Systems, methods, and related aspects for nucleic acid size selection

Abstract

Provided herein are methods of size selecting nucleic acid molecules that include contacting a sample that includes nucleic acid fragments with a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to solid supports and / or to charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, and separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample. Related systems, devices, compositions, and kits are also provided.

Description

SYSTEMS, METHODS, AND RELATED ASPECTS FOR NUCLEIC ACID SIZE SELECTIONCROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 63 / 651 ,731 , filed May 24, 2024, the disclosure of which is incorporated herein by reference.BACKGROUND

[0002] Library preparation for most long-read sequencing technologies follows a similar workflow. First, HMW DNA (50 kb - Mb+) must be isolated. Next, the DNA is prepared for sequencing using various enzymatic steps. During enzymatic processing, size selection is used to remove smaller background molecules from the desired library products. This is done almost exclusively with Beckman Coulter AMPURE® beads. However, AMPURE® has low recovery efficiency (<25%) for HMW DNA due to bead entanglement, preferentially losing the longest, most desired DNA molecules. This problem is exacerbated as libraries grow longer and in library preparations requiring multiple AMPURE® steps. Furthermore, the size selection cutoffs for AMPURE® (150 bp - 800 bp) are too low for most long-read libraries. Thus, a follow-up size selection is often performed using a pulsed field gel electrophoresis (PFGE) instrument such as Sage Science's BLUEPIPPIN™ (Beverly, MA) to enhance read lengths by isolating only the highest molecular weight library products. While BLUEPIPPIN™ can size select large DNA (4 kb - 40 kb), it is slow (8 hours) and also damages DNA during the long PFGE process, necessitating subsequent enzymatic repair.

[0003] Accordingly, there remains a need in the art for technologies capable of rapid size selection of both small and large nucleic acid molecules that does not require separate AMPURE® and PFGE purification steps and which does not damage the nucleic acids during processing.SUMMARY

[0004] The present disclosure relates, in certain aspects, to devices, systems, compositions, kits, and methods for selecting nucleic acids having desired sizes or lengths from pools of variable sized nucleic acid fragments. In some embodiments,mixtures of affinity tagged nucleic acid binding compounds and untagged nucleic acid binding compounds are contacted with samples that comprise the nucleic acid fragment pools to form nucleic acid fragment-bound compounds. In some embodiments, capture reagents attached to solid supports are used to select nucleic acid fragments having a minimum desired length from the nucleic acid fragment-bound compounds. These and other aspects will be apparent upon a complete review of the present disclosure, including the accompanying figures.

[0005] In one aspect, the present disclosure provides a method of size selecting nucleic acid molecules. The method includes contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds. The mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds. In addition, a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds. The method also includes contacting the set of nucleic acid fragmentbound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds (e.g., supercharged synthetic compounds or the like) under conditions sufficient to produce captured nucleic acid fragment-bound compounds. The set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds. In addition, an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound. The method also includes separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample, thereby size selecting the nucleic acid molecules in the sample.

[0006] In some embodiments, the one or more charge modulation compounds alter a net charge associated with a given nucleic acid fragment-bound compound ina captured nucleic acid fragment-bound compound that comprises the given nucleic acid fragment-bound compound. In some embodiments, the set of capture reagents are attached to the one or more charge modulation compounds and the separating step comprises separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample using an applied electric field. In some embodiments, the method further comprises separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of the captured nucleic acid fragment-bound compounds. In some embodiments, the method comprises eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds. In some embodiments, the method comprises eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents. In some embodiments, the method comprises eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using one or more elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

[0007] In some embodiments, the method comprises adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds. In some embodiments, the method comprises adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds. In some embodiments, the method comprises adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution. In some embodiments, the method comprises adjusting the conditions sufficient to produce the set of nucleic acid fragment-bound compounds and / or the conditionssufficient to produce the captured nucleic acid fragment-bound compounds, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

[0008] In some embodiments, the method comprises repeating the method at least one time under different conditions. In some embodiments, the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

[0009] In some embodiments, the method further comprises preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique. In some embodiments, the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides. In some embodiments, most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds. In some embodiments, the nucleic acid molecules comprise DNA molecules. In some embodiments, the nucleic acid molecules comprise RNA molecules. In some embodiments, the nucleic acid molecules are double-stranded. In some embodiments, the nucleic acid molecules are single-stranded.

[0010] In some embodiments, an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment. In some embodiments, the amount of the affinity tagged nucleic acid bindingcompounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds. In some embodiments, when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases. In some embodiments, the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen. In some embodiments, the method comprises estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio using an equation as follows: f(n / p) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.

[0011] In some embodiments, the capture reagents are selected from the group consisting of: a biotin molecule (e.g., standard biotin or modified biotin, such as 2-iminobiotin and biotin sulfoxide, among many others), a streptavidin molecule (e.g., standard streptavidin or modified streptavidin), an avidin molecule (e.g., standard avidin or modified avidin), an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen. In some embodiments, the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

[0012] In some embodiments, the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner. In some embodiments, the nucleic acid binding compounds comprise one or more synthetic compounds. In some embodiments, the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides (e.g., non-specifically binding poly-lysines and the like). In some embodiments, the nucleic acid binding proteins comprise RNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise double-stranded DNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise single-stranded DNA-binding proteins. In some embodiments, the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

[0013] In some embodiments, the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound. In some embodiments, the method comprises flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber. In some embodiments, the step of joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber comprises concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length.

[0014] In some embodiments, the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and, flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane. In some embodiments, one or more of the nucleic acid fragments to be cleaved are above a maximum desired length. In some embodiments, the conditions comprise mixing conditions. In some embodiments, the first and second solid supports comprise magnetic beads and wherein the method comprising creating the mixing conditions using a magnet. In some embodiments, the method comprises using an agitation technique to create the mixing conditions.

[0015] In another aspect, the present disclosure provides a system for size selecting nucleic acid molecules in a sample. The system includes a sample container receiving area structured to receive at least one sample container; a fluid handling subassembly configured to selectively convey fluid to and / or from the sample container when the sample container is received in the sample container receiving area and optionally, an electrophoresis subassembly configured to apply an electric field to the sample container when the sample container is received in the sample container receiving area; a controller operably connected at least to the fluid handling subassembly and to the electrophoresis subassembly, if present, wherein the controller comprises a processor, and a memory communicatively directly or remotely coupled to the processor, the memory storing non-transitory computer executableinstructions which, when executed by the processor with the sample container received in the sample container receiving area, perform operations comprising: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds in the sample container under conditions sufficient to produce a set of nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds in the sample container under conditions sufficient to produce captured nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound; and separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample container using the fluid handling subassembly and the electrophoresis subassembly, if present.

[0016] In some embodiments, the set of capture reagents are attached to the one or more charge modulation compounds, the electrophoresis subassembly is present, and the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample under an applied electric field using the electrophoresis subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operationscomprising: separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents using the fluid handling subassembly and / or another operably connected system subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly and one or more elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

[0017] In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly. In some embodiments, the non- transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate the conditionssufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution using the fluid handling subassembly and / or another operably connected system subassembly. In some embodiments, the non- transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the conditions sufficient to produce the set of nucleic acid fragment-bound compounds and / or the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

[0018] In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: repeating the operations at least one time under different conditions using the fluid handling subassembly and / or another operably connected system subassembly. In some embodiments, the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

[0019] In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique using the fluid handling subassembly. In some embodiments, the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at leastabout 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides. In some embodiments, most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds.

[0020] In some embodiments, an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment. In some embodiments, the amount of the affinity tagged nucleic acid binding compounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds. In some embodiments, when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio using an equation as follows: f(n / p) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment. In some embodiments, the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0021] In some embodiments, the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen. In some embodiments, the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

[0022] In some embodiments, the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner. In some embodiments, the nucleic acid binding compounds comprise one or more synthetic compounds. In some embodiments, the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides (e.g., poly-lysines and the like). In some embodiments, the nucleic acid binding proteins comprise RNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise double-stranded DNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise single-stranded DNA- binding proteins. In some embodiments, the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

[0023] In some embodiments, the nucleic acid molecules comprise DNA molecules. In some embodiments, the nucleic acid molecules comprise RNA molecules. In some embodiments, the nucleic acid molecules are double-stranded. In some embodiments, the nucleic acid molecules are single-stranded.

[0024] In some embodiments, the sample is disposed in a chamber of the sample container that comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber is configured to comprise a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having theminimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber and using the fluid handling subassembly; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length and using the fluid handling subassembly; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound and using the fluid handling subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber using the fluid handling subassembly. In some embodiments, the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length using the fluid handling subassembly.

[0025] In some embodiments, the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber is further configured to comprise set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acidfragments that are below the minimum desired length using the fluid handling subassembly; and flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane using the fluid handling subassembly. In some embodiments, one or more of the nucleic acid fragments to be cleaved are above a maximum desired length. In some embodiments, the conditions comprise mixing conditions.

