DNA insert size selection

Abstract

Methods for obtaining a target median deoxyribonucleic acid (DNA) insert size utilizes several solid-phase reversible immobilization (SPRI) bead solutions. At different steps of the method workflows, different ratios of the SPRI bead solution to DNA solution are utilized. Each method includes back-to-back DNA fragment removal / retention steps, where different ratios of the SPRI bead solution to DNA solution are utilized in the back-to-back steps.

Description

DNA INSERT SIZE SELECTIONCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 63 / 686,016, filed August 22, 2024, the contents of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Double-stranded DNA (dsDNA) target molecules can be fragmented to form shorter strands, referred to as insert strands. These insert strands are often short, ranging from about 300 bp to about 450 bp. These insert strands can be tagged with adapter sequences and dehybridized to generate a library of smaller, single-stranded DNA molecules (ssDNA). These smaller, single-stranded DNA molecules may be used as templates in DNA sequencing reactions. The templates may enable short read lengths to be obtained, and then during data analysis, overlapping short sequence reads can be aligned to reconstruct the longer nucleic acid sequences.SUMMARY

[0003] Methods are disclosed herein to generate insert strands having a median size ranging from about 800 base pairs (bp) to about 1500 bp. Larger (i.e. , longer) inserts lead to the formation of larger template strands that are ultimately sequenced. The larger template strands help when aligning reads back to the reference genome, as the longer reads map uniquely and span large variants. This helps to reduce gaps in the genome coverage. Larger inserts and larger template strands will enhance the mappability in genome regions that are traditionally challenging to map, resulting in improved accuracy of variant calling. Additionally, larger inserts and larger template strands will enable a user to fully leverage the advantages of long sequencing-by- synthesis (SBS) reads by reducing the readl and read2 overlap and increasing the usable yield.

[0004] A first example method uses a clean-up process at the outset to remove potential contaminants and very small fragmented DNA, followed by a double size selection process, i.e., back-to-back DNA fragment removal / retention. A second example method uses the double size selection process instead of the clean-up process. This method removes potential contaminants and small fragmented DNA of a predetermined size at the outset. The initial processes of both methods generate a DNA solution containing a higher concentration of DNA fragments of a minimum onset size, e.g., ranging from about 500 bp to about 700 bp. A solution containing DNA fragments with the minimum onset size, in turn, increases the probability that that final library will have a higher concentration of larger fragments / insert strands. With the second method, minimum onset size can be pushed to higher base pairs without jeopardizing loss of larger library material, which can otherwise occur when double size selection processes are executed exclusively at the end of the preparation. The second method may also lead to more efficient ligation, thus potentially increasing the yield of the larger inserts strand in the final library.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

[0006] Fig. 1 A is a schematic illustration of an example of the method disclosed herein;

[0007] Fig. 1 B a schematic illustration of another example of the method disclosed herein;

[0008] Fig. 2A is a graph depicting fluorescence units (Y axis, [FU]) versus the fragment insert size (X axis, base pairs) for example samples exposed to an example of the method disclosed herein;

[0009] Fig. 2B is a graph depicting fluorescence units (Y axis, [FU]) versus the fragment insert size (X axis, base pairs) for comparative samples exposed to a comparative method;

[0010] Fig. 2C is a graph depicting an enlarged view of one sample in the circled portion of Fig. 2A and one example sample in the circled portion of Fig. 2B versus the fragment insert size (X axis, base pairs), illustrating the presence of smaller fragments in the comparative sample;

[0011] Fig. 3 is a graph depicting the number of fragments (Y axis) versus the insert size of the fragments (X axis, base pairs) for fragments selected in accordance with an example of the method disclosed herein;

[0012] Fig. 4 is a graph depicting fluorescence units (Y axis, [FU]) versus the fragment insert size (X axis, base pairs) for two different example samples exposed to an example of the method disclosed herein;

[0013] Fig. 5 is a graph depicting fluorescence units (Y axis, [FU]) versus the fragment insert size (X axis, base pairs) for an example sample exposed to an example of the method disclosed herein and for a comparative sample exposed to a comparative method; and

[0014] Fig. 6A and Fig. 6B are graphs depicting fluorescence units (Y axis, [FU]) versus the fragment insert size (X axis, base pairs) for an example sample exposed to a first example of the methods disclosed herein and for another example sample exposed to a second example of the methods disclosed herein.DETAILED DESCRIPTION

[0015] The methods disclosed herein enables a user to obtain a library containing a target median deoxyribonucleic acid (DNA) insert size ranging from about 800 bp to about 1500 bp. Each method utilizes several solid-phase reversible immobilization (SPRI) bead solutions at different steps of the method workflow. Moreover, at the different steps, different ratios of the SPRI bead solution to DNA solution are utilized. Additionally, each method includes back-to-back DNA fragment removal / retention steps, each of which utilizes a specific ratio. In each method, the combination of the workflow, the ratios at the specific steps, and the back-to-back DNA fragmentremoval / retention steps have been found to yield larger insert sizes without having to perform a polymerase chain reaction (PCR).

[0016] Definitions

[0017] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0018] As used herein, the singular forms “a,” “an,” and “the” refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0019] Reference throughout the specification to “one example,” “another example,” “an example,” and so forth, means that a particular element (e.g., feature, structure, composition, configuration, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

[0020] The terms “substantially” and “about” used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, these terms can refer to less than or equal to ±5% from a stated value, such as less than or equal to ±2% from a stated value, such as less than or equal to ±1 % from a stated value, such as less than or equal to ±0.5% from a stated value, such as less than or equal to ±0.2% from a stated value, such as less than or equal to ±0.1 % from a stated value, such as less than or equal to ±0.05% from a stated value.

[0021] Adapter. A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation. Suitable adapter lengths may range from about 10 nucleotides to about 100 nucleotides, or from about 12 nucleotides to about 60 nucleotides, or from about 15 nucleotides to about 50 nucleotides. The adaptermay include any combination of nucleotides and / or nucleic acids. In some examples, the adapter can include an amplification domain, e.g., having a universal nucleotide sequence, such as a P5 or P7 sequence, that can serve as a starting point for template amplification and cluster generation. In other examples, the adapter can include a sequence that is complementary to at least a portion of a flow cell surface bound primer (which includes the universal nucleotide sequence). In the latter example, the adapter sequence can hybridize to the complementary flow cell surface bound primer during amplification and cluster generation. In some examples, the adapter can also include a sequencing primer sequence (i.e. , sequencing binding site) or a sequencing sample index (i.e., a barcode sequence).

[0022] DNA Sample: A polymeric form of nucleotides that includes deoxyribonucleotides, deoxyribonucleotide analogs, or complementary deoxyribonucleotides derived from an RNA (ribonucleic acid) sample. The DNA sample is double stranded. The DNA sample may include naturally occurring DNA, which includes a nitrogen containing heterocyclic base (a nucleobase such as adenine, thymine, cytosine and / or guanine), a sugar (specifically deoxyribose, i.e., a sugar lacking a hydroxyl group that is present at the 2’ position in ribose), and a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety known in the art.

