical shearing or enzymatic fragmentation is an important step in the library construction for techniques such as next generation sequencing (NGS) applications.

The ability to capture and sequence large contiguous DNA fragments is a key step towards the comprehensive characterization of complex genomic regions. Long-read sequencing results can be maximized by loading only long fragments onto the sequencer, thereby eliminating any issues with preferential sequencing of smaller fragments. This can be achieved through size selection to exclude the portion of the sample below a specified threshold. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kb.
Technologies such as Next-Generation Sequencing (NGS) technology are highly important in the field of genetics, enabling large-scale, high throughput genetic studies for a variety of research and diagnostic applications. NGS is used for economically sequencing entire genomes, and is also an important tool for several research and diagnostic applications which require targeted DNA sequencing of specific genomic loci.

Several methods have been developed for the enrichment of specific genomic DNA fragments for use in sequencing for clinical and research applications. Almost all of these DNA enrichment methods rely on fragmentation of genomic DNA prior to amplification, resulting in relatively short (less than 1000 base-pair) sequencing templates. Hence, the currently used methods for genomic DNA isolation and enrichment are a major limitation for comprehensively characterizing complex genomic loci because they cannot provide the larger size fragments that are required to successfully span complex sequences, such as extended repeats, or resolve sections of unknown or unexpected sequence that have been inserted or rearranged within the targeted region.
In NGS, sequencing is performed by repeated cycles of polymerase-mediated nucleotide extensions or, in one format, by iterative cycles of oligonucleotide ligation. As a massively parallel process, NGS generates hundreds of megabases to gigabases of nucleotide-sequence output in a single instrument run, depending on the platform. NGS technologies have become a major driving force in genetic research. Several NGS technology platforms have found widespread use and include, for example, the following NGS platforms: Roche/454, Illumina Solexa Genome Analyzer, the Applied Biosystems SOLiD™ system, Ion Torrent™ semiconductor sequence analyzer, PacBio® real-time sequencing and Helicos™ Single Molecule Sequencing (SMS) (Refs 1 and 2: Harismendy, O., et al; Hodzic, J., et al).
All these NGS technology platforms require the preparation of a sequencing library which is suitable for massive parallel sequencing, which in turn relies on efficient library preparation methods for generating high quality sequencing data. Hence obtaining long genomic DNA fragments is a basic requisite for such sequencing platforms. Many methods have been developed for obtaining long genomic fragments, but they either have limitations of being time consuming , costly, or do not yield genomic DNA above 10 kb with high efficiency and purity. Some methods such as purification by cutting out bands from gels is highly time consuming and inefficient.
Another widely used technology is the size selective precipitation with a poly(alkylene oxide) polymer containing buffer, in particular polyethylene glycol based buffers.
Such Solid-phase reversible immobilization (SPRI) beads have become core components in high throughput DNA/RNA cleanup and size selection in recently developed methods. But typical application of such commercial beads has been limited to the size interval of 150 to 800 bp of fragmented DNA. Gel based selections have been used for targeting longer DNA fragments, for example, in mate-pair sequencing NGS libraries production (3–6 Kbp), 10x Genomics-linked reads (10 Kbp), and in the third generation sequencing methods such as Pacific Biosciences (Menlo Park, CA) (.6 Kbp) and Oxford Nanopore Technologies (Oxford, United Kingdom) (.10 Kbp). Gel-based solutions are limited by long duration, expensive equipment, and poor sample recovery. Stortchevoi, demonstrated the repurposing of these beads to enrich 1.5- 7kb genomic DNA fragments (Ref 5: Stortchevoi et al).