[0026] In another aspect, the present disclosure provides a device that comprises at least one chamber that comprises: a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

[0027] In some embodiments, the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, andwherein conditions in the chamber are such that: two or more nucleic acid fragments in the sample having less than the minimum desired length are joined together to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber, a nucleic acid fragment-bound compound is produced in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length, and a captured nucleic acid fragmentbound compound is produced in the first section of the chamber using the nucleic acid fragment-bound compound.

[0028] In some embodiments, the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein conditions in the chamber are such that: the sample that comprises the nucleic acid fragments flows over the first and second solid supports in the first section of the chamber so that at least some of the nucleic acid fragments are cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and at least some of the cleaved nucleic acid fragments that are below the minimum desired length flow from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane.

[0029] In another aspect, the present disclosure provides a composition that comprises a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired lengtheach bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

[0030] In another aspect, the present disclosure provides a kit comprising a set of affinity tagged nucleic acid binding compounds; a set of untagged nucleic acid binding compounds; and, a set of capture reagents that selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds. In some embodiments, one or more components of the kit are disposed in one or more containers.

[0031] In some embodiments, the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen. In some embodiments, the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0032] In some embodiments, the kit further comprises one or more first solid supports and / or to one or more charge modulation compounds that are attached, or attachable, to the set of capture reagents. In some embodiments, the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate. In some embodiments, the kit further comprises a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of ligase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution. In some embodiments, the kit furthercomprises a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of nuclease and / or transposase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution.

[0033] In some embodiments, a mixture comprises the set of affinity tagged nucleic acid binding compounds and the set of untagged nucleic acid binding compounds. In some embodiments, a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of nucleic acid fragments in a sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds when the sample is contacted with the mixture under conditions sufficient to produce a set of nucleic acid fragment-bound compounds. In some embodiments, an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound when the set of nucleic acid fragment-bound compounds is contacted with the set of capture reagents under conditions sufficient to produce captured nucleic acid fragment-bound compounds.

[0034] In some embodiments, the kit further comprises one or more reagents for eluting nucleic acid fragments having a minimum desired length from other components of captured nucleic acid fragment-bound compounds. In some embodiments, the kit further comprises one or more reagents for eluting nucleic acid fragments having less than a minimum desired length from other components of captured nucleic acid fragment-bound compounds. In some embodiments, the kit further comprises one or more of the affinity tags that are free in solution. In some embodiments, the kit further comprises one or more additional reagents selected from the group consisting of: a molecular crowder, a chaotropic salt, a monovalent salt, a divalent salt, an alcohol, a polyamine, and a denaturing agent.

[0035] In some embodiments, the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner. In some embodiments, thenucleic acid binding compounds comprise one or more synthetic compounds. In some embodiments, the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides (e.g., poly-lysines and the like). In some embodiments, the nucleic acid binding proteins comprise RNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise double-stranded DNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise single-stranded DNA- binding proteins. In some embodiments, the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the devices, systems, methods, kits, and related computer readable media disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings which are included byway of example and not byway of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.

[0037] FIG. 1 schematically shows a strategy for DNA size selection via sso7d protein binding according to an exemplary embodiment.

[0038] FIG. 2 is a flow chart that schematically shows exemplary method steps of size selecting nucleic acid molecules according to some aspects disclosed herein.

[0039] FIG. 3A is a schematic diagram of an exemplary device suitable for use with some aspects disclosed herein.

[0040] FIG. 3B is a schematic diagram of an exemplary device suitable for performing methods of size-controlled concatenation according to some aspects disclosed herein.

[0041] FIG. 3C is a schematic diagram of an exemplary device suitable for performing methods of non-exponential insert size selection in a nuclease and / or transposase system according to some aspects disclosed herein.

[0042] FIG. 4 is a schematic diagram of an exemplary system suitable for use with some aspects disclosed herein.

[0043] FIG. 5 schematically shows aspects of a sso7D 7kDa thermostable, nonspecific DNA-binding protein and related constructs according to an exemplary embodiment.

[0044] FIG. 6: Size selection response to varying sso7d mixture ratios. Focusing on the 1 pM and 1.5 pM streptag-sso7d condition at different non-tagged ratios, the resulting selected libraries were analyzed on the Agilent Femto Pulse and the data were converted into a molecule density plot. The two FemtoPulse size distribution plots above demonstrate an increase in size selection ability as the ratio of nontagged is increased.

[0045] FIG. 7: Size distribution profiles comparing sso7d size selection against current methods. The indicated dashed line (WL012423_No Size Selection) represents the non-selected starting library, containing numerous short inserts of gDNA below 10K. In contrast, the solid lines (denoted as WL012423_0.5uM_300x and WL012423_0.7uM_300x, respectively) represent results achieved using the sso7d protein-based method and exhibited decent recovery and effectiveness in size selection. A comparison with current size selection protocols placed the sso7d-based method between the SRE-diluted AMPure method (dashed line labeled as WL012423_SRE_2ug) and gel-based PippinHT (dashed line labeled as WL012423_PippinHT) in terms of size selection performance.

[0046] FIG. 8: Summary table of sso7d size selection against current methods. Comparison of various parameters across the different size selection methods which illustrates potential advantages and disadvantages.

[0047] FIGS. 9A-9D: Size selection performance is reflected in Revio sequencing HiFi read length results. Revio is very sensitive to loading of smaller libraries and a prominent fraction is observed in the current standard SRE Diluted AMPure method. Conversely, sso7d-streptag appears to effectively remove fragments under 10kb compared to SRE-DilAMPure in the context of this experiment. Although gel-based size selection using Sage Science Pippin HT offers the best size selection, it poses greater risk of sample loss and does not support high-throughput processing.DEFINITIONS

[0048] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0049] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0050] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, devices, kits, compositions, systems, and computer readable media, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0051] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0052] About: As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In some embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% of a possible value or element).

[0053] Affinity level. As used herein, the term “affinity level” or sometimes simply “affinity” refers to a value that reflects a measure of the strength of a binding interaction between two or more reversibly bound molecules. In some embodiments, an affinity level is reported as an equilibrium dissociation constant (KD), among other metrics.

[0054] Affinity tag: As used herein, the term “affinity tag” refers to a portion of a chemical compound or structure that selectively or preferentially binds to another chemical compound or structure, such as a capture reagent or recognition moiety. Some exemplary affinity tags include histone-tags, streptavidin-tags, avidin-tags, aptamers, oligonucleotides, peptides, antibodies or antigen binding portions thereof, and antigens, among others. In some embodiments, an affinity tag can be adapted for use as a capture reagent.

[0055] Affinity tagged nucleic acid binding compound: As used herein, the term “affinity tagged nucleic acid binding compound” refers to a nucleic acid binding compound that is attached to an affinity tag.

[0056] Binding: As used herein, the term “binding” typically refers to a non- covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; “indirect” binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0057] Capture reagent: As used herein, the term “capture reagent” refers to a portion of a chemical compound or structure that selectively or preferentially binds to another chemical compound or structure, such as an affinity tag or recognition moiety. In some embodiments, a capture reagent includes biotin, which selectively or preferentially binds to an affinity tag, such as streptavidin or avidin. In some embodiments, a capture reagent can be adapted for use as an affinity tag.

[0058] Concatenated nucleic acid fragment. As used herein, the term “concatenated nucleic acid fragment” refers to a nucleic acid formed by joining two or more nucleic acid fragments together with one another. In some embodiments, nucleic acid fragments are joined together with one another using ligase enzymes.

[0059] In some embodiments: As used herein, the term “in some embodiments” refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0060] Mixture: As used herein, “mixture” refers to a combination of two or more different components (e.g., chemical compounds or reagents) .

[0061] Nucleic acid: As used herein, the term “nucleic acid” refers to at least two nucleotides covalently linked together. Nucleic acids include naturally occurring, modified, or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which are capable of hybridization to a complementary nucleic acid by Watson-Crick basepairing. Nucleic acids can also include nucleotide analogs (e.g., bromodeoxyuridine (BrdU)), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, cfDNA, ctDNA, or any combination thereof.

[0062] Nucleic acid binding compound: As used herein, the term “nucleic acid binding compound” refers to a molecular compound that comprises one or more nucleic acid binding domains that bind to nucleic acids in a specific or non-specific manner. In some embodiments, a nucleic acid binding compound is a nucleic acid binding protein that includes one or more DNA or RNA binding domains. Some exemplary nucleic acid binding proteins suitable for use with embodiments of the present disclosure include histone proteins, sso7d protein, and DNA stabilizing stress response proteins, among many others. In some embodiments, nucleic acid binding compounds are partially or completely synthetic compounds.

[0063] Sample: As used herein, the term “sample” means anything capable of being processed and / or analyzed using a device or system disclosed herein. Exemplary sample types include environmental samples and biological samples. In some embodiments, a sample includes a tissue or organ obtained from a subject; acell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a ceil lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a nucleic acid), which is processed or analyzed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells, cell components, or non-cellular fractions.

[0064] Selectively binds: The term “selectively binds” in the context of biomolecules refers to a ligand that binds preferentially to a given biomolecule rather than to other biomolecules. In some embodiments, for example, the capture reagents disclosed herein (e.g., a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, an antigen, or the like) preferentially bind to corresponding affinity tags (e.g., a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, an antigen, or the like), but not to other biomolecules.