[0023] The DNA sample may be genomic DNA (gDNA) that can be isolated from one or more cells, bodily fluids (e.g., whole blood, blood spots, saliva) or tissues. gDNA can be prepared by lysing a cell that contains the DNA. The cell may be lysed under conditions that substantially preserve the integrity of the cell's gDNA. In one particular example, thermal lysis may be used to lyse a cell. In another particular example, exposure of a cell to alkaline pH can be used to lyse a cell while causing relatively little damage to gDNA. Any of a variety of basic compounds can be used for lysis including, for example, potassium hydroxide, sodium hydroxide, and the like. Additionally, relatively undamaged gDNA can be obtained from a cell lysed by an enzyme that degrades the cell wall. Cells lacking a cell wall either naturally or due to enzymatic removal can also be lysed by exposure to osmotic stress. Other conditions that can be used to lyse a cell include exposure to detergents, mechanical disruption,sonication heat, pressure differential such as in a French press device, or Dounce homogenization. Agents that stabilize gDNA can be included in a cell lysate or isolated gDNA sample including, for example, nuclease inhibitors, chelating agents, salts, buffers and the like.

[0024] Each'. When used in reference to a collection of items, each identifies an individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0025] Flow Cell: A vessel having an open or enclosed flow channel where a reaction can be carried out, an inlet for delivering reagent(s) to the flow channel, and an outlet for removing reagent(s) from the flow channel. In some examples, the flow cell enables the detection of the reaction that occurs therein. For example, the flow cell can include one or more transparent surfaces allowing for the optical detection of arrays, optically labeled molecules, or the like.

[0026] Fragment: A portion or piece of the DNA sample.

[0027] Insert Size: The length, i.e. , number of base pairs, in a double stranded DNA (dsDNA) fragment that is present in a library of fragments. The target median DNA insert size is the goal for the middle size in a library containing a plurality of fragments of different sizes.

[0028] Insert Strands: The dsDNA fragments present in a library of fragments.

[0029] Primer. A single stranded nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a fragment. As one example, a flow cell surface bound primer can serve as a starting point for fragment amplification and cluster generation. As another example, a primer (e.g., a sequencing primer) may be introduced that can hybridize to fragments or fragment amplicons in order to prime synthesis of a new strand that is complementary to the fragments or fragment amplicons. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases long. In an example, each of the flow cell surface bound primer and the sequencing primer is a short strand, ranging from 10 to 60 bases, or from 20 to 40 bases.

[0030] Template Strands: The single stranded DNA (ssDNA) fragments that are generated after dsDNA fragments in a library are denatured, seeded, and clustered. The template strands are sequenced.

[0031] Volume ratio: The quantitative relationship between the volume of a homogeneous solid-phase reversible immobilization (SPRI) bead solution and the volume of a DNA solution. The volume ratio is sometimes depicted as “x” in the notation #x (e.g., 1x, 1.5x, etc.). Thus, 1x is a 1 :1 volume ratio of SPRI bead solution:DNA solution, 0.7x is a 0.7:1 volume ratio of SPRI bead solution:DNA solution, etc.

[0032] First Method

[0033] The first method disclosed herein includes a specific workflow, during which specific solutions and solution ratios are utilized. The first method enables the size selection of relatively long DNA fragments, thus enabling a library with long insert strands (having a median insert size ranging from about 800 bp to about 1500 bp) to be generated for sequencing operations without having to perform PCR. Thus, the following method is performed, in order, without preforming PCR.

[0034] Fig. 1 A schematically illustrates an example of the first method, which generally includes removing fragments from a DNA solution using a first solid-phase reversible immobilization (SPRI) bead solution at a first volume ratio of the first SPRI bead solution:the DNA solution (Fig. 1A, A.); performing end repair on remaining fragments from the DNA solution, thereby forming end repaired fragments (Fig. 1 A, C.); generating a second DNA solution with the end repaired fragments; sequentially: removing first remaining fragments from the second DNA solution using a second SPRI bead solution at a second volume ratio of the second SPRI bead solution:the second DNA solution that is less than the first volume ratio (Fig. 1A, D.) and retaining second remaining fragments in the second DNA solution using a third SPRI bead solution at a third volume ratio of the third SPRI bead solution:the second DNA solution that is less than the first volume ratio and greater than the second volume ratio (Fig. 1A, E.); exposing the second remaining fragments to A-tailing and ligation to form sample fragments (Fig. 1 A, G.); generating a third DNA solution with the samplefragments; and sequentially: retaining first sample fragments in the third DNA solution using a fourth SPRI bead solution at a fourth volume ratio of the fourth SPRI bead solution:the third DNA solution that is less than the first volume ratio and greater than the third volume ratio (Fig. 1 A, H), releasing the first sample fragments, generating a fourth DNA solution with the first sample fragments, and retaining second sample fragments in the fourth DNA solution using a fifth SPRI bead solution at a fifth volume ratio of the fifth SPRI bead solution:the fourth DNA solution that is less than the first volume ratio and greater than or equal to the fourth volume ratio (Fig. 1 A, I.). Throughout the first method, each of the SPRI bead solutions includes SPRI beads targeting a library fragment size greater than the target median DNA insert size. While specific examples of the ratios are described herein, the first method may be used to target other median insert sizes by utilizing differently sized SPRI beads 16 and by varying the ratios used throughout the method, as long as the two back-to-back SPRI processes are used and the ratios follow this protocol: the second volume ratio is less than the first volume ratio, the third volume ratio is less than the first volume ratio and greater than the second volume ratio, the fourth volume ratio is less than the first volume ratio and greater than the third volume ratio, and the fifth volume ratio is less than the first volume ratio and greater than or equal to the fourth volume ratio.

[0035] The DNA sample used in the first method may be genomic DNA (gDNA) that can be isolated from one or more cells, bodily fluids (e.g., whole blood, blood spots, saliva) or tissues. At the outset of the first method, the DNA sample is fragmented using any suitable technique. Fragmentation breaks the double stranded DNA into pieces (i.e., fragments), and can be performed using physical or enzymatic shearing methods.

[0036] Examples of physical (or mechanical) shearing methods include acoustic shearing (e.g., sonication), hydrodynamic shearing, or nebulization.

[0037] Acoustic sonication uses ultrasound waves to break apart chemical bonds. Acoustic sonication is performed with an ultrasonicator, which shears the DNA sample by applying bursts of ultrasound. The ultrasound waves cleave hydrogen bonds and causes single- and double-strand ruptures of the DNA helix. With hydrodynamic shearing, the DNA sample is exposed to hydrodynamic shear forces when it is forcedthrough a small orifice of a syringe. Nebulization involves forcing the DNA sample through a small hole in a nebulizer unit using compressed air or nitrogen.

[0038] Examples of enzymatic shearing methods use transposons, restriction enzymes, or nicking enzymes to fragment the DNA sample.

[0039] Prior to removing fragments from the DNA solution, the first method may further comprise: shearing a DNA sample; and generating the DNA solution with the sheared DNA sample.

[0040] The DNA fragments generated from the DNA sample are then used in the first method, which will be described in more detail in reference to Fig. 1A. The DNA fragments 10 are the initial fragments generated from the DNA sample, and variations of reference numeral 10, such as 10A, 10B, etc. are used to identify specific fragments that are removed or retained or modified at a particular step in the first method workflow.