Though all these above methods disclose the use of beads/ nanoparticles for purifying DNA, the DNA sizes purified by these methods are approximately 3 to 7 kb. For example, U.S. Pat. No. 5,898,071 discloses use of beads for purifying plasmid DNA such as pUC plasmids which are approximately 2.5 kb- 3kb in size.
The current invention discloses the use of magnetic beads, to enrich and purify very long genomic DNA fragments, above 7 to 8kb. The method disclosed herein can be used for purifying genomic DNA fragments as long as 20kb- 45kb. The current invention discloses a two-step nanoparticle bead-based technology consisting of reagents in two separate fractions to together accomplish a very precise cut off to remove smaller fragments. Moreover, the proportions of reagents can be manipulated to obtain varied size cut offs. A macromolecular crowding agent in the presence of a salt is used to drive binding of DNA onto the surface of carboxyl-coated magnetic beads.
SUMMARY
Embodiments:
The current invention discloses a two-step nanoparticle bead-based method for enriching and purifying long genomic DNA fragments. The compositions and method disclosed herein can be used to generate long size DNA fragments with very high purity which can be directly employed for any sequencing platforms.
One embodiment of the current invention is a two-step treatment kit for enrichment of high molecular weight DNA (HMW) molecules from a heterogeneous genomic DNA sample wherein the kit comprises a first solution S1 comprising 200-8000 MW 20%-80% (w/v) polyethylene glycol (PEG), a second solution S2 comprising 30%-60% (v/v) an inorganic salt and 40%-70% magnetic nanoparticles (w/v).
In one embodiment, the solution S1 is provided in a first container and the solution S2 is provided in a second container.
In one embodiment, the solutions S1 and S2 of the kit are added in two separate steps to the genomic DNA sample.
In one embodiment, the inorganic salt in the kit is selected from the group consisting of MgCl2, NaCl and CaCl2.
In one embodiment, the concentration of the inorganic salt is in the range 0.01- 1M.
In one embodiment, the kit further comprises an elution buffer.
In one embodiment, the elution buffer is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.
In one embodiment, the spherical magnetic nanoparticles comprise carboxyl, methyl, amino, thiol, and phosphoric acid groups on the surface.
In one embodiment, the kit enriches for high molecular weight DNA molecules larger than 10 kb.
In one embodiment, the kit enriches for high molecular weight DNA molecules larger than 40kb.
In one embodiment, the spherical magnetic nanoparticles are comprised of a superparamagnetic ferric oxide core encapsulated by a polyacrylic acid coated silica layer.
One embodiment of the current invention is a method for enrichment of high molecular weight DNA molecules from a heterogenous genomic DNA sample, wherein the method comprises the steps of:
a. Adding a first solution S1 to a genomic DNA sample (G) in the ratio of 0.6x -1x v/ v of the genomic DNA sample volume to obtain S1: G mix;
b. Adding a second solution S2 to the S1:G mix obtained in step (a) in the ratio of 0.5 – 0.8x v/v of the S1:G mix to obtain the S2:S1:G mix;
c. Incubating the S2:S1:G mix for 20- 60 minutes at room temperature;
d. Precipitating the genomic DNA bound to the magnetic nanoparticles in the S2:S1:G mix by using a magnetic force to keep the magnetic nanoparticles stationary ; and
e. Eluting the DNA bound to the magnetic nanoparticles by adding 1-10 mM elution buffer.
wherein the first solution S1 comprises 200-8000 polyethylene glycol (PEG), and the second solution S2 comprises 30%-60% (v/v) low molecular weight inorganic salt and 40%-70% (w/v) magnetic nanoparticle beads.
In one embodiment, the elution buffer used in the method disclosed above is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.
In one embodiment, the magnetic force for precipitating the magnetic bead bound DNA in the method disclosed herein provided by a magnetic stand or a magnetic particle mover.