[0065] Sequencing: As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, epigenetics sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, sequencing by binding (SBB), metagenome sequencing, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high- throughput sequencing, massively parallel signature sequencing, emulsion PCR, coamplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, long-read sequencing, single-molecule sequencing, sequencing-by-synthesis (SBS), real-time sequencing, reverse-terminator sequencing, SMRT sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performed by a sequencing system such as, for example, sequencing systems commercially available from Pacific Biosciences, Inc., Illumina, Inc., Oxford Nanopore Technologies, Inc., or Applied B iosystem s / Thermo Fisher Scientific, among many others.

[0066] Solid support: As used herein, the term “solid support” refers to a solid material which can be derivatized with, or otherwise attached to, a chemical moiety, such as a capture reagent. Exemplary solid supports include a microplate, a resin (e.g., an agarose resin), a bead, a microbead, a fiber, a whisker, a ceramic layer, a comb, a membrane, a crystal, and a self-assembling monolayer, among others.

[0067] System: As used herein, the term "system" in the context of medical or scientific instrumentation refers a group of objects, subassemblies, and / or devices that form a network for performing a desired objective.

[0068] Untagged nucleic acid binding compounds: As used herein, the term “untagged nucleic acid binding compounds” refers to a nucleic acid binding compound that lacks an attached affinity tag.DETAILED DESCRIPTIONI. EXEMPLARY METHODS AND RELATED DEVICES

[0069] The present disclosure relates, in certain aspects, to methods and related devices for selecting nucleic acid molecules having desired lengths or sizes (e.g., large nucleic acid molecules having about 1 ,000 or more bases or short nucleic acid molecules having less than about 1 ,000 bases) from pools of variable length nucleic acid fragments. In some embodiments, the methods disclosed herein are suitable for use under physiological conditions, thereby minimizing the risk of damaging nucleic acid template molecules. In some embodiments of the size selection methods of the present disclosure, proteins that bind cooperatively to dsDNA in a non- sequence-specific manner are purified in two forms: a tag-free version and a version carrying an affinity tag (e.g., a his-tag, a strep-tag, an inactivated SpyTag, of the like). Exemplary DNA binding proteins of use in these methods include sso7d protein (Sulfolobus solfataricus), histones and DNA stabilizing stress response proteins, among numerous others. In fact, essentially any nucleic acid binding protein that cooperatively or non-cooperatively binds with nucleic acids can be adapted for usewith the methods and other aspects of the present disclosure. In some embodiments, a solid support such as a microwell plate well surface or resin carrying a capture reagent with a selected affinity level (e.g., a moderate affinity) to the selected tag is used for the capture of the dsDNA binding protein. Immobile solid supports such as resins or plate wells are typically more amenable to high-throughput methods as disclosed herein, than many currently available options for the selection of large DNA molecules. In other exemplary embodiments, RNA-binding or single-stranded DNA (ssDNA) binding proteins are instead of dsDNA binding proteins to make the methods of the present disclosure applicable to other types of polynucleotides (e.g., RNA or ssDNA). In some embodiments, DNA binding compounds are mixed with synthetic analogs bearing an affinity tag for a similar chemistry-based approaches. These and other attributes of the present disclosure will be apparent upon a complete review of the specification, including the accompanying figures.

[0070] To illustrate, FIG. 1 schematically shows a strategy for DNA size selection via sso7d protein binding according to an exemplary embodiment. As shown, a pool of DNA molecules having different template sizes is contacted with a mixture of DNA binding proteins that either have an affinity tag (e.g., streptag sso7d) or lack the affinity tag (e.g., untagged sso7d). Statistically, larger DNA templates in the mixture will tend to have more bound affinity tagged DNA binding proteins than shorter DNA templates in the mixture. As further shown, magnetic beads having attached capture reagents (e.g., streptavidin) are then used to capture the larger templates in the mixture in some embodiments. The captured DNA templates are then typically separated from non-captured DNA templates and other reagents in the mixture.

[0071] When a relatively small fraction of the tagged variant of the DNA binding protein (e.g., < 1 %) is mixed with a pool of DNA molecules, the number of affinity tags present on a given molecule becomes a function of the number of base pairs. As the fraction of tagged DNA binding protein is reduced, the size of DNA molecules that are likely to have multiple tags increases. For example, for a DNA binding protein with a footprint of p bases, a mixture with f tagged DNA binding protein molecules to untagged DNA binding protein molecules, and a DNA molecule of n base pairs, the mean number of tags present will be f*(n / p). For any given DNA size, the fraction containing k tags will generally follow the Poisson distribution function. With sufficiently low affinity for individual binding events between the tag and theimmobilized capture reagent, one can require the presence of multiple tags for capture in the binding step. For example, by reducing f, one can control the minimum DNA size n that is likely to bind multiple affinity tags.

[0072] As a further illustration, FIG. 2 is a flow chart that schematically shows exemplary method steps of size selecting nucleic acid molecules according to some aspects disclosed herein. As shown, method 200 includes contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds (step 202). The mixture includes a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds. Examples of suitable affinity tags and nucleic acid binding compounds in addition to conditions sufficient to produce the nucleic acid fragment-bound compounds are described further herein. A ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds. Methods of adjusting this ratio are described further herein.

[0073] Method 200 also includes contacting the set of nucleic acid fragmentbound compounds with a set of capture reagents that are attached to one or more first solid supports under conditions sufficient to produce captured nucleic acid fragmentbound compounds (step 204). Examples of suitable capture reagents and solid supports binding compounds as well as conditions sufficient to produce the captured nucleic acid fragment-bound compounds are described further herein. In some embodiments, a set of capture reagents is attached to one or more charge modulation compounds (e.g., super-charged synthetic compounds or the like). The set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds. In addition, an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragmentbound compound that includes a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

[0074] In addition, method 200 further includes separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample to effect the size selection of nucleic acid molecules in the sample. In some embodiments, method 200 also includes separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of the captured nucleic acid fragment-bound compounds (e.g., unbound solid support attached capture reagents or the like). In some embodiments, method 200 includes eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds. In some embodiments, method 200 includes eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents. In other words, undesired nucleic acid fragments are eluted while the desired nucleic acid fragments remain bound to solid support attached capture reagents. In some embodiments, method 200 includes eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using one or more elution strategies that involve, for example, a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition, among other conditions.

[0075] In some embodiments, capture reagents are attached to charge modulation compounds (e.g., super-charged synthetic compounds or the like). Typically, the charge modulation compounds alter a net charge associated with a given nucleic acid fragment-bound compound in a captured nucleic acid fragmentbound compound that comprises the given nucleic acid fragment-bound compound. In some of these embodiments, method 200 includes separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample using an applied electric field.

[0076] Method 200 also typically includes preparing the nucleic acid fragments having the minimum desired length (i.e., the size selected nucleic acid molecules) foruse in performing a nucleic acid sequencing or another nucleic acid analytical technique. The particular sample preparation protocol for a given nucleic acid sequencing application will depend on the specific sequencing platform intended for use. For example, if a PacBio HiFi sequencing strategy is contemplated, then a SMRTbell library preparation protocol may be utilized, or if a PacBio Onso sequencing strategy is intended for use, then an Onso library preparation protocol may be used. Kits for SMRTbell library preparation for HiFi sequencing, HiFi barcoding, Onso library preparation, and Onso barcoding and other consumables are commercially available from Pacific Biosciences, Inc. Other sequencing strategies and related sample preparation protocols / kits might include those commercially available from Illumina, Inc., Oxford Nanopore Technologies, Inc., and Applied Biosystems / Thermo Fisher Scientific, among many others.

[0077] The conditions used when performing the methods of the present disclosure can be readily adjusted or tuned to size select nucleic acids of many different desired lengths. In some embodiments, for example, the minimum desired length of the nucleic acid fragments to be selected from a given sample is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or even more nucleotides in length. As described herein, in a given application of the methods of the present disclosure, most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are typically each bound to one or fewer affinity tagged nucleic acid binding compounds. This facilitates selecting longer nucleic acids from a pool of variable length nucleic acid fragments, because longer nucleic acids are statistically more likely to be bound by more affinity tagged nucleic acid binding compounds, than shorter nucleic acids, as described further herein. Methods of estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment are also described herein.

[0078] Many different types of nucleic acid binding compounds are optionally utilized when performing the methods of the present disclosure depending on the type of nucleic acid to be sized selected. In some embodiments, for example, the nucleicacid molecules to be size selected in a given sample are DNA and / or RNA molecules. In addition, those nucleic acid molecules are double-stranded or single-stranded. Nucleic acid binding compounds used in a given application are selected accordingly. In some embodiments, the nucleic acid binding compounds are nucleic acid binding proteins. The nucleic acid binding proteins can be RNA-binding proteins, doublestranded DNA-binding proteins, or single-stranded DNA-binding proteins, again, depending on the type of nucleic acid to be size selected in a given application. Some exemplary nucleic acid binding proteins can be histone proteins, sso7d proteins, and DNA stabilizing stress response proteins, among many others. An example utilizing sso7d proteins is provided further herein. In some embodiments, nucleic acid binding compounds can include one or more synthetic compounds capable of binding a particular nucleic acid type targeted for size selection.