[0041] Several DNA solutions 12, 12’, 12”, 12”’ and SPRI bead solutions 14A, 14B, 14C, 14D, 14E are used throughout the first method.

[0042] In each DNA solution 12, 12’, 12”, 12’” some DNA fragments 10 are incorporated into a liquid carrier. As shown in Fig. 1A, the type of fragments, e.g., 10A, 10B, etc. (e.g., small or large) that remains in solution from one step of the workflow to another may change. As such, the amount of DNA fragments 10 present in any of the DNA solutions 12, 12’, 12”, 12’” may differ from one step of the workflow to another.

[0043] The liquid carrier in each DNA solution 12, 12’, 12”, 12’” is an aqueous buffer. The aqueous buffer includes water and a buffer salt (e.g., Tris(hydroxymethyl) aminomethane (Tris or TRIS) acetate salt, pH 7.6). The buffer salt may be present in a concentration ranging from about 7 mM to about 12 mM.

[0044] Each example of the SPRI bead solution 14A, 14B, etc. used throughout the first method includes paramagnetic beads (i.e. , SPRI beads 16) in a binding buffer. Each SPRI bead 16 is 1 pm ± 8% in size, and includes a buoyant polystyrene core surrounded by a thin layer of magnetite. On the surface, the SPRI beads 16 are coated with carboxyl molecules, which provide charge groups for DNA fragment binding. While SPRI beads 16 are referenced, it is to be understood that othercommercially available size selection beads may be used, such as Illumina Purification Beads (Illumina Inc.) or AMPure XP beads (Beckman).

[0045] The SPRI beads 16 are selected to target a fragment size that is greater than the target median DNA insert size. In one example, the target median DNA insert size ranges from about 800 base pairs (bp) to about 1300 bp; and the SPRI beads 16 target a library fragment size of 1500 bp.

[0046] The binding buffer of each SPRI bead solution 14A, 14B, etc. provides salt ions and adjusts the pH in order to facilitate the binding of some of the fragments 10 and the SPRI beads 16. As an example, the binding buffer may include lithium chloride ions, a buffer salt, and water. Each of the salt ions and buffer salt may be present in the SPRI bead solution 14A, 14B, etc. in a concentration of 2.15 M + / - 5%. Water makes up a balance of the solution 14A, 14B, etc. The SPRI beads 16 are present in an amount ranging from about 0.8 wt% to about 1.5 wt%, based on the total weight of the binding buffer. In one example, the SPRI beads 16 are present in an amount of about 1 % in the SPRI bead solution 14A, 14B. In an example, about 10 mg of SPRI beads 16 are present per 1 ml of binding buffer.

[0047] The SPRI bead solutions 14A, 14B, etc. may also include a crowding agent to aid in forcing fragments 10 of a particular size out of the solution 14A, 14B, etc. An example of a suitable crowding agent is poly(ethylene glycol) having a weight average molecular weight ranging from about 300 g / mol to about 35,000 g / mol. The crowding agent may be present in the SPRI bead solution 14A, 14B, etc. in an amount of about 18 wt% + / - 0.05%.

[0048] Specific examples of the SPRI bead solution 14A, 14B, etc. that may be used to retain larger fragments includes SPRI beads 16 that target a fragment size of 1500 bp, from about 10 wt% to about 18 wt% of poly(ethylene glycol) having a weight average molecular weight ranging from about 8,000 g / mol to about 20,000 g / mol, from about 2 M to about 4 M of LiCI or NaCI, about 10 mM TRIS buffer, and water.

[0049] The first DNA solution 12 used in the first method includes fragments 10 of the DNA sample in an example of the liquid carrier. The fragments 10 may be present in an amount ranging from about 2 pg to about 3 pg.

[0050] As shown in Fig. 1 A, at A., the first DNA solution 12 is exposed to a first SPRI bead solution 14A. The first SPRI bead solution 14A is used to remove very small fragments (e.g., 100 bp or less), and thus the first volume ratio (SPRI bead solution 14A:DNA solution 12) that is used is relatively high. In an example, the first volume ratio is 1.6x ± 0.5%. Within the mixture of 12 and 14A, the smaller fragments 10A remain unattached from the SPIR beads 16, and larger fragments 10B (greater than 100 bp) attach to the SPRI beads 16.

[0051] At B. in Fig. 1A, the unattached smaller fragments 10A are removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 16 and the larger fragments 10B attached thereto within the container than holds the mixture 12, 14A, thus effectively pulling them out of solution. While the beads 16 and larger fragments 10B are retained by the magnetic field, the unattached smaller fragments and solution are removed. These components can be removed via aspiration.

[0052] The magnetic field can be then removed, leaving the SPRI beads 16 and the larger fragments 10B attached thereto in the container. At B. in Fig. 1A, the larger fragments 10B are eluted from the SPRI beads 16. Elution can take place under aqueous conditions, and thus water or TRIS buffer (e.g., 100 mM, pH 8) may be added to the container housing the SPRI beads 16 and the larger fragments 10B attached thereto. Elution can take place at a temperature ranging from about 10°C to room temperature (e.g., from about 18°C to about 22°C). Once the larger fragments 10B are eluted, the SPRI beads 16 can be removed using a magnet. More specifically, the beads 16 are attracted to the magnet, and the supernatant solution is collected.

[0053] At this step in the workflow, the larger fragments 10B may be referred to as the remaining fragments from the DNA solution 14A.

[0054] As shown in Fig. 1A, at C., end repair is performed on the remaining fragments 10B. An end repair mix is added to the container housing the remaining fragments 10B. This mix converts the damaged or incompatible 5’- and / or 3’- protruding ends of the fragments 10B to 5’-phosphorylated, blunt-ended DNA. In one example, the end repair mix includes DNA Polymerase I, Large (Klenow) Fragment, and end repair takes place at 25°C for 15 minutes. In another example, the end repairmix includes T4 DNA Polymerase, and end repair takes place at 15°C for 12 minutes. In still another example, the end repair mix includes T4 Polynucleotide Kinase, and end repair takes place at 37°C for 30 minutes.

[0055] The end repaired fragments 10Cs and 10CL (also referred to as remaining fragments) are then exposed to back-to-back removal and retaining steps. Thus, sequentially performed SPRI steps are then performed, as shown schematically in D. and E. of Fig. 1A. The end repaired fragments 10Cs and 10CL are used to generate a second DNA solution 12’, which is used in the sequentially performed SPRI steps.

[0056] As shown in Fig. 1A, at D., the second DNA solution 12’ is exposed to a second SPRI bead solution 14B. The second SPRI bead solution 14B is used to remove very large fragments 10CL (e.g., greater than 1500 bp), and thus the second volume ratio (SPRI bead solution 14B:DNA solution 12’) is relatively low. In an example, the second volume ratio is 0.42x ± 0.5%. Within the mixture of 12’ and 14B, the larger fragments 10CL (greater than 1500 bp) attach to the SPRI beads 16, and the smaller fragments 10Cs remain unattached from the SPRI beads 16.

[0057] In this step of the workflow, the SPRI beads 16 and the larger fragments 10CL attached thereto are removed from the mixture 12’, 14B, e.g., using a magnet. The unattached smaller fragments 10Cs remain in the solution. Thus, the first of the back-to-back steps removes first remaining fragments (i.e., the larger fragments 10CL) from the mixture 12’, 14B.