BRIEF DESCRIPTION OF FIGURES:
Fig.1 shows an agarose gel image of DNA elutes obtained from varying the reagent mixture concentrations as mentioned in Table 1, for one step method using different ratios of solutions S1 and S2.
Fig.2 shows an agarose gel image of DNA elutes obtained from varying the S1 and S2 reagent mixture concentrations as mentioned in Table 2, with varying concentrations of S1 and S2 relative to the sample volume, with two step method.
Fig.3 shows a graph wherein the proportion of different sized fragments recovered (10kb, 15kb, 20kb, 48.5kb) in the elute is compared across conditions 1-5 and the SPRI condition. The size of fragments selected across the conditions mentioned in Table 3.
DETAILED DESCRIPTION OF THE INVENTION
The current invention discloses a two-step nanoparticle bead-based method for enriching and purifying long genomic DNA fragments. The compositions and method disclosed herein can be used to generate long size DNA fragments with very high purity which can be directly employed for any sequencing platforms.
The current invention discloses a bead based technology which comprises reagents in two separate fractions to together accomplish very precise cut off to remove smaller fragments. By manipulating the proportions of reagents, the current method can be engineered to obtain varied size cut offs of purified genomic DNA. In the current invention, a macromolecular crowding agent in the presence of a salt is used to drive binding of DNA onto the surface of carboxyl-coated magnetic beads. The core principle behind size separation is that there is an inverse relationship between the concentration of the crowding agent and the size of bound DNA fragments.
DEFINITIONS
As used herein, the term “enrichment” refers to a method in which the concentration of a specific target DNA molecule is increased relative to the concentration of total DNA in a sample, where the sample has heterogenous DNA molecules. In the current invention, the target DNA molecules are the ones that are bigger than 5kb in size. In some embodiments, the target DNA molecules are bigger than 10kb, 30kb or 40 kb in size. The total DNA sample is genomic DNA sample.
As used herein, the term heterogenous DNA sample contains genomic DNA fragments of heterogenous or different sizes.
In genome sequencing, and as used herein “coverage” or “sequence coverage” refers to the number of reads that are uniquely mapped to a reference genome. –The larger the length of the DNA fragments, the lesser the number of reads to be sequenced and mapped to the reference genome. Larger reads generally contain more genetic information, which means that fewer reads are needed to cover the entire genome. As a result, larger reads lead to higher coverage, ensuring that each region of the genome is sequenced multiple times. Higher coverage increases confidence in the accuracy of the sequencing data and improves the detection of genetic variations, including single nucleotide polymorphisms (SNPs) and structural variants.

As used herein, “Depth” refers to the average number of times a specific nucleotide in the genome is sequenced by individual reads. Currently in the industry, the depth of coverage required to sequence an entire genome using Oxford Nanopore Technologies is approximately 30x. (Ref 4: Khrenova et al.; Ref. 3: Jain, M., et al)
For samples with shorter reads (with mean lengths of 2 kbp), a higher coverage depth of 50x is required. As a consequence of increased coverage with a lesser number of reads, we reduce the depth of coverage required to map the entire genome.

One of the most commonly used metrics to assess the quality of a sequenced genome is N50. The N50 value is calculated as follows: first, the contigs are sorted based on length. Then, the cumulative length of each contig/read length is calculated and a graph is plotted for the number of read lengths against the number of bases of that read length. The N50 value is determined by considering the middle value of the data - with each half containing equal amounts of information. If the N50 value is towards the greater side, the quality of the sequenced genome is better - since it denotes that the genome was sequenced from longer fragments (of higher read lengths), with reduced depth of coverage.

As a result, there would be higher genome coverage with a shorter depth of coverage.