[0079] Various types of affinity tags and corresponding capture reagents that selectively bind with one another are optionally utilized in performing embodiments of the methods disclosed herein. In some embodiments, for example, affinity tags and capture reagents are independently selected from biotin molecules, streptavidin molecules, avidin molecules, aptamers, oligonucleotides, peptides, antibodies or antigen binding portions thereof, and antigens, among others. In some embodiments, an amount of affinity tagged nucleic acid binding compounds in a given mixture is selected such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment. In some embodiments, for example, the amount of the affinity tagged nucleic acid binding compounds includes less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds. In some embodiments, when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases. As described herein, an exemplary method of estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio uses an equation as follows: f(n / p)where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.

[0080] In some embodiments, method 200 includes adjusting a density of a set of capture reagents that are attached to solid supports to modulate the conditions that are sufficient to produce the captured nucleic acid fragment-bound compounds. In some embodiments, method 200 includes adjusting a binding capacity of a set of capture reagents that are attached to solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds. In some of these embodiments, method 200 includes adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the solid supports with a selected amount of the affinity tags that are free in solution (e.g., when performing step 204 of method 200). Any suitable solid support is optionally utilized to perform method 200. In some embodiments, for example, a solid support can be a resin, a bead, a substrate, a well of a microwell plate, or the like.

[0081] As mentioned herein, the conditions for selecting nucleic acid fragments having a desired size according to the methods of the present disclosure can be adjusted or tuned. In some embodiments, for example, method 200 includes adjusting the conditions sufficient to produce a set of nucleic acid fragment-bound compounds and / or the conditions sufficient to produce a captured nucleic acid fragment-bound compounds. In some of these embodiments, the conditions that are adjusted are selected from a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof. In some embodiments, method 200 is repeated one or more times under different conditions. In some embodiments, the different conditions include different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in a given mixture.

[0082] The present disclosure also provides various devices of use in performing the size selection methods disclosed herein. In overview, FIG. 3A is a schematic diagram of an exemplary device suitable for use with some aspects disclosed herein. As shown, device 300 includes a chamber (e.g., a well of a microwell plate or the like) that includes a set of nucleic acid fragment-bound compounds. The nucleic acid fragment-bound compounds include nucleic acid fragments 302 from a sample pool of variable length nucleic acid fragments that are bound to affinity tagged nucleic acid binding compounds 304 (e.g., affinity tagged sso7d protein) and untagged nucleic acid binding compounds 306 (e.g., untagged sso7d protein). As described further herein, a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in a given mixture is typically selected such that a plurality of the nucleic acid fragments in a sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds 304, whereas nucleic acid fragments in the sample having less than the minimum desired length each bind to a nucleic acid binding compound of no more than one of the affinity tagged nucleic acid binding compounds 304. As additionally shown, device 300 also includes set of capture reagents 310 that are attached to a solid support (e.g., a magnetic bead or the like) 308 prior to forming captured nucleic acid fragment-bound compounds, in which affinity tags of the affinity tagged nucleic acid binding compounds 304 bind to capture reagents 310.

[0083] Sequencing platforms (e.g., SMRT Sequencing platforms or the like) typically have an optimal size for nucleic acid template molecules to maximize throughput. That optimal size is generally significantly larger than a given sample of interests (e.g., PCR products or cDNA samples from transcript libraries). Thus, various concatenation strategies have been used to generate templates having desired sizes. Some methods for insert concatenation, however, use defined sequences to drive a controlled number and order of concatenation events. These protocols work well when exact control of the order and number of steps is a desired feature, but typically involve the use of a large number of oligonucleotide reagents and molecular biology techniques. These protocols also provide less control of total insert size when the inserts are heterogenous in nature. Accordingly, controlling the number of inserts in a concatenation reaction through, for example, sequence-specific recombination and / orligation allows for the programming of the number of inserts that can be linked together in a manner that is not directly related to the total size of the given insert produced. FIG. 3B is a schematic diagram of an exemplary device suitable for performing such methods of size-controlled concatenation according to some aspects disclosed herein. As shown, device 312 includes a chamber divided by a filter or membrane 316 that is permeable to solution but impermeable to beads. As also shown, one side of the divided chamber includes capture reagents attached to beads 314, as described herein, while the other side of the divided chamber includes a ligase enzyme immobilized or attached to a set of beads 318.

[0084] As described herein, dsDNA binding proteins can be used in sizeselection by diluting a small fraction of affinity tagged proteins into a background of untagged proteins and using affinity or capture reagent resin directed towards the tag. In some embodiments, a bead-immobilized ligase enzyme and the affinity or capture reagent resin are separated by a buffer-permeable membrane. In these embodiments, the solution typically contains the DNA sample of interest, the dsDNA binding protein size-selection reagents, solution components necessary for the ligation reaction of interest and any adapters required to drive concatenation of the sample molecules.

[0085] As part of a given method for size-controlled concatenation, nucleic acid molecules that are smaller than the target size of the dsDNA binding protein reagents will have access to the chamber of device 312 containing the bead- immobilized ligase. As concatenation proceeds, the larger concatenated molecules are captured in the side of the chamber containing the affinity resin for the tagged dsDNA binding protein. Upon completion of the reaction, the affinity beads are collected and the concatenated products of target size are eluted from the affinity resin for library preparation. Thus, this exemplary method only uses a single marker for separating concatenated inserts and generates size-selected products in a “one-pot” synthesis reaction using a device, such as device 312. The insert size distribution resulting from this size-controlled concatenation method is typically much less sensitive to the distribution of fragment sizes and is applicable to heterogenous or mixed sample types.

[0086] In contrast to the size-controlled concatenation methods described herein, the process can be inverted by mixing the affinity beads with an immobilized nuclease or transposase enzyme to provide a method that produces non-exponentialsize distributions in enzymatically fragmented DNA. Given that the reactive sites of nucleases and transposase are quite small relative to the sizes of nucleic acid inserts of interest, the probability of cleavage is constant at a given DNA site independent of the fragment it is contained within. Accordingly, by flowing the reaction buffer over bead-immobilized enzyme and the size-selection bead support under mixing conditions, fragments can be removed from exposure to the fragmenting enzyme once they fall below the size threshold of interest.

[0087] To illustrate, FIG. 3C is a schematic diagram of an exemplary device suitable for performing methods of non-exponential insert size selection in a transposase system according to some aspects disclosed herein. As shown, device 320 includes a chamber having membrane 326 that divides the chamber into two sections. A first section of the chamber includes a set of capture reagents that are attached to first solid supports 324. As also shown, the first section of the chamber also includes a set of nuclease and / or transposase enzymes attached to second solid supports 322. The nucleic acid sample and the mixture of nucleic acid binding compounds are also disposed in the first section of the chamber. Device 320 also includes a second section of the chamber on the other side of membrane 326 that includes an outlet. Membrane 326 is impermeable to the first and second solid supports (e.g., beads) but is permeable to reagents in solution. As the non-exponential insert size selection method proceeds, beads 322 and 324 are mixed with one another (e.g., magnetically and / or with agitation) while the reactions occurs under flow conditions. Nucleic acid molecules in the sample that are at or above the size of interest are retained in the first section of the chamber and exposed to the nuclease and / or transposase enzymes, while nucleic acid molecules that are below the sizeselection threshold are eluted from the system, flowed through the membrane and out of the outlet of device 320, and collected in vessel 328.II. EXEMPLARY SYSTEMS AND COMPUTER READABLE MEDIA

[0088] The present disclosure also provides various systems and computer program products or machine readable media of use in performing the nucleic acid size selection methods disclosed herein. In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer programproducts, machine readable media, electronic storage media, software (e.g., machineexecutable code or logic instructions) and / or the like. To illustrate, FIG. 4 provides a schematic diagram of an exemplary system suitable for use with implementing at least aspects of the methods disclosed in this application. As shown, system 400 includes at least one controller or computer, e.g., server 402 (e.g., a search engine server), which includes processor 404 and memory, storage device, or memory component 406, and one or more other communication devices 414, 416, (e.g., client-side computer terminals, mobile phones, tablets, laptops, other mobile devices, etc. (e.g., for receiving data sets, etc.) in communication with the remote server 402, through electronic communication network 412, such as the Internet or other internetwork. Communication devices 414, 416 typically include an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 402 computer over network 412 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), a web-based user interface, and / or the like) for displaying results or status upon implementing the methods described herein. In certain aspects, communication networks also encompass the physical transfer of data from one location to another, for example, using a hard drive, thumb drive, or other data storage mechanism. System 400 also includes program product 408 (e.g., for performing the nucleic acid size selection methods as described herein) stored on a computer or machine readable medium, such as, for example, one or more of various types of memory, such as memory 406 of server 402, that is readable by the server 402, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 414 (schematically shown as a desktop or personal computer). In some aspects, system 400 optionally also includes at least one database server, such as, for example, server 410 associated with an online website having data stored thereon (e.g., entries corresponding to sample data, etc.) searchable either directly or through search engine server 402. System 400 optionally also includes one or more other servers positioned remotely from server 402, each of which are optionally associated with one or more database servers 410 located remotely or located local to each of the other servers. The other servers can beneficially provide service to geographically remote users and enhance geographically distributed operations, if desired.