[0058] The remainder of the second DNA solution 12’ (containing the smaller fragments 10Cs) is exposed to a third SPRI bead solution 14C. The third SPRI bead solution 14C is used to remove the smallest of the remaining smaller fragments 10Cs (e.g., 700 bp or less, identified by 10Css), and thus the third volume ratio (SPRI bead solution 14C: DNA solution 12’) that is used is smaller than the first volume ratio and greater than the second volume ratio. In an example, the third volume ratio is 0.61 x ± 0.5%. In another example, the third volume ratio is 0.55x ± 0.5%. Within the mixture of 12’ and 14C, the larger of the smaller fragments 10Cs (greater than 700 bp, identified by 10CSL) attach to the SPRI beads 16, and the smallest of the smaller fragments 10Css (700 bp or less) remain in the solution and unattached from the SPRI beads 16.

[0059] At F. in Fig. 1 A, the unattached smallest of the smaller fragments 10Css are removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 16 and the larger of the smaller fragments 10CSL attached thereto within the container than holds the mixture 12’, 14C, thus effectively pulling them out of solution. While the beads 16 and larger of the smaller fragments 10CSL are retained by the magnetic field, the unattached smaller fragments 10Css and solution are removed. These components can be removed via aspiration. Thus, the second of the back-to-back steps retains second remaining fragments (i.e. , the larger of the smaller fragments 10CSL) from the mixture 12’, 14C.

[0060] The magnetic field can be then removed, leaving the SPRI beads 16 and the larger of the smaller fragments 10CSL attached thereto in the container. At F. in Fig. 1A, the larger of the smaller fragments 10CSL are eluted from the SPRI beads 16.Elution can take place under aqueous conditions, and thus water may be added to the container housing the SPRI beads 16 and the larger of the smaller fragments 10CSL attached thereto. Once the larger of the smaller fragments 10CSL are eluted, the SPRI beads 16 can be removed using a magnet. More specifically, the beads 16 are attracted to the magnet, and the supernatant solution is collected.

[0061] At G. in Fig. 1 A, the larger of the smaller fragments 1 OCSL (i.e. , the second remaining fragments) are then exposed to A-tailing and ligation to form sample fragments 10D (shown at H. in Fig. 1A). A-tailing can be performed with one or combination of the following enzymes: Taq Polymerase (active at 72°C for about 20 minutes) and Klenow Fragment (3'— 5' exo-) (active at 37°C for about 30 minutes). The adapters that are to be ligated to the A-tailed fragments each contain a 'T'-base overhang, which provides a complementary overhang for ligating the adapter to the A- tailed fragmented DNA. Ligase enzymes are used to connect the adapters to both ends of the fragments 1 OCSL.

[0062] The sample fragments 10D are used to generate a third DNA solution 12”, which is used in a second set of sequentially performed SPRI steps.

[0063] As shown in Fig. 1 , at H., the third DNA solution 12” is exposed to a fourth SPRI bead solution 14D. The fourth SPRI bead solution 14D is used to remove the smaller of the remaining sample fragments 10D (e.g., 750 bp or less, or 400 bp orless), and thus the fourth volume ratio (SPRI bead solution 14D:DNA solution 12”) is less than the first volume ratio and greater than the third volume ratio. In an example, the fourth volume ratio is 0.7x ± 0.5%. In another example, the fourth volume ratio is 0.85x ± 0.5%. Within the mixture of 12” and 14D, the larger fragments 1 ODL. (greater than 750 bp) attach to the SPRI beads 16, and the smaller fragments 10D remain in the mixture unattached from the SPRI beads 16.

[0064] At I. in Fig. 1 , the unattached smaller fragments 10D have been removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 16 and the larger fragments 10DL attached thereto within the container than holds the mixture 12”, 14D, thus effectively pulling them out of solution. While the beads 16 and larger fragments 1 ODL are retained by the magnetic field, the unattached smaller fragments 10D and solution are removed. These components can be removed via aspiration. After this, first sample fragments (i.e. , larger fragments 10DL) are retained and used in the second of the back-to-back SPRI steps.

[0065] The first sample fragments / larger sample fragments 1 ODL are released / eluted from the SPRI beads 16, and the latter are removed, e.g., using a magnet. Elution may be performed as described herein.

[0066] A fourth DNA solution 12”’ is then generated with the first sample fragments / larger sample fragments 10DL. Second sample fragments 10DL2 are retained in the fourth DNA solution 12”’ using a fifth SPRI bead solution 14E at a fifth volume ratio of the fifth SPRI bead solution 14E:the fourth DNA solution 12’”. The fifth SPRI bead solution 14E is used to remove the smaller of the remaining sample fragments 10D (e.g., 400 bp or less), and thus the fifth volume ratio (SPRI bead solution 14E:DNA solution 12’”) is less than the first volume ratio and greater than or equal to the fourth volume ratio. In an example, the fifth volume ratio is 0.85x ± 0.5%. Within the mixture of 12’” and 14E, the larger fragments 10DL2 (greater than 400 bp) attach to the SPRI beads 16, and the smaller fragments 10DL remain in the solution and unattached from the SPRI beads 16.

[0067] The unattached fragments 10DL are then removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads16 and the larger of the smaller fragments 10DL2 attached thereto within the container than holds the mixture 12”’, 14F, thus effectively pulling them out of solution. While the beads 16 and fragments 10DL2 are retained by the magnetic field, the unattached smaller fragments 10DL and solution are removed. These components can be removed via aspiration. The magnetic field can be then removed, leaving the SPRI beads 16 and the fragments 10DL2 attached thereto in the container. The fragments 10DL2 are then eluted and separated from the SPRI beads 16.

[0068] These fragments 10DL2 have library sizes ranging from about 800 bp to about 2000 bp, and can be used in clustering and sequencing on a flow cell surface. Suitable flow cells include those sold by Illumina, Inc. for sequencing-by-synthesis (SBS). During SBS, shorter fragments within the library may preferentially cluster, and thus the median insert size of the generated template strands may range from about 800 bp to about 1500 bp.

[0069] In one example of the first method, the following volume ratios are used: the first volume ratio is 1.6x ± 0.5%; the second volume ratio is 0.42x ± 0.5%; the third volume ratio is 0.61 x ± 0.5%; the fourth volume ratio is 0.7x ± 0.5%; and the fifth volume ratio is 0.85x ± 0.5%. In another example of the first method, the following volume ratios are used: the first volume ratio is 1 ,6x ± 0.5%; the second volume ratio is 0.42x ± 0.5%; the third volume ratio is 0.55x ± 0.5%; the fourth volume ratio is 0.85x ± 0.5%; and the fifth volume ratio is 0.85x ± 0.5%.

[0070] Second Method

[0071] The second method disclosed herein also includes a specific workflow, during which specific solutions and solution ratios are utilized. Like the first method, the second method enables the size selection of relatively long DNA fragments, thus enabling a library with long insert strands (having a median insert size ranging from about 800 bp to 1500 bp) to be generated for sequencing operations without having to perform PCR. Thus, the following method is performed, in order, without preforming PCR.