Magnetic nanoparticles which bind DNA and have sufficient surface area to permit efficient binding can be used in the present invention.
As used herein “nanoparticles”, “magnetic nanoparticles” “beads” are used interchangeably.
As used herein, "magnetic nanoparticles" are magnetic particles with diameter of approximately 300-800 nm, which are attracted by a magnetic field. The magnetic nanoparticles used in the method of the present invention comprise a magnetic metal oxide core, which is generally surrounded by an adsorptively or covalently bound silane coat which has a polymer coating such as polyacrylic acid.
The magnetic nanoparticle further comprising a surface functional group selected from the group consisting of carboxylic acid/carboxylate, amino/imine, methyl, methylene, thiol, anhydride, phosphoric acid, sulfuric acid/sulfonate, sulfonamide, and phosphatide.
Such particles and methods of making them have already been described in prior art such as US2020124592A1 (Magnetic nanoparticle); which are incorporated herein.
The magnetic metal oxide core is preferably iron oxide, and is in the form of Fe3O4. Suitable silanes useful to coat the nanoparticle surfaces include Tetraethyl orthosilicate (TEOS), Aminopropyl triethoxysilane (APTES), Amino propyl trimethoxy silane (APTMS), triethoxysilylpropyl succinic anhydride (TESPSA).
As used herein, the term "functional group-coated surface" refers to a surface which is coated with moieties which each have a free functional group which is bound to the amine group on the nanoparticle; as a result, the surfaces of the microparticles are coated with the functional group containing moieties. The functional group binds to DNA in solution.
As used herein, “percentage recovery” of the purified HMW DNA molecules is defined as the percentage of HMW DNA recovered in the elute when compared to the percentage of HMW DNA present in the genomic DNA sample.
Superparamagnetism is a property exhibited by iron oxide nanoparticles, such as Fe2O3 or Fe3O4 nanoparticles containing an organic or inorganic coating. The term is used to bring in an analogy between the small magnetic moment of a single paramagnetic atom and the larger magnetic moment of nano-sized magnetic particles that arise from coupling of spins. In the absence of magnetic field, the particles no longer show magnetic interaction - which contributes to their usability. Ref 6: Tobias et al).
EMBODIMENTS:
One embodiment of the present invention is a method of enriching and purifying DNA fragments of long size from a solution containing DNA.
The current invention discloses a two-step nanoparticle bead-based method for enriching and purifying long genomic DNA fragments. The compositions and method disclosed herein can be used to generate long size DNA fragments with very high purity which can be directly employed for any sequencing platforms.
One embodiment of the current invention is a two-step treatment kit for enrichment of high molecular weight DNA (HMW) molecules from a heterogeneous heterogenous genomic DNA sample wherein the kit comprises a first solution S1 comprising 200-8000 MW 20%-80% (w/v) polyethylene glycol (PEG), a second solution S2 comprising 30%-60% (v/v) low molecular weight inorganic salt and 40%-70% (w/v) magnetic nanoparticles.
In one embodiment, the solution S1 is provided in a first container and the solution S2 is provided in a second container. In one embodiment, the solutions S1 and S2 of the kit are added in two separate steps to the genomic DNA sample. In one embodiment, the kit further comprises an elution buffer. In one embodiment, the elution buffer is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.

In one embodiment, the inorganic salt in the solution S2 in the kit is selected from the group consisting of MgCl2, NaCl and CaCl2.

In one embodiment, the concentration of the inorganic salt is in the range 0.01- 1M.
In one embodiment, the spherical magnetic nanoparticles comprise carboxyl, methyl, amino, thiol, and phosphoric acid groups on the surface.
In one embodiment, the kit enriches for high molecular weight DNA molecules larger than 10 kb.
In one embodiment, the kit enriches for high molecular weight DNA molecules larger than 40kb.
In one embodiment, the spherical magnetic nanoparticles are comprised of a superparamagnetic ferric oxide core encapsulated by a polyacrylic acid coated silica layer.