[0089] As understood by those of ordinary skill in the art, memory 406 of the server 402 optionally includes volatile and / or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. It is also understood by those of ordinary skill in the art that although illustrated as a single server, the illustrated configuration of server 402 is given only by way of example and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 402 shown schematically in FIG. 4, represents a server or server cluster or server farm and is not limited to any individual physical server. The server site may be deployed as a server farm or server cluster managed by a server hosting provider. The number of servers and their architecture and configuration may be increased based on usage, demand and capacity requirements for the system 400. As also understood by those of ordinary skill in the art, other user communication devices 414, 416 in these aspects, for example, can be a laptop, desktop, tablet, personal digital assistant (PDA), cell phone, server, or other types of computers. As known and understood by those of ordinary skill in the art, network 412 can include an internet, intranet, a telecommunication network, an extranet, or world wide web of a plurality of computers / servers in communication with one or more other computers through a communication network, and / or portions of a local or other area network.

[0090] As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 408 is optionally in the form of microcode, programs, cloud computing format, routines, and / or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 408, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.

[0091] As further understood by those of ordinary skill in the art, the term "computer-readable medium" refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term "computer-readable medium" encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 408 implementing the functionality or processesof various aspects of the present disclosure, for example, for reading by a computer. A "computer-readable medium" may take many forms, including but not limited to, nonvolatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.

[0092] Program product 408 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 408, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects disclosed herein. All such operations are well known to those of ordinary skill in the art of, for example, computer systems.

[0093] In some aspects, program product 408 includes non-transitory computer-executable instructions which, when executed by electronic processor 404, perform at least: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds in the sample container under conditions sufficient to produce a set of nucleic acid fragment-bound compounds using a fluid handling subassembly, contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to solid supports in the sample container under conditions sufficient to produce captured nucleic acid fragment-bound compounds using the fluid handling subassembly, and separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample container using the fluid handling subassembly.

[0094] In some embodiments, system 400 also includes a sample container receiving area 420 structured to receive sample container 422 (e.g., a nucleic acid size selection device as described herein). System 400 also typically includes operably connected fluid handling subassembly 418 configured to selectively convey fluid to and / or from sample container 422 when sample container 422 is received in sample container receiving area 420.III. EXEMPLARY COMPOSITIONS AND KITS

[0095] Aspects of the present disclosure also provide various compositions and kits related to the nucleic acid size selection methods described herein. In some embodiments, for example, the present disclosure provides a composition that includes a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds as described herein. The composition also includes a set of capture reagents that are attached to solid supports under conditions sufficient to produce captured nucleic acid fragment-bound compounds.

[0096] In some embodiments, the kits of the present disclosure include a set of affinity tagged nucleic acid binding compounds, a set of untagged nucleic acid binding compounds, and a set of capture reagents that selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds. In some embodiments, kits also include first and optionally, second solid supports (e.g., a resin, a bead, a substrate, a well of a microwell plate, or the like) that are attached, or attachable, to the set of capture reagents. In some embodiments, the capture reagents and the corresponding affinity tags are independently selected from, for example, a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen. In some embodiments, the components of a given kit are packaged in separate containers. In some embodiments, at least some components of a given kit are combined in a single container.

[0097] In some embodiments, the kits of the present disclosure also include a device as described herein. To illustrate, in some embodiments, the kits further include a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of ligaseenzymes attached, or attachable, to the one or more second solid supports, in which the membrane is impermeable to the first and second solid supports, and in which the membrane is permeable to reagents in solution. In some embodiments, the kits further include a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of nuclease and / or transposase enzymes attached, or attachable, to the one or more second solid supports, in which the membrane is impermeable to the first and second solid supports, and in which the membrane is permeable to reagents in solution.

[0098] In some embodiments, the kits of the present disclosure include a mixture that comprises the set of affinity tagged nucleic acid binding compounds and the set of untagged nucleic acid binding compounds. In some embodiments, a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of nucleic acid fragments in a sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds when the sample is contacted with the mixture under conditions sufficient to produce a set of nucleic acid fragment-bound compounds. In some embodiments, an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound when the set of nucleic acid fragmentbound compounds is contacted with the set of capture reagents under conditions sufficient to produce captured nucleic acid fragment-bound compounds.

[0099] In some embodiments, the kits of the present disclosure also include reagents for eluting nucleic acid fragments having a minimum desired length from other components of captured nucleic acid fragment-bound compounds. In some embodiments, the kits also include reagents for eluting nucleic acid fragments having less than a minimum desired length from other components of captured nucleic acid fragment-bound compounds. In some embodiments, the kits also include affinity tags that are free in solution. In some embodiments, the kits of the present disclosure also include one or more additional reagents, such as a molecular crowder, a chaotropicsalt, a monovalent salt, a divalent salt, an alcohol, a polyamine, and a denaturing agent, among other reagents.

[0100] In some embodiments, the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner. In some embodiments, the nucleic acid binding compounds comprise one or more synthetic compounds. In some embodiments, the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides (e.g., poly-lysines and the like). In some embodiments, the nucleic acid binding proteins comprise RNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise double-stranded DNA-binding proteins. In some embodiments, the nucleic acid binding proteins comprise single-stranded DNA- binding proteins. In some embodiments, the nucleic acid binding proteins are selected from, for example, a histone protein, an sso7d protein, and a DNA stabilizing stress response protein, among other nucleic acid binding proteins.

[0101] In addition to above-referenced components, the kits of the present disclosure can further include instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper, plastic, or the like. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or subpackaging) or the like. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, or the like. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.IV. EXAMPLE: SSO7D PROTEIN-BASED SIZE SELECTION STRATEGY

[0102] INTRODUCTION

[0103] Size selection for large molecules of DNA may be accomplish by utilizing various ratios of tagged and nontagged DNA binding proteins (e.g., sso7d,among many others) to achieve a sample which contains a statistically higher probability of tags being present on the larger molecules. Samples which contain a sufficient amount of tags can then be captured onto solid supports (e.g., functionalized magnetic beads) to select for the largest fragments. Various proteins, tags, and capturing solid supports with different properties and affinities can be used to achieve this effect. This example illustrates the concept using sso7d with either strep-tag or biotin-tag, and magnetic streptavidin beads for capture.

[0104] MATERIALS - SSO7D PROTEIN PURIFICATION

[0105] Sso7d is a thermostable, nonspecific DNA-binding protein. The strep tag, GGWSHPQFEK (SEQ ID NO:1 ), is attached at the C-terminus of Sso7d construct. Both non-tagged and streptag-Sso7d variants feature a 6His tag at the C-terminus for convenient purification. The plasmid construct is cloned into BL21 * cells, which were cultured in auto-induced LB media at 37°C for 4-5 hours before being shifted to 18°C overnight. To eliminate DNA binding, cell lysis was carried out in the presence of 1 M salt. The resulting clear lysate underwent a 70°C incubation in a hot water bath for 30 minutes, followed by filtration before passing through the initial HisTrap column on the AKTA FPLC. Elution from the HisTrap column was subsequently applied to a Heparin column, and the eluate underwent buffer exchange to reach the final storage conditions of 50mM NaCI, 50mM Tris pH 8, and 0.01 % Tween-20.

[0106] MATERIALS - PREPARATION OF NONTAGGED:TAGGED MIXTURE

[0107] The tagged and untagged sso7d stocks were stored at a concentration between 1.5 mM to 3 mM in a buffer containing 50 mM NaCI, 50 mM Tris pH 8, and 0.01 % Tween-20. To prepare the protein mix for size selection, the tagged sso7d protein was diluted 100-fold with a binding solution containing 10 mM NaCI, 10 mM Tris pH 8, and 0.0625% Tween 20 to a final concentration of 15 pM to 30 pM, measured by Thermo Scientific NanoDrop. The nontagged and tagged sso7d proteins were next mixed at various stoichiometric ratios to achieve the desired size selection results. This ratio tends to change for different purification lots of the protein and often needs recalibration to achieve desired results. For example, we have defined that a final concentration of 150 pM sso7d-nontag and 0.5 pM sso7d-tag performs well for sso7d-nontag Batchi , while a final concentration of 80 pM sso7d-nontag and 1 pM ofsso7d-tag will work for sso7d-nontag Batch3 as shown in Table 1 below. The sso7d mixtures were stored at a 6x Protein Mix concentration to control carryover of salt and buffer components into the binding step.Table 1: 6X Protein Mix CompositionTarget Final Cone. (1X) 6x Protein Mix

[0108] Materials - Preparation of gDNA library

[0109] High-molecular-weight (HMW) gDNA was extracted from HG002 cells and were mechanically sheared by the Megaruptor® 3 to an average length of 15kb to 20kb. The gDNA fragments were converted into sequencing libraries using the PacBio SMRTbell® prep kit 3.0. The library size distribution was analyzed using the Agilent Femto Pulse system with the Genomic DNA 165kb kit, and the library concentration was measured using the Qubit 1X dsDNA HS Assay Kit. Depending on the quality and shear of the extracted genome, the final libraries may contain between 10% to 30% of smaller undesired fragments with a length below 10kb. With 3 pg input of HMW genome, the recovery after SMRTbell library preparation is expected to be between 0.7 pg to 1.5 pg, which then proceeds directly into protein-based size selection.