[0072] Fig. 1 B schematically illustrates an example of the second method, which generally includes sequentially: removing first fragments from a DNA solution using afirst solid-phase reversible immobilization (SPRI) bead solution at a first volume ratio of the first SPRI bead solution:the DNA solution (Fig. 1 B, A.) and retaining second fragments in the DNA solution using a second SPRI bead solution at a second volume ratio of the second SPRI bead solution:the DNA solution that is greater than the first volume ratio (Fig. 1 B, B.); performing end repair on the retained second fragments in the DNA solution (Fig. 1 B, D.); generating a second DNA solution with the end repaired retained second fragments; sequentially: removing third fragments from the second DNA solution using a third SPRI bead solution at a third volume ratio of the third SPRI bead solution:the second DNA solution that is less than the second volume ratio (Fig 1 B, E.) and retaining fourth fragments in the second DNA solution using a fourth SPRI bead solution at a fourth volume ratio of the fourth SPRI bead solution:the second DNA solution that is greater than the third volume ratio (Fig. 1 B, F); exposing the fourth fragments to A-tailing and ligation to form sample fragments (Fig. 1 B, H.); generating a third DNA solution with the sample fragments; and sequentially: retaining first sample fragments in the third DNA solution using a fifth SPRI bead solution at a fifth volume ratio of the fifth SPRI bead solution:the third DNA solution that is greater than the fourth volume ratio (Fig. 1 B, I), releasing the first sample fragments, generating a fourth DNA solution with the first sample fragments, and retaining second sample fragments in the fourth DNA solution using the fifth SPRI bead solution at a sixth volume ratio of the fifth SPRI bead solution:the fourth DNA solution that is equal to the fourth volume ratio (Fig. 1 B, J); wherein each of the SPRI bead solutions includes SPRI beads 16 targeting a fragment size greater than the target median DNA insert size.

[0073] While specific examples of the ratios are described herein, the second method may be used to target other insert sizes by utilizing differently sized SPRI beads 16 and by varying the ratios used throughout the method, as long as the three back-to-back SPRI processes are used and the ratios follow this protocol: the second volume ratio is greater than the first volume ratio, the third volume ratio is less than the second volume ratio, the fourth volume ratio is greater than the third volume ratio, and the fifth volume ratio is greater than the fourth volume ratio.

[0074] The DNA sample used in the second method may be genomic DNA (gDNA) that can be isolated from one or more cells, bodily fluids (e.g., whole blood, blood spots, saliva) or tissues. At the outset of the second method, the DNA sample is fragmented using any suitable technique described herein with respect to the first method. In one specific example, prior to removing fragments from the DNA solution, the second method further comprises: shearing a DNA sample; and generating the DNA solution with the sheared DNA sample.

[0075] The DNA fragments generated from the DNA sample are used in the second method, which will be described in more detail in reference to Fig. 1 B. In this figure, the DNA fragments 20 are the initial fragments generated from the DNA sample, and variations of reference numeral 20, such as 20A, 20B, etc. are used to identify specific fragments that are removed or retained or modified at a particular step in the second method workflow.

[0076] Several DNA solutions 22, 22’, 22” and SPRI bead solutions 24A, 24B, 24C, 24D, 24E, 24F are used throughout the second method.

[0077] In each DNA solution 22, 22’, 22” some DNA fragments 20 are incorporated into a liquid carrier. As shown in Fig. 1 B, the type of fragments, e.g., 20A, 20B, etc. (e.g., small or large) that remains in solution from one step of the workflow to another may change. As such, the amount of DNA fragments 20 present in any of the DNA solutions 22, 22’, 22” may differ from one step of the workflow to another.

[0078] The liquid carrier in each DNA solution 22, 22’, 22” is an aqueous buffer. Any of the example aqueous buffers set forth herein may be used.

[0079] Each example of the SPRI bead solution 24A, 24B, etc. used throughout the second method includes paramagnetic beads (i.e. , SPRI beads 26) in a binding buffer. Each SPRI bead 26 is 1 pm ± 8% in size, and includes a buoyant polystyrene core surrounded by a thin layer of magnetite. On the surface, the SPRI beads 26 are coated with carboxyl molecules, which provide charge groups for DNA fragment binding. While SPRI beads 26 are referenced, it is to be understood that other commercially available size selection beads may be used, such as Illumina Purification Beads (Illumina Inc.) or AMPure XP beads (Beckman).

[0080] The SPRI beads 26 are selected to target a fragment size that is greater than the target median DNA insert size. In one example, the target median DNA insert size ranges from about 800 base pairs (bp) to about 1300 bp; and the SPRI beads 26 target a library fragment size of 1500 bp.

[0081] The binding buffer of each SPRI bead solution 24A, 24B, etc. provides salt ions and adjusts the pH in order to facilitate the binding of some of the fragments 20 and the SPRI beads 26. Any of the example binding buffer set forth herein may be used.

[0082] The SPRI bead solutions 24A, 24B, etc. may also include a crowding agent to aid in forcing fragments 20 of a particular size out of the solution 24A, 24B, etc. Any of the example crowding agent set forth herein may be used in any of the amounts set forth herein.

[0083] Specific examples of the SPRI bead solution 24A, 24B, etc. that may be used to retain larger fragments includes SPRI beads 26 that target a fragment size of 1500 bp, from about 10 wt% to about 18 wt% of poly(ethylene glycol) having a weight average molecular weight ranging from about 8,000 g / mol to about 20,000 g / mol, , from about 2 M to about 4 M of LiCI or NaCI, about 10 mM TRIS buffer, and water.

[0084] The first DNA solution 22 used in the second method includes fragments 20 of the DNA sample in an example of the liquid carrier. The fragments 20 may be present in an amount ranging from about 2 pg to about 3 pg. The fragments 20 are exposed to back-to-back removal and retaining steps, as shown in Fig. 1 B, A. and B.

[0085] As shown in Fig. 1 B, at A., the first DNA solution 22 is exposed to a first SPRI bead solution 24A. The first SPRI bead solution 24A is used to remove first (very large) fragments 1 OAL (e.g., those greater than 1500 bp), and thus a first volume ratio (SPRI bead solution 24A:DNA solution 22) is used that is relatively low. In an example, the first volume ratio used in the second method is 0.44x ± 0.5%. Within the mixture of 22 and 24A, the larger fragments 20AL attach to the SPRI beads 26, and the smaller fragments 2OAs remain unattached from the SPRI beads 26.

[0086] In this step of the workflow, the SPRI beads 26 and the larger fragments 10AL attached thereto are removed from the mixture 22, 24A, e.g., using a magnet. The unattached smaller fragments 10As remain in the solution. Thus, the first of theback-to-back steps removes first fragments (i.e. , the larger fragments 10AL) from the mixture 22, 24A.