One embodiment of the current invention is a method for enrichment of high molecular weight DNA molecules from a heterogenous genomic DNA sample, wherein the method comprises the steps of: (a) adding a first solution S1 to a genomic DNA sample (G) in the ratio of 0.6x -1x v/v of the genomic DNA sample volume to obtain S1: G mix; (b) adding a second solution S2 to the S1:G mix obtained in step (a) in the ratio of 0.5 – 0.8x v/v of the S1:G mix to obtain the S2:S1:G mix; (c) incubating the S2:S1:G mix for 20- 60 minutes at room temperature; (d) Precipitating the genomic DNA bound to the magnetic nanoparticles in the S2:S1:G mix by using a magnetic force to keep the magnetic nanoparticles stationary ; and (e) Eluting the DNA bound to the magnetic nanoparticles by adding 1-10 mM elution buffer,
wherein the first solution S1 comprises 200-8000 polyethylene glycol (PEG), and the second solution S2 comprises 30%-60% (v/v) low molecular weight inorganic salt and 40%-70% (w/v) magnetic nanoparticle beads.
In one embodiment, the elution buffer used in the method disclosed above is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.
In one embodiment, the magnetic force for precipitating the magnetic bead bound DNA in the method disclosed herein provided by a magnetic stand or a magnetic particle mover.
In one embodiment, the current invention encompasses a kit and compositions for a two-step purification of long-size genomic DNA fragments.
In one embodiment, the polyethylene glycol is present in S1 in a concentration range of 7% to 50% (w/v). Suitable concentrations in the S1 solution may be in a range selected from 10% to 45%, such as 12% to 40% and 15% to 35%.( All % with respect to the polymer / crowding agent ) are indicated as (w/v).
PEG may have a molecular weight that in the range of 3000 to 30000, e.g. selected from 4000 to 25000, 5000 to 25000, 6000 to 20000 and 6000 to 16000.
In one embodiment, the inorganic salt in the S2 solution may be NaCl, MgCl2, or CaCl2 . The inorganic salt, preferably alkali metal salt, may be in a concentration that lies in the range of 0.05M to 1M.
In one embodiment, the salt may be in a concentration in the range of 0.01M-0.5M. In one embodiment, the salt may be in a concentration that lies in the range of 0.025M -0.1M.
In one embodiment, one or more functional groups are bound/ adsorbed to the nanoparticle surface. Examples of functional groups include carboxylic acid/carboxylate groups, imine/amino groups, methyl groups, methylene groups, thiol groups, anhydride groups, phosphoric acid groups, sulfuric acid/sulfonate groups, sulfonamide or phosphatide groups.
In one embodiment, the functional group is a carboxylic acid.
In one embodiment, a suitable moiety with a free carboxylic acid functional group is an amine moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the magnetic nanoparticle.
In one embodiment, the magnetic nanoparticles at the end of the process are separated from the elution buffer by, for example, filtration or applying a magnetic field to draw down the nanoparticles.
In one embodiment, by manipulating the proportions of reagents, the current method can be engineered to obtain varied size cut offs of purified genomic DNA.
EXAMPLES
Example I: One-step method using magnetic nanoparticles
Based on previous research and experimentation, a protocol was developed to perform size selection of long reads. This protocol was performed using magnetic nanoparticles with diameter 200-800nm, and attached to amine moieties that are functionalized with carboxyl groups and New England Biolab’s Quick-load 1kb Extend DNA Ladder (Catalog number: N3239S) as the sample.

Materials and Methods
The reagent mixture S1+S2 is comprised of 200-8000 MW polyethylene glycol (PEG) from SRL Labs, 30%-60% (v/v) low molecular weight magnesium chloride and 40%-70% (w/v) magnetic nanoparticles. The New England Biolab’s Quick-load 1kb Extend DNA Ladder was used as the DNA sample to select fragments from. . This ladder contains 13 bands of sizes ranging from 0.5kbp to 10kbp (Fig. 1) .
1. 30µL of the sample was taken in a sterile microcentrifuge tube and “y” µL of the reagent mixture (S1+S2) was added to it to obtain a reaction mixture. (Please note: “y” µL refers to reagent volumes mentioned in Table 1)
2. The reaction mixture was lightly vortexed for 30 seconds and incubated for 30 minutes at room temperature.
3. The tube with the reaction mixture was placed on a magnetic microcentrifuge tube stand (Magnetic Stand) from Cambrian Biotech wherein the magnetic beads were immobilized and the supernatant was discarded.
4. The magnetic beads were washed with 70% ethanol for 1 minute.
5. The beads were dried at room temperature for a period of 10 minutes.
6. 30 µL of the elution buffer, 1mM Tris HCl was added and the mixture was incubated at room temperature for 10 minutes.
7. The resulting elutes were transferred to another sterile microcentrifuge tube.
Table 1 shows the reagent volume (S1+S2) mentioned is with reference to sample volume taken. For eg. “0.2x” indicates that the volume of the reagent is 0.2 times the sample(G) volume taken (30µL).
Table 1: Reagent concentrations across varying concentrations of PEG
Sl. No Concentration of PEG S1+S2
Volume taken “y”µL
I PEG 200
20-60% 0.2 x
0.5x
1.25x
2x
II PEG 200
20-40% 0.75x
1.25x
1.75x
III PEG 1500
10-70% 0.2x
0.5x
1.25x
2x
IV PEG 1500
10-60% 0.2x
0.5x
0.75x
1x