[0110] Method - Size Selection Procedure

[0111] The size selection process can be summarized into three main steps: 1 ) protein binding to DNA library, 2) capture of largest DNA fragments containing sufficient tags with streptavidin beads, and 3) elution of captured DNA to recover the sample. Up to 800 ng of initial SMRTbell library input can be size selected in each 60pL reaction.

[0112] A brief overview of the entire process is as follows. During the binding step, sso7d protein was bound to SMRTbell libraries at 37°C for 15 minutes in a binding buffer containing 10 mM NaCI, 10 mM Tris pH 8, and 0.0625% Tween 20. Thelarger fragments of DNA with a higher probability of containing tags were captured using Dynabeads MyOne Streptavidin T1 beads at room temperature for 15 minutes. Once the sso7d-tag-library complex was captured by the streptavidin bead, the supernatant containing the shorter fragments was removed. The captured libraries were recovered by incubating the streptavidin beads with high salt elution buffer (1 M of NaCI, 10mM Tris pH8, and 0.0625% Tween) for 30 minutes at room temperature. The eluted sample was then put through 1 2X Ampure cleanup with two rounds of 80% ethanol wash prior to EB elution.

[0113] Size selection can be performed on up to 800 ng of SMRTbell material in a volume of 50 pL. If the volume is less than 50uL, Binding Buffer can be added to achieve the 50 pL volume. 10 pL of 6x Protein Mix was then added to the sample, rapidly mixed, and incubated for 15 minutes at 37°C. Table 2 below describes the sso7d binding conditions for a typical reaction.Table 2: Binding Reaction ConditionsIncubate at 37C for 15 minutes.

[0114] The sample was next applied to Dynabeads MyOne Streptavidin T1 to capture molecules which contain sufficient tags. Prior to usage, 30 pL of the beads were washed twice with 300 pL binding reagent. The washed beads were magnetized and the supernatant discarded leaving a dry bead pellet. Immediately, 60 pL of SMRTbell-sso7d sample was applied directly to the dry beads, rapidly mixed to resuspend the beads, and incubated for 15 minutes at room temperature.

[0115] The Steptavidin beads which contained the capture samples were magnetized and the supernatant containing smaller fragments was discarded. Immediately, 50 pL of high salt Elution Buffer (1 M of NaCI, 10mM Tris pH8, and 0.0625% Tween) was applied to the sample, thoroughly mixed to resuspend the beads, and allowed to incubate for 30 minutes at room temperature. 1.2X Ampure cleanup of the eluted sample was performed to remove the remnant sso7d proteinsand consisted of two rounds of 80% ethanol wash followed by elution into standard EB (10 mM Tris pH8.5).

[0116] RESULTS - SIZE SELECTION RESPONSE TO VARIOUS CONCENTRATIONS AND RATIOS OF [SS07D-NONTAGGED] : [SSO7D- TAGGED]

[0117] Various ratios and concentration of the sso7d protein mix (sso7d- nontag batch #1 and sso7d-streptag batch #1 ) were explored to ascertain size selection performance and total sample recovery. We tested 4 fixed concentrations of the sso7d-tagged (0.5, 0.7, 1.0, and 1.5 pM) against 5 competing ratios of the sso7d- nontagged (100x, 150x, 200x, 280x, 350x). Detailed concentrations of the sso7d mixtures can be found in Table 3 below.

[0118] One important observation is that a particular concentration of sso7d- tagged should be present, with the lowest 0.5 pM condition producing very low bead capture % and final yield. When working at sufficient concentration of sso7d-tagged, the capture %, final yield, and GQN10K (Genome Quality Number at 10K cutoff) generally responded in a predictable fashion. As the competing ratio of sso7d- nontagged is increased, the bead capture% and final yield decreased as the GQN10k value improved which indicated more stringent size selection as shown in Table 3. While the most aggressive ratio of nontagged-sso7d produced the best size selection results, this usually came at the cost of reduced final sample yield. Aspects of the results are also further summarized in FIGS. 7 to 9A-9D.Table3: Summary of nontagged: tagged sso7d titration sweep

[0119] Some further aspects are defined in the following clauses:

[0120] Clause 1 : A method of size selecting nucleic acid molecules, the method comprising: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of agiven nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound; and, separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample, thereby size selecting the nucleic acid molecules in the sample.

[0121] Clause 2: The method of Clause 1 , wherein the one or more charge modulation compounds alter a net charge associated with a given nucleic acid fragment-bound compound in a captured nucleic acid fragment-bound compound that comprises the given nucleic acid fragment-bound compound.

[0122] Clause 3: The method of Clause 1 or Clause 2, wherein the set of capture reagents are attached to the one or more charge modulation compounds and wherein the separating step comprises separating the captured nucleic acid fragmentbound compounds from the other nucleic acid fragments in the sample using an applied electric field.

[0123] Clause 4: The method of any one of the preceding Clauses 1 -3, further comprising separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of the captured nucleic acid fragment-bound compounds.

[0124] Clause 5: The method of any one of the preceding Clauses 1 -4, comprising eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds.

[0125] Clause 6: The method of any one of the preceding Clauses 1 -5, comprising eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents.

[0126] Clause 7: The method of any one of the preceding Clauses 1 -6, comprising eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using one ormore elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

[0127] Clause 8: The method of any one of the preceding Clauses 1 -7, comprising adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds.

[0128] Clause 9: The method of any one of the preceding Clauses 1 -8, comprising adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds.

[0129] Clause 10: The method of any one of the preceding Clauses 1-9, comprising adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution.

[0130] Clause 11 : The method of any one of the preceding Clauses 1 -10, comprising adjusting the conditions sufficient to produce the set of nucleic acid fragment-bound compounds and / or the conditions sufficient to produce the captured nucleic acid fragment-bound compounds, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

[0131] Clause 12: The method of any one of the preceding Clauses 1 -11 , comprising repeating the method at least one time under different conditions.

[0132] Clause 13: The method of any one of the preceding Clauses 1 -12, wherein the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

[0133] Clause 14: The method of any one of the preceding Clauses 1 -13, further comprising preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique.

[0134] Clause 15: The method of any one of the preceding Clauses 1 -14, wherein the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides.

[0135] Clause 16: The method of any one of the preceding Clauses 1 -15, wherein most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds.

[0136] Clause 17: The method of any one of the preceding Clauses 1 -16, wherein an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment.

[0137] Clause 18: The method of any one of the preceding Clauses 1 -17, wherein the amount of the affinity tagged nucleic acid binding compounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds.

[0138] Clause 19: The method of any one of the preceding Clauses 1 -18, wherein when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases.

[0139] Clause 20: The method of any one of the preceding Clauses 1 -19, comprising estimating a number of affinity tagged nucleic acid binding compounds thatwill bind to a given nucleic acid fragment to select the ratio using an equation as follows:Knlp) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.

[0140] Clause 21 : The method of any one of the preceding Clauses 1 -20, wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0141] Clause 22: The method of any one of the preceding Clauses 1 -21 , wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

[0142] Clause 23: The method of any one of the preceding Clauses 1 -22, wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

[0143] Clause 24: The method of any one of the preceding Clauses 1 -23, wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

[0144] Clause 25: The method of any one of the preceding Clauses 1 -24, wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

[0145] Clause 26: The method of any one of the preceding Clauses 1 -25, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

[0146] Clause 27: The method of any one of the preceding Clauses 1 -26, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

[0147] Clause 28: The method of any one of the preceding Clauses 1 -27, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

[0148] Clause 29: The method of any one of the preceding Clauses 1 -28, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

[0149] Clause 30: The method of any one of the preceding Clauses 1 -29, wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0150] Clause 31 : The method of any one of the preceding Clauses 1 -30, wherein the nucleic acid molecules comprise DNA molecules.

[0151] Clause 32: The method of any one of the preceding Clauses 1 -31 , wherein the nucleic acid molecules comprise RNA molecules.

[0152] Clause 33: The method of any one of the preceding Clauses 1 -32, wherein the nucleic acid molecules are double-stranded.

[0153] Clause 34: The method of any one of the preceding Clauses 1 -33, wherein the nucleic acid molecules are single-stranded.

[0154] Clause 35: The method of any one of the preceding Clauses 1 -34, wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acidfragment having the minimum desired length; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound.

[0155] Clause 36: The method of any one of the preceding Clauses 1 -35, comprising flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber.

[0156] Clause 37: The method of any one of the preceding Clauses 1 -36, wherein the step of joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber comprises concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length.

[0157] Clause 38: The method of any one of the preceding Clauses 1 -37, wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and, flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length fromthe first section of the chamber and out of the outlet of the second section of the chamber through the membrane.

[0158] Clause 39: The method of any one of the preceding Clauses 1 -38, wherein one or more of the nucleic acid fragments to be cleaved are above a maximum desired length.

[0159] Clause 40: The method of any one of the preceding Clauses 1 -39, wherein the conditions comprise mixing conditions.