[0087] The remainder of the DNA solution 22 (containing the smaller fragments 10As) is exposed to a second SPRI bead solution 24B. This is shown in Fig. 1 B, at B. The second SPRI bead solution 24B is used to retain second fragments 20ASL, which are the larger of the remaining smaller fragments 2OAs. The second fragments 20ASL are greater than 800 bp. In other words, the second SPRI bead solution 24B is used to remove the smallest of the remaining smaller fragments 2OAs, which are 800 bp or less and are identified by 10As2. Thus, the second volume ratio (SPRI bead solution 24B:DNA solution 22) is greater than the first volume ratio. In an example, the second volume ratio used in the second method is 0.50x ± 0.5%. Within the mixture of 22 and 24B, the larger fragments 20ASL of the remaining smaller fragments 2OAs attach to the SPRI beads 26, and the smallest fragments 20As2 of the remaining smaller fragments 10As remain in the solution and unattached from the SPRI beads 26.

[0088] At C. in Fig. 1 B, the unattached smallest fragments 20As2 are removed from the mixture 22, 24B. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 26 and the larger fragments 20ASL attached thereto within the container than holds the mixture 22, 24B, thus effectively pulling them out of solution. While the beads 26 and larger fragments 20ASL are retained by the magnetic field, the unattached smaller fragments 20As2 and solution are removed. These components can be removed via aspiration. Thus, the second of the back-to-back steps in the second method retains the larger fragments 20ASL from the mixture 22, 24B.

[0089] The magnetic field can be then removed, leaving the SPRI beads 26 and the larger fragments 20ASL attached thereto in the container. At C. in Fig. 1 B, the larger fragments 20ASL are eluted from the SPRI beads 26. Elution can take place as described herein, e.g., under aqueous conditions. Thus, water or TRIS buffer (e.g., 100 mM, pH 8) may be added to the container housing the SPRI beads 26 and the attached larger fragments 20ASL. Elution can take place at a temperature ranging from about 10°C to room temperature (e.g., from about 18°C to about 22°C). Once the larger fragments 20ASL are eluted, the SPRI beads 26 can be removed using amagnet. More specifically, the beads 26 are attracted to the magnet, and the supernatant solution is collected.

[0090] As shown in Fig. 1 B, at D., end repair is performed on the retained second fragment (i.e., the larger fragments 20ASL), generating fragments 20B. An end repair mix is added to the container housing the larger fragments 20ASL. This mix converts the damaged or incompatible 5’- and / or 3’-protruding ends of the fragments 20ASL to 5’-phosphorylated, blunt-ended DNA (i.e., fragments 20B). In one example, the end repair mix includes DNA Polymerase I, Large (Klenow) Fragment, and end repair takes place at 25°C for 15 minutes. In another example, the end repair mix includes T4 DNA Polymerase, and end repair takes place at 15°C for 12 minutes. In still another example, the end repair mix includes T4 Polynucleotide Kinase, and end repair takes place at 37°C for 30 minutes.

[0091] The end repaired fragments 20B are then exposed to a second set of back- to-back removal and retaining steps. Thus, sequentially performed SPRI steps are then performed, as shown schematically in E. and F. of Fig. 1 B. The end repaired fragments 20B are used to generate a second DNA solution 22’, which is used in the sequentially performed SPRI steps.

[0092] As shown in Fig. 1 B, at E., the second DNA solution 22’ is exposed to a third SPRI bead solution 24C. The third SPRI bead solution 24C is used to remove third fragments, which are very large fragments 20BL, i.e., those greater than 1500 bp. Thus, the third SPRI bead solution 24C has a third volume ratio (SPRI bead solution 24C:DNA solution 22’) that is relatively low. In an example, the third volume ratio used in the second method is 0.44x ± 0.5%. Within the mixture of 22’ and 24C, the larger fragments 1 OBL. attach to the SPRI beads 26, and the smaller fragments 2OBs remain unattached from the SPRI beads 26.

[0093] In this step of the workflow, the SPRI beads 26 and the larger fragments 20BL attached thereto are removed from the mixture 22’, 24C, e.g., using a magnet. The unattached smaller fragments 2OBs remain in the solution. Thus, the first of these back-to-back steps removes the larger fragments 20BL) from the mixture 22’, 24C.

[0094] The remainder of the second DNA solution 22’ (containing the smaller fragments 2OBs) is exposed to a fourth SPRI bead solution 24D. The fourth SPRIbead solution 24D is used to remove the smallest of the remaining smaller fragments 2OBs (e.g., 750 bp or less, identified by 20Bss), and thus the fourth volume ratio (SPRI bead solution 24D:DNA solution 22’) is greater than the third volume ratio. In an example, the fourth volume ratio in the second method is 0.53x ± 0.5%. Within the mixture of 22’ and 24D, the larger of the smaller fragments 10Bs, which are greater than 750 bp and identified by 20BSL, attach to the SPRI beads 26, and the smallest fragments 20Bss (750 bp or less) remain in the solution and unattached from the SPRI beads 26.

[0095] At G. in Fig. 1 B, the unattached smallest fragments 20Bss are removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 26 and the larger fragments 20BSL attached thereto within the container than holds the mixture 22’, 24D, thus effectively pulling them out of solution. While the beads 26 and attached larger fragments 20BSL are retained by the magnetic field, the unattached smallest fragments 20Bss and solution are removed. These components can be removed via aspiration. Thus, the second of these back-to- back steps retains fourth fragments (i.e. , the larger of the smaller fragments 10Bs) from the mixture 22’, 24D.

[0096] The magnetic field can be then removed, leaving the SPRI beads 26 and the attached larger fragments 20BSL in the container. At G. in Fig. 1 B, the larger fragments 20BSL are eluted from the SPRI beads 26. Elution can take place as described herein. Once the larger fragments 20BSL are eluted, the SPRI beads 26 can be removed using a magnet. More specifically, the beads 26 are attracted to the magnet, and the supernatant solution is collected.

[0097] At H. in Fig. 1 B, the larger fragments 20BSL (i.e., the fourth remaining fragments) are then exposed to A-tailing and ligation to form sample fragments 20C (shown at I. in Fig. 1 B). A-tailing can be performed with one or combination of the following enzymes: Taq Polymerase (active at 72°C for about 20 minutes) and Klenow Fragment (3'— >5' exo-) (active at 37°C for about 30 minutes). The adapters that are to be ligated to the A-tailed fragments each contain a 'T'-base overhang, which provides a complementary overhang for ligating the adapter to the A-tailed fragmented DNA.Ligase enzymes are used to connect the adapters to both ends of the fragments 20BSL.

[0098] The sample fragments 20C are used to generate a third DNA solution 22”, which is used in a third set of sequentially performed SPRI steps.

[0099] As shown in Fig. 1 B, at I., the third DNA solution 22” is exposed to a fifth SPRI bead solution 24E. The fifth SPRI bead solution 24E is used to remove the smaller of the remaining sample fragments 20C (e.g., 400 bp or less), and thus has a fifth volume ratio (SPRI bead solution 24E:DNA solution 22”) that is greater than the fourth volume ratio. In an example, the fifth volume ratio is 0.85x ± 0.5%. Within the mixture of 22” and 24E, the larger fragments 20CL (greater than 400 bp) attach to the SPRI beads 26, and the smaller fragments 2OCs remain in the mixture unattached from the SPRI beads 26.

[0100] Prior to J. in Fig. 1 B, the unattached smaller fragments 2OCs have been removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 26 and the larger fragments 20CL attached thereto within the container than holds the mixture 22”, 24E, thus effectively pulling them out of solution. While the beads 26 and larger fragments 20CL are retained by the magnetic field, the unattached smaller fragments 2OCs and solution are removed. These components can be removed via aspiration. After this, first sample fragments (i.e., larger fragments 20CL) are retained and used in the second of these back-to-back SPRI steps.