Results
Fig.1 shows an agarose gel image of DNA elutes obtained from varying the reagent mixture concentrations as mentioned in Table 1, for one step method using different ratios of solutions S1 and S2.
The agarose gel image depicts the various band sizes that were selected as a result of the experiment. As it can be seen, reagent volumes 0.2x and 0.5x of conditions I and III do not result in any bands. On the contrary, the concentrations 1.25x and 2x from condition III; and concentrations 0.75x and 1x select all the band sizes from the sample. Since we aim to select only bands of higher sizes (~10kb), another two-step protocol described in Example 2 was performed.

Example 2
Materials and Methods
Based on the results from Example 1, a new method was developed wherein the reagents consisted of a first solution S1 having 20%-80% (w/v) of 200-8000 MW polyethylene glycol (PEG), a second solution S2 containing 30%-60% (v/v) low molecular weight magnesium chloride (MgCl2) and 40%-70% (w/v) magnetic nanoparticles.
The sample used was DNA extracted from human whole blood. For reference, the sizes were compared with the New England Biolab’s Quick-load 1kb Extend DNA Ladder. The ladder contains 13 bands of sizes ranging from 0.5kbp to 10kbp. It is important to note that in this two-step method, the reagents S1 and S2 were added separately in the mentioned concentration ratios.
The following protocol was adhered to:
1. 30µL (Volume of genomic DNA samples denoted as “x” in Table 2) of the genomic DNA sample was taken in a sterile microcentrifuge tube and varying volumes of S1 were added to the sample, denoted as “m” µL; to obtain a reaction mixture S1:G. (Please note: “m” µL refers to reagent volumes given in column 2 in Table 2)
2. The reaction mixture S1:G was lightly vortexed for 30 seconds.
3. Varying volumes of the solution S2 was added to the reaction mixture to obtain S1:S2:G mix (S2 volumes relative to the sample volume are given in column 3 of Table 2) . This mix was vortexed for 30 seconds and incubated for 30 minutes at room temperature.
4. The tube with the mixture S1:S2:G was placed on a magnetic stand wherein the magnetic beads (nanoparticles) were immobilized and the supernatant was discarded.
5. The magnetic beads were washed with 70% ethanol for 1 minute.
6. The beads were dried at room temperature for a period of 10 minutes.
7. 30 uL of the elution buffer, 1mM Tris-HCl was added and the mixture was incubated at room temperature for 10 minutes.
8. The resulting elutes were transferred to another sterile microcentrifuge tube.
Table 2: Volumes of S1(m) and S2 (n) tested with respect to genomic DNA sample volumes (x). The S1 and S2 volumes given below are with reference to sample volume taken. For eg. “0.6x” indicates that the volume of the reagent is 0.6 times the sample volume taken (30uL).

Condition S1 (m) S2 (n) Elution Time (min)
I 0.6x 0.8x 5
II 0.7x 0.7x
III 0.8x 0.6x 10
IV 1x 0.5x

Results
Fig.2 shows an agarose gel image of DNA elutes obtained from varying the S1 and S2 reagent mixture concentrations as mentioned in Table 2, with varying concentrations of S1 and S2 relative to the sample volume.
As depicted in the agarose gel image shown in Fig. 2, the various ratios of sample volume, S1 and S2 mentioned in Table.2 were able to successfully bind and purify selectively the large fragments (~10kb) and the smaller fragments were not bound. It can be seen that increasing the time of elution to 10 minutes increased the number of higher fragments selected (that are near the 10kb size), while the elution time of 5 minutes resulted in lesser number of selected fragments.
Example 3
Based on the data from Examples 1 and 2, six new combinations were developed with varying concentrations of S1 and S2 in each method (as listed in Table 3), and these methods were tested against the performance of SPRI beads. The New England Biolabs 1kb Extend DNA ladder was used as the DNA sample with heterogeneous sizes, whose band sizes ranged from 0.5kb to 48.5kb.
SPRI beads, also known as solid-phase reversible immobilization beads, are small, spherical paramagnetic particles used in various biological and biochemical applications (Ref 7: SPRI select User Guide PN B24965AA (October 2012).
In nucleic acid purification, SPRI beads are used to selectively capture and isolate particular DNA base pair sizes from a complex mixture. The beads are functionalized with complementary sequences or specific binding agents that recognize and bind to the target nucleic acids. By applying a magnetic field, the SPRI beads can be easily separated from the rest of the sample, allowing for efficient purification of the desired genetic material.