[0160] Clause 41 : The method of any one of the preceding Clauses 1 -40, wherein the first and second solid supports comprise magnetic beads and wherein the method comprising creating the mixing conditions using a magnet.

[0161] Clause 42: The method of any one of the preceding Clauses 1 -41 , comprising using an agitation technique to create the mixing conditions.

[0162] Clause 43: A system for size selecting nucleic acid molecules in a sample, comprising: a sample container receiving area structured to receive at least one sample container; a fluid handling subassembly configured to selectively convey fluid to and / or from the sample container when the sample container is received in the sample container receiving area and optionally, an electrophoresis subassembly configured to apply an electric field to the sample container when the sample container is received in the sample container receiving area; a controller operably connected at least to the fluid handling subassembly and to the electrophoresis subassembly, if present, wherein the controller comprises a processor, and a memory communicatively directly or remotely coupled to the processor, the memory storing non-transitory computer executable instructions which, when executed by the processor with the sample container received in the sample container receiving area, perform operations comprising: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds in the sample container under conditions sufficient to produce a set of nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the mixture comprises a first set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having aminimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds in the sample container under conditions sufficient to produce captured nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound; and separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample container using the fluid handling subassembly and / or the electrophoresis subassembly, if present.

[0163] Clause 44: The system of Clause 43, wherein the set of capture reagents are attached to the one or more charge modulation compounds, wherein the electrophoresis subassembly is present, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample under an applied electric field using the electrophoresis subassembly.

[0164] Clause 45: The system of Clause 43 or Clause 44, wherein the non- transitory computer executable instructions which, when executed by the processor, further perform operations comprising: separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly.

[0165] Clause 46: The system of any one of the preceding Clauses 43-45, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length from the other components of thecaptured nucleic acid fragment-bound compounds using the fluid handling subassembly.

[0166] Clause 47: The system of any one of the preceding Clauses 43-46, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents using the fluid handling subassembly and / or another operably connected system subassembly.

[0167] Clause 48: The system of any one of the preceding Clauses 43-47, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly and one or more elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

[0168] Clause 49: The system of any one of the preceding Clauses 43-48, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly.

[0169] Clause 50: The system of any one of the preceding Clauses 43-49, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compoundsusing the fluid handling subassembly and / or another operably connected system subassembly.

[0170] Clause 51 : The system of any one of the preceding Clauses 43-50, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution using the fluid handling subassembly and / or another operably connected system subassembly.

[0171] Clause 52: The system of any one of the preceding Clauses 43-51 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the conditions sufficient to produce the set of nucleic acid fragment-bound compounds and / or the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

[0172] Clause 53: The system of any one of the preceding Clauses 43-52, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: repeating the operations at least one time under different conditions using the fluid handling subassembly and / or another operably connected system subassembly.

[0173] Clause 54: The system of any one of the preceding Clauses 43-53, wherein the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

[0174] Clause 55: The system of any one of the preceding Clauses 43-54, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique using the fluid handling subassembly.

[0175] Clause 56: The system of any one of the preceding Clauses 43-55, wherein the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides.

[0176] Clause 57: The system of any one of the preceding Clauses 43-56, wherein most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds.

[0177] Clause 58: The system of any one of the preceding Clauses 43-57, wherein an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment.

[0178] Clause 596: The system of any one of the preceding Clauses 43-58, wherein the amount of the affinity tagged nucleic acid binding compounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds.

[0179] Clause 60: The system of any one of the preceding Clauses 43-59, wherein when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases.

[0180] Clause 61 : The system of any one of the preceding Clauses 43-60, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio using an equation as follows: f(n / p) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.

[0181] Clause 62: The system of any one of the preceding Clauses 43-61 , wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0182] Clause 63: The system of any one of the preceding Clauses 43-62, wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

[0183] Clause 64: The system of any one of the preceding Clauses 43-63, wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

[0184] Clause 65: The system of any one of the preceding Clauses 43-64, wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

[0185] Clause 66: The system of any one of the preceding Clauses 43-65, wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

[0186] Clause 67: The system of any one of the preceding Clauses 43-66, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

[0187] Clause 68: The system of any one of the preceding Clauses 43-67, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

[0188] Clause 69: The system of any one of the preceding Clauses 43-68, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

[0189] Clause 70: The system of any one of the preceding Clauses 43-69, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

[0190] Clause 71 : The system of any one of the preceding Clauses 43-70, wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0191] Clause 72: The system of any one of the preceding Clauses 43-71 , wherein the nucleic acid molecules comprise DNA molecules.

[0192] Clause 73: The system of any one of the preceding Clauses 43-72, wherein the nucleic acid molecules comprise RNA molecules.

[0193] Clause 74: The system of any one of the preceding Clauses 43-75, wherein the nucleic acid molecules are double-stranded.

[0194] Clause 75: The system of any one of the preceding Clauses 43-74, wherein the nucleic acid molecules are single-stranded.

[0195] Clause 76: The system of any one of the preceding Clauses 43-75, wherein the sample is disposed in a chamber of the sample container that comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber is configured to comprise a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computer executable instructions which, when executed by the processor, furtherperform operations comprising: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber and using the fluid handling subassembly; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length and using the fluid handling subassembly; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound and using the fluid handling subassembly.

[0196] Clause 77: The system of any one of the preceding Clauses 43-76, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber using the fluid handling subassembly.

[0197] Clause 78: The system of any one of the preceding Clauses 43-77, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length using the fluid handling subassembly.

[0198] Clause 79: The system of any one of the preceding Clauses 43-78, wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber is further configured to comprise set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computerexecutable instructions which, when executed by the processor, further perform operations comprising: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length using the fluid handling subassembly; and flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane using the fluid handling subassembly.

[0199] Clause 80: The system of any one of the preceding Clauses 43-79, wherein one or more of the nucleic acid fragments to be cleaved are above a maximum desired length.

[0200] Clause 81 : The system of any one of the preceding Clauses 43-80, wherein the conditions comprise mixing conditions.

[0201] Clause 82: A device, comprising at least one chamber that comprises: a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragmentbound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in theset of capture reagents in order to capture the given nucleic acid fragment-bound compound.

[0202] Clause 83: The device of Clause 82, wherein the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein conditions in the chamber are such that: two or more nucleic acid fragments in the sample having less than the minimum desired length are joined together to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber, a nucleic acid fragment-bound compound is produced in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length, and a captured nucleic acid fragment-bound compound is produced in the first section of the chamber using the nucleic acid fragment-bound compound.

[0203] Clause 84: The device of Clause 82 or Clause 83, wherein the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein conditions in the chamber are such that: the sample that comprises the nucleic acid fragments flows over the first and second solid supports in the first section of the chamber so that at least some of the nucleic acid fragments are cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and at least some of the cleaved nucleic acid fragments that are below the minimumdesired length flow from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane.

[0204] Clause 85: A composition, comprising: a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

[0205] Clause 86: A kit, comprising: a set of affinity tagged nucleic acid binding compounds; a set of untagged nucleic acid binding compounds; and, a set of capture reagents that selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds.

[0206] Clause 87: The kit of Clause 86, wherein one or more components of the kit are disposed in one or more containers.

[0207] Clause 88: The kit of Clause 86 or Clause 87, wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0208] Clause 89: The kit of any one of the preceding Clauses 86-88, wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

[0209] Clause 90: The kit of any one of the preceding Clauses 86-89, further comprising one or more first solid supports and / or to one or more charge modulation compounds that are attached, or attachable, to the set of capture reagents.

[0210] Clause 91 : The kit of any one of the preceding Clauses 86-90, wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

[0211] Clause 92: The kit of any one of the preceding Clauses 86-91 , further comprising a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of ligase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution.

[0212] Clause 93: The kit of any one of the preceding Clauses 86-92, further comprising a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of nuclease and / or transposase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution.

[0213] Clause 94: The kit of any one of the preceding Clauses 86-93, wherein a mixture comprises the set of affinity tagged nucleic acid binding compounds and the set of untagged nucleic acid binding compounds.

[0214] Clause 95: The kit of any one of the preceding Clauses 86-94, wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of nucleic acid fragments in a sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds when the sample is contacted with the mixture under conditions sufficient to produce a set of nucleic acid fragment-bound compounds.

[0215] Clause 96: The kit of any one of the preceding Clauses 86-95, wherein an affinity level between the set of capture reagents and the affinity tags is selectedsuch that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound when the set of nucleic acid fragmentbound compounds is contacted with the set of capture reagents under conditions sufficient to produce captured nucleic acid fragment-bound compounds.

[0216] Clause 97: The kit of any one of the preceding Clauses 86-96, further comprising one or more reagents for eluting nucleic acid fragments having a minimum desired length from other components of captured nucleic acid fragment-bound compounds.

[0217] Clause 98: The kit of any one of the preceding Clauses 86-97, further comprising one or more reagents for eluting nucleic acid fragments having less than a minimum desired length from other components of captured nucleic acid fragmentbound compounds.

[0218] Clause 99: The kit of any one of the preceding Clauses 86-98, further comprising one or more of the affinity tags that are free in solution.

[0219] Clause 100: The kit of any one of the preceding Clauses 86-99, further comprising one or more additional reagents selected from the group consisting of: a molecular crowder, a chaotropic salt, a monovalent salt, a divalent salt, an alcohol, a polyamine, and a denaturing agent.