[0101] The first sample fragments / larger sample fragments 20CL are released / eluted from the SPRI beads 26, and the latter (i.e., the beads 26) are removed, e.g., using a magnet. Elution may be performed as described herein.

[0102] A fourth DNA solution 22”’ is then generated with the first sample fragments / larger sample fragments 20CL. The fourth DNA solution 22”’ includes both larger sample fragments 20CLL and smaller sample fragments 20CLS. Second sample fragments, i.e., the larger fragments 20CL2, are retained in the fourth DNA solution 22’” using the fifth SPRI bead solution 24E at a sixth volume ratio of the fifth SPRI bead solution 24E:the fourth DNA solution 12’”. The sixth volume ratio is equal to the fifth volume ratio. Thus, the fifth SPRI bead solution 14E is again used to removeremaining smaller fragment 20CLS, which are 400 bp or less. In an example, the sixth volume ratio is 0.85x ± 0.5%. Within the mixture of 22”’ and 24F, the larger fragments 20CLL (greater than 400 bp) attach to the SPRI beads 26, and the smaller fragments 20CLS remain in the solution and unattached from the SPRI beads 26.

[0103] The unattached smaller fragments 20CLS are then removed. While not illustrated, it is to be understood that a magnetic field can be used to consolidate the SPRI beads 26 and the larger fragments 20CLL attached thereto within the container, thus effectively pulling them out of solution. While the beads 26 and larger fragments 20CLL are retained by the magnetic field, the smaller fragments 20CLS and solution are removed. These components can be removed via aspiration. The magnetic field can be then removed, leaving the SPRI beads 26 and the fragments 20CLL attached thereto in the container. The fragments 20CLL are then eluted and separated from the SPRI beads 26.

[0104] These fragments 20CLL have library sizes ranging from about 800 bp to about 2000 bp, and can be used in clustering and sequencing on a flow cell surface. Suitable flow cells include those sold by Illumina, Inc. for sequencing-by-synthesis (SBS). During SBS, shorter fragments within the library may preferentially cluster, and thus the median insert size of the generated template strandsmay range from about 800 bp to about 1500 bp.

[0105] In one example of the second method, the following volume ratios are used: the first volume ratio is 0.44x ± 0.5%; the second volume ratio is 0.50x ± 0.5%; the third volume ratio is 0.44x ± 0.5%; the fourth volume ratio is 0.53x ± 0.5%; and the fifth and sixth volume ratios are each 0.85x ± 0.5%.

[0106] The use of SPRI beads 16, 26 in the first and second methods disclosed herein provides several advantages. The methods can be automated and can tolerate both low and high concentrations of the DNA sample, which is unlike gel electrophoresis methods. The methods provide consistency and a high library yield.

[0107] To further illustrate the present disclosure, examples are given herein. It is to be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure.NON-LIMITING WORKING EXAMPLES

[0108] Example 1

[0109] An example of the first method disclosed herein (referred to as Method A) and a comparative method (referred to as Comparative Method B) were performed to determine the effect of the workflow, the ratio, and the presence of back-to-back SPRI steps. Four samples (1 -4) and four comparative examples (5-8) were respectively prepared using the method A and comparative method B. The example workflow (Method A) and comparative workflow (Comparative Method B) are shown in Table 1.

[0110] For each SPRI step in the workflow(s), the SPRI bead solution included: 10 mg / ml SPRI beads, 18 wt% poly(ethylene glycol) having a weight average molecular weight of about 8,000 g / mol, 2.15 M NaCI, 10 mM TRIS buffer, and a balance of water.TABLE 1

[0111] For quality control analysis, Bioanalyzer analysis was used for DNA sample size analysis, and qPCR was used for concentration analysis. The Bioanalyzer analysis was performed by injecting a small amount (1 :50 dilution factor) of therespective library DNA sample (generated via Method A or Comparative Method B) into an Agilent Bioanalyzer.

[0112] The Bioanalyzer analysis results for the example samples (generated with Method A) are shown in Fig. 2A and the Bioanalyzer analysis results for the comparative samples (generated with Comparative Method B) are shown in Fig. 2B. The comparative samples exhibited a higher noise level from 50 bp to 700 bp. Fig. 2C depicts respective curves from Fig. 2A and Fig. 2B within the time (seconds, X axis) that the two overlapped. This comparison illustrates that smaller fragments were removed via Method A, but not using Comparative Method B.

[0113] The qPCR (concentration) results are shown in Table 2.TABLE 2

[0114] The variation observed between samples 1 -4 (prepared via Method A) is within the normal range of multi-step workflow. Comparing the results of Example Samples 1 -4 with Comparative Samples 5-8, these results clearly demonstrate that the particular order of Method A contributes to a higher yield of fragments of the targeted size.

[0115] The sample fragments for one of the Example Samples was introduced into a patterned flowcell used with the NOVASEQ™ 6000 Sequencing System (Illumina Inc.) and exposed to clustering in accordance with the system. The clusters were sequenced using the system. The number of fragments at a particular insert size from the readl and read2 positions was determined from sequencing. The number of fragments versus the insert size is shown in Fig. 3. As depicted, the predominant insert size ranges from about 750 bp to about 1200 bp. The results in Fig. 2C indicate that non-trivial amounts of small fragments were present in the Comparative Samples, and thus these sample were not qualified for sequencing.

[0116] Example 2

[0117] A portion of Method A was performed twice to illustrate the effect of the ratio or SPRI bead solution: DNA sample solution. These methods are referred to as Method C and Method D. Two samples (9 and 10) were prepared using Methods C and D, respectively, as shown in Table 3.TABLE 3

[0118] After workflow step #4, the insert size of the fragments was determined using Bioanalyzer analysis as described in Example 1 , and the results are shown in Fig. 4. As depicted, the insert size of the fragments (sample 10) generated with the smaller ratio (0.4x used in Method D) was significantly increased compared to the insert size of the fragments (sample 9) generated with the larger ratio (0.44x used in Method C). The results in Fig. 4 illustrate the importance of the ratio selection.

[0119] Example 3

[0120] A portion of Method A (referred to as Method E) and a portion of Comparative Method B (referred to as Comparative Method F) were performed to illustrate the effect the back-to-back SPRI steps. One sample (11) and one comparative sample (12) were respectively prepared using Method E and Comparative Method F. The example workflow (Method E) and comparative workflow (Comparative Method F) are shown in Table 4.

[0121] For each SPRI step in the workflow(s), the SPRI bead solution was the same as described in Example 1.TABLE 4

[0122] Bioanalyzer analysis was performed as described in Example 1. The bioanalyzer analysis results for the example samples are shown in Fig. 5. The insert size of the comparative sample 12 (formed with Comparative Method F) was about 824 bp, while the insert size of the example sample 11 was about 1700 bp (formed with Method E). The results in Fig. 5 illustrate the importance of the back-to-back SPRI steps.