Materials and Methods
The same protocol as Example 2 was employed in this experiment, with the reagent ratios used as listed in Table.3
The New England Biolab’s Quick-load 1kb Extend DNA Ladder was used as the sample. The ladder contains 13 bands of sizes ranging from 0.5kbp to 48.5kbp.
Table 3: Volumes of solutions S1 (m) and S2 (n) used in a two step reaction with reference to DNA sample volume (x) . The reagent volume mentioned is with reference to sample volume taken. For eg. “0.6x” indicates that the volume of the reagent is 0.6 times the sample volume taken (30uL).

Conditions S1 S2
1 0.75x 0.5x
2 0.55x 0.5x
3 0.8x 0.6x
4 0.6x 0.8x
5 1x 0.5x

Results
The results were assessed by Qubit™ dsDNA Quantification Assay Kits (Catalog number: Q32851). Qubit assays are fluorometric quantification assays used to accurately measure the concentration of nucleic acids or protein samples. The Qubit assays utilize specific fluorescent dyes that selectively bind to the target molecules of interest. When the dye binds to the biomolecule, it undergoes a change in fluorescence intensity and emission wavelength. The Qubit™ Fluorometer instrument then measures the fluorescence signal and translates it into a concentration value.
Fig.3 shows a graph wherein the proportion of different sized fragments recovered (10kb, 15kb, 20kb, 48.5kb) in the elute is compared across conditions 1-5 and the SPRI condition. The size of fragments selected across the conditions mentioned in Table 3.
Results from the above done experiment demonstrated that there was a superior depletion of <3kb fragments with the methods employed. The combination of the reagents of S1 and S2 in two steps as mentioned in Table.3 were capable of selectively binding only to the larger fragments.
As compared to the SPRI beads, which selected <5% of the large fragments (~48.5kb) fragments, there was a significantly higher selection of larger size fragments at >15% using the S1 and S2 reagents by condition-4. While conditions 2, 3 and 5 significantly selected fragments >15kb, condition 4 was able to select only the largest fragments (48.5kb) while dropping fragments smaller than that.

Example 4
Materials and Methods
The MagAttract DNA Kit (Catalog number: 67563) was used to extract DNA from human whole blood to be used as the DNA sample to select long fragments.
The protocol as mentioned in Example 3 was employed for the size selection process.
Results
Table 4: Percentage recovery and N50 obtained from SPRI and Condition-4, wherein the volume of S1 is 0.6x and S2 is 0.8x(refer to Table 3)
Extraction Size Selection Recovery (%) N50 (kb)
MagAttract None - 9
SPRI 93 13
Condition-4 83 22

While the percentage of HMW DNA recovered from the SPRI condition is higher at 93%, the N50 value is 13kb. However, while Condition-4 has 83% recovery of HMW DNA, the N50 value is higher - 22kb, which indicates that there are more long fragments of that size. This is an indication of better quality of genome sequenced, and also the reduction in depth of coverage while maintaining the length of coverage.