[0123] Example 4

[0124] Examples of the first and second methods (respectively referred to as Method G and Method H) described herein were performed to determine the effect of the workflow, the ratio, and the presence of back-to-back SPRI steps. Seven samples (13-19) were exposed to the first method (Method G) and four samples (20-23) were exposed to the second method (Method H). The workflows are shown in Table 5.

[0125] For each SPRI step in the workflow(s), the SPRI bead solution included: 10 mg / ml SPRI beads, 18 wt% poly(ethylene glycol) having a weight average molecular weight of about 8,000 g / mol, 2.15 M NaCI, 10 mM TRIS buffer, and a balance of water.TABLE 5

[0126] For quality control analysis, Bioanalyzer analysis was used for DNA sample size analysis, and qPCR was used for concentration analysis. The Bioanalyzer analysis was performed by injecting a small amount (1 :50 dilution factor) of the respective library DNA sample (generated via Method G or Example H) into an Agilent Bioanalyzer.

[0127] The Bioanalyzer analysis results for the Method G samples are shown in Fig. 6A and the Bioanalyzer analysis results for the Method H samples are shown in Fig. 6B. These results illustrate that the minimum onset size for the libraries generated with both methods was above 500 bp. For the samples generated using Method G, the minimum onset size was about 550 bp, and the samples generated using Method H, the minimum onset size was about 700 bp. The median insert size for the templates of samples 13-19 (prepared with Method G) was about 800 bp, and the median insert size for the templates of of samples 20-23 (prepared with Method H) was about 940 bp. Both of the methods are suitable for obtaining the higher insert sizes.

[0128] Additional Notes

[0129] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matterdisclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0130] Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

[0131] While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

Claims

What is claimed is:1 . A method for obtaining a target median deoxyribonucleic acid (DNA) insert size, comprising: removing fragments from a DNA solution using a first solid-phase reversible immobilization (SPRI) bead solution at a first volume ratio of the first SPRI bead solution:the DNA solution; performing end repair on remaining fragments from the DNA solution, thereby forming end repaired fragments; generating a second DNA solution with the end repaired fragments; sequentially: removing first remaining fragments from the second DNA solution using a second SPRI bead solution at a second volume ratio of the second SPRI bead solution:the second DNA solution that is less than the first volume ratio; and retaining second remaining fragments in the second DNA solution using a third SPRI bead solution at a third volume ratio of the third SPRI bead solution:the second DNA solution that is less than the first volume ratio and greater than the second volume ratio; exposing the second remaining fragments to A-tailing and ligation to form sample fragments; generating a third DNA solution with the sample fragments; and sequentially: retaining first sample fragments in the third DNA solution using a fourth SPRI bead solution at a fourth volume ratio of the fourth SPRI bead solution:the third DNA solution that is less than the first volume ratio and greater than the third volume ratio; releasing the first sample fragments; generating a fourth DNA solution with the first sample fragments; and retaining second sample fragments in the fourth DNA solution using a fifth SPRI bead solution at a fifth volume ratio of the fifth SPRI bead solution:thefourth DNA solution that is less than the first volume ratio and greater than or equal to the fourth volume ratio; wherein each of the SPRI bead solutions includes SPRI beads targeting a library fragment size greater than the target median DNA insert size.

2. The method as defined in claim 1 , wherein: the first volume ratio is 1 ,6x ± 0.5%; the second volume ratio is 0.42x ± 0.5%; the third volume ratio is 0.61 x ± 0.5%; the fourth volume ratio is 0.7x ± 0.5%; and the fifth volume ratio is 0.85x ± 0.5%.

3. The method as defined in claim 1 , wherein: the first volume ratio is 1 ,6x ± 0.5%; the second volume ratio is 0.42x ± 0.5%; the third volume ratio is 0.55x ± 0.5%; the fourth volume ratio is 0.85x ± 0.5%; and the fifth volume ratio is 0.85x ± 0.5%.

4. The method as defined in any one of claims 1-3, wherein each of the SPRI bead solutions includes about 18 wt% of poly(ethylene glycol) having a molecular weight of about 8,000 g / mol, 2.15 M NaCI, 10 mM TRIS buffer, and water.

5. The method as defined in any one of claims 1-4, wherein: the target median deoxyribonucleic acid (DNA) insert size ranges from about 800 base pairs (bp) to about 1300 bp; and the SPRI beads target a library fragment size of 1500 bp.

6. The method as defined in any one of claims 1-5, further comprising performing the method in order without preforming a polymerase chain reaction.

7. The method as defined in any one of claims 1-6, wherein prior to removing fragments from the DNA solution, the method further comprises: shearing a DNA sample; and generating the DNA solution with the sheared DNA sample.

8. A method for obtaining a target median deoxyribonucleic acid (DNA) insert size, comprising: sequentially: removing first fragments from a DNA solution using a first solid-phase reversible immobilization (SPRI) bead solution at a first volume ratio of the first SPRI bead solution:the DNA solution; and retaining second fragments in the DNA solution using a second SPRI bead solution at a second volume ratio of the second SPRI bead solution:the DNA solution that is greater than the first volume ratio; performing end repair on the retained second fragments in the DNA solution; generating a second DNA solution with the end repaired retained second fragments; sequentially: removing third fragments from the second DNA solution using a third SPRI bead solution at a third volume ratio of the third SPRI bead solution:the second DNA solution that is less than the second volume ratio; and retaining fourth fragments in the second DNA solution using a fourth SPRI bead solution at a fourth volume ratio of the fourth SPRI bead solution:the second DNA solution that is greater than the third volume ratio; exposing the fourth fragments to A-tailing and ligation to form sample fragments; generating a third DNA solution with the sample fragments; and sequentially: retaining first sample fragments in the third DNA solution using a fifth SPRI bead solution at a fifth volume ratio of the fifth SPRI bead solution:the third DNA solution that is greater than the fourth volume ratio;releasing the first sample fragments; generating a fourth DNA solution with the first sample fragments; and retaining second sample fragments in the fourth DNA solution using the fifth SPRI bead solution at a sixth volume ratio of the fifth SPRI bead solution:the fourth DNA solution that is equal to the fourth volume ratio; wherein each of the SPRI bead solutions includes SPRI beads targeting a library fragment size greater than the target median DNA insert size.

9. The method as defined in claim 8, wherein: the first volume ratio is 0.44x ± 0.5%; the second volume ratio is 0.50x ± 0.5%; the third volume ratio is 0.44x ± 0.5%; the fourth volume ratio is 0.53x ± 0.5%; and the fifth and sixth volume ratios are each 0.85x ± 0.5%.

10. The method as defined in any one of claims 8 or 9, wherein each of the SPRI bead solutions includes about 18 wt% of poly(ethylene glycol) having a molecular weight of about 8,000 g / mol, 2.15 M NaCI, 10 mM TRIS buffer, and water.11 . The method as defined in any one of claims 8-10, wherein: the target median deoxyribonucleic acid (DNA) insert size ranges from about 900 base pairs (bp) to about 1300 bp; and the SPRI beads target a library fragment size of 1500 bp.

12. The method as defined in any one of claims 8-11 , further comprising performing the method in order without preforming a polymerase chain reaction.

13. The method as defined in any one of claims 8-12, wherein prior to removing fragments from the DNA solution, the method further comprises: shearing a DNA sample; and generating the DNA solution with the sheared DNA sample.

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