References:
1. Harismendy, O., Ng, P. C., Strausberg, R. L., Wang, X., Stockwell, T. B., Beeson, K. Y., Schork, N. J., Murray, S. S., Topol, E. J., Levy, S., & Frazer, K. A. (2009). Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome biology, 10(3), R32
2. Hodzic, J., Gurbeta, L., Omanovic-Miklicanin, E., & Badnjevic, A. (2017). Overview of Next-generation Sequencing Platforms Used in Published Draft Plant Genomes in Light of Genotypization of Immortelle Plant (Helichrysium Arenarium). Medical archives (Sarajevo, Bosnia and Herzegovina), 71(4), 288–292.
3. Jain, M., Koren, S., Miga, K. H., Quick, J., Rand, A. C., Sasani, T. A., Tyson, J. R., Beggs, A. D., Dilthey, A. T., Fiddes, I. T., Malla, S., Marriott, H., Nieto, T., O'Grady, J., Olsen, H. E., Pedersen, B. S., Rhie, A., Richardson, H., Quinlan, A. R., Snutch, T. P., … Loose, M. (2018). Nanopore sequencing and assembly of a human genome with ultra-long reads. Nature biotechnology, 36(4), 338–345.
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, Claims:1. A two-step treatment kit for enrichment of high molecular weight DNA molecules from a heterogenous genomic DNA sample wherein the kit comprises a first solution S1 comprising 200-8000 MW 20%-80% (w/v) polyethylene glycol (PEG), a second solution S2 comprising 30%-60% (v/v) inorganic salt and 40%-70% (w/v) magnetic nanoparticles.

2. The kit as claimed in claim 1, wherein the solution S1 is provided in a first container and the solution S2 is provided in a second container.

3. The kit as claimed in claim 1, wherein the inorganic salt is selected from the group consisting of MgCl2, NaCl and CaCl2.

4. The kit as claimed in claim 1, wherein the concentration of the inorganic salt is in the range 0.01-1M.

5. The kit as claimed in claim 1, wherein the solutions S1 and S2 are added in two separate steps to the genomic DNA sample.

6. The kit as claimed in claim 1, wherein it further comprises an elution buffer.

7. The kit as claimed in claim 2, wherein the elution buffer is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.

8. The kit as claimed in claim 1, wherein the kit provides a recovery of 50%-90% of high molecular weight DNA from the genomic DNA sample.

9. The kit as claimed in claim 1, wherein the kit provides a recovery of 60%-90% of high molecular weight DNA from the genomic DNA sample.
10. The kit as claimed in claim 1, wherein the spherical magnetic nanoparticles contain carboxyl, methyl, amino, thiol, and phosphoric acid groups on the surface.

11. The kit as claimed in claim 1, wherein the kit enriches for high molecular weight DNA molecules larger than 10 kb.

12. The kit as claimed in claim 1, wherein it enriches for high molecular weight DNA molecules larger than 40kb.

13. The kit as claimed in claim 1, wherein the magnetic nanoparticles are comprised of a superparamagnetic ferric oxide core encapsulated by a polyacrylic acid coated silica layer.

14. A method for enrichment of high molecular weight DNA molecules from a heterogenous genomic DNA sample, wherein the method comprises the steps of:
a. Adding a first solution S1 to a genomic DNA sample (G) in the ratio of 0.6x -1x v/ v of the genomic DNA sample volume to obtain S1: G mix;
b. Adding a second solution S2 to the S1:G mix obtained in step (a) in the ratio of 0.5 – 0.8x v/v of the S1:G mix to obtain the S2:S1:G mix;
c. Incubating the S2:S1:G mix for 20- 60 minutes at room temperature;
d. Precipitating the genomic DNA bound to the magnetic nanoparticles in the S2:S1:G mix by using a magnetic force to keep the magnetic nanoparticles stationary ; and
e. Eluting the DNA bound to the magnetic nanoparticles by adding 1-10 mM elution buffer.
wherein the first solution S1 comprises 200-8000 polyethylene glycol (PEG), and the second solution S2 comprises 30%-60% (v/v) low molecular weight inorganic salt and 40%-70% (w/v) magnetic nanoparticles.
15. The method as claimed in claim 10, wherein the elution buffer is selected from the group comprising of Tris hydrochloric acid, Nuclease Free Water, and Tris-EDTA.

16. The method as claimed in claim 10, wherein the magnetic force for precipitating the magnetic bead bound DNA is a magnetic stand or a magnetic particle mover.