[0220] Clause 101 : The kit of any one of the preceding Clauses 86-100, wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

[0221] Clause 102: The kit of any one of the preceding Clauses 86-101 , wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

[0222] Clause 103: The kit of any one of the preceding Clauses 86-102, wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

[0223] Clause 104: The kit of any one of the preceding Clauses 86-103, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

[0224] Clause 105: The kit of any one of the preceding Clauses 86-104, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

[0225] Clause 106: The kit of any one of the preceding Clauses 86-105, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

[0226] Clause 107: The kit of any one of the preceding Clauses 86-106, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

[0227] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, systems, and / or computer readable media or other aspects thereof can be used in various combinations. All patents, patent applications, websites, other publications or documents, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

WHAT IS CLAIMED IS:1 . A method of size selecting nucleic acid molecules, the method comprising: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound; and, separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample, thereby size selecting the nucleic acid molecules in the sample.

2. The method of claim 1 , wherein the one or more charge modulation compounds alter a net charge associated with a given nucleic acid fragment-bound compound in a captured nucleic acid fragment-bound compound that comprises the given nucleic acid fragment-bound compound.

3. The method of claim 1 , wherein the set of capture reagents are attached to the one or more charge modulation compounds and wherein theseparating step comprises separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample using an applied electric field.

4. The method of claim 1 , further comprising separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragmentbound compounds from other components of the captured nucleic acid fragmentbound compounds.

5. The method of claim 4, comprising eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds.

6. The method of claim 4, comprising eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents.

7. The method of claim 4, comprising eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using one or more elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

8. The method of claim 1 , comprising adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds.

9. The method of claim 1 , comprising adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate theconditions sufficient to produce the captured nucleic acid fragment-bound compounds.

10. The method of claim 9, comprising adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution.11 . The method of claim 1 , comprising adjusting the conditions sufficient to produce the set of nucleic acid fragment-bound compounds and / or the conditions sufficient to produce the captured nucleic acid fragment-bound compounds, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence of other additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

12. The method of claim 1 , comprising repeating the method at least one time under different conditions.

13. The method of claim 12, wherein the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

14. The method of claim 4, further comprising preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique.

15. The method of claim 1 , wherein the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, atleast about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides.

16. The method of claim 1 , wherein most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds.

17. The method of claim 1 , wherein an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment.

18. The method of claim 17, wherein the amount of the affinity tagged nucleic acid binding compounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds.

19. The method of claim 17, wherein when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases.

20. The method of claim 1 , comprising estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio using an equation as follows: f(n / p) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.21 . The method of claim 1 , wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

22. The method of claim 1 , wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

23. The method of claim 1 , wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

24. The method of claim 1 , wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

25. The method of claim 1 , wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

26. The method of claim 25, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

27. The method of claim 25, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

28. The method of claim 25, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

29. The method of claim 25, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

30. The method of claim 1 , wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, anoligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.31 . The method of claim 1 , wherein the nucleic acid molecules comprise DNA molecules.

32. The method of claim 1 , wherein the nucleic acid molecules comprise RNA molecules.

33. The method of claim 1 , wherein the nucleic acid molecules are doublestranded.

34. The method of claim 1 , wherein the nucleic acid molecules are singlestranded.

35. The method of claim 1 , wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound.

36. The method of claim 35, comprising flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber.

37. The method of claim 35, wherein the step of joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber comprises concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length.

38. The method of claim 1 , wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the method comprises: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and, flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane.

39. The method of claim 38, wherein one or more of the nucleic acid fragments to be cleaved are above a maximum desired length.

40. The method of claim 38, wherein the conditions comprise mixing conditions.41 . The method of claim 40, wherein the first and second solid supports comprise magnetic beads and wherein the method comprising creating the mixing conditions using a magnet.

42. The method of claim 40, comprising using an agitation technique to create the mixing conditions.

43. A system for size selecting nucleic acid molecules in a sample, comprising: a sample container receiving area structured to receive at least one sample container; a fluid handling subassembly configured to selectively convey fluid to and / or from the sample container when the sample container is received in the sample container receiving area and optionally, an electrophoresis subassembly configured to apply an electric field to the sample container when the sample container is received in the sample container receiving area; a controller operably connected at least to the fluid handling subassembly and to the electrophoresis subassembly, if present, wherein the controller comprises a processor, and a memory communicatively directly or remotely coupled to the processor, the memory storing non-transitory computer executable instructions which, when executed by the processor with the sample container received in the sample container receiving area, perform operations comprising: contacting a sample that comprises nucleic acid fragments with a mixture of nucleic acid binding compounds in the sample container under conditions sufficient to produce a set of nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the mixture comprises a first set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untaggednucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; contacting the set of nucleic acid fragment-bound compounds with a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds in the sample container under conditions sufficient to produce captured nucleic acid fragment-bound compounds using the fluid handling subassembly, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound; and separating the captured nucleic acid fragment-bound compounds from other nucleic acid fragments in the sample container using the fluid handling subassembly and the electrophoresis subassembly, if present.

44. The system of claim 43, wherein the set of capture reagents are attached to the one or more charge modulation compounds, wherein the electrophoresis subassembly is present, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: separating the captured nucleic acid fragment-bound compounds from the other nucleic acid fragments in the sample under an applied electric field using the electrophoresis subassembly.

45. The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: separating the nucleic acid fragments having the minimum desired length in the captured nucleic acid fragment-bound compounds from other components of thecaptured nucleic acid fragment-bound compounds using the fluid handling subassembly.

46. The system of claim 45, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly.

47. The system of claim 45, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds under conditions sufficient for the nucleic acid fragments having the minimum desired length to remain bound to the corresponding capture reagent in the set of capture reagents using the fluid handling subassembly and / or another operably connected system subassembly.

48. The system of claim 45, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: eluting the nucleic acid fragments having the minimum desired length or nucleic acid fragments having less than the minimum desired length from the other components of the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly and one or more elution strategies selected from the group consisting of: a selected salt condition, a selected heat condition, a selected pH condition, a selected competitive affinity group condition, a selected protease condition, and a denaturant condition.

49. The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:adjusting a density of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly.

50. The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting a binding capacity of the set of capture reagents that are attached to the first solid supports to modulate the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly.51 . The system of claim 50, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the binding capacity of the set of capture reagents by contacting the set of capture reagents that are attached to the first solid supports with a selected amount of the affinity tags that are free in solution using the fluid handling subassembly and / or another operably connected system subassembly.

52. The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: adjusting the conditions sufficient to produce the set of nucleic acid fragmentbound compounds and / or the conditions sufficient to produce the captured nucleic acid fragment-bound compounds using the fluid handling subassembly and / or another operably connected system subassembly, wherein the conditions are selected from the group consisting of: a molecular crowder concentration, a molecular crowder molecular weight, a molecular crowder type, presence or absence of chaotropic salts, presence or absence of monovalent and / or divalent salts, salt concentration and type, alcohol type and concentration, presence or absence of polyamines, presence or absence of denaturing agents, presence or absence ofother additive molecules, pH, binding time, temperature, binding volume, and combinations thereof.

53. The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: repeating the operations at least one time under different conditions using the fluid handling subassembly and / or another operably connected system subassembly.

54. The system of claim 53, wherein the different conditions comprise different ratios of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture.

55. The system of claim 45, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: preparing the nucleic acid fragments having the minimum desired length for use in performing at least one nucleic acid sequencing technique using the fluid handling subassembly.

56. The system of claim 43, wherein the minimum desired length of at least one strand of the nucleic acid fragments is at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 5000 nucleotides, at least about 6000 nucleotides, at least about 7000 nucleotides, at least about 8000 nucleotides, at least about 9000 nucleotides, at least about 10000 nucleotides, at least about 15000 nucleotides, at least about 20000 nucleotides, or more nucleotides.

57. The system of claim 43, wherein most of the nucleic acid fragments in the sample having lengths that are less than the minimum desired length are each bound to one or fewer affinity tagged nucleic acid binding compounds.

58. The system of claim 43, wherein an amount of the affinity tagged nucleic acid binding compounds in the mixture is such that a number of the affinity tagged nucleic acid binding compounds bound to a given nucleic acid fragment in a nucleic acid fragment-bound compound is a function of a length of the given nucleic acid fragment.

59. The system of claim 58, wherein the amount of the affinity tagged nucleic acid binding compounds comprises less than about 1 % weight per volume (w / v) of the mixture of nucleic acid binding compounds.

60. The system of claim 58, wherein when the amount of the affinity tagged nucleic acid binding compounds in the mixture is decreased, a likelihood that multiple affinity tagged nucleic acid binding compounds are bound to the given nucleic acid fragment increases as the length of the given nucleic acid fragment increases.61 . The system of claim 43, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: estimating a number of affinity tagged nucleic acid binding compounds that will bind to a given nucleic acid fragment to select the ratio using an equation as follows: f(n / p) where f is a ratio of a number of molecules in the first set to a number of molecules in the second set, n is a number of nucleotides in a strand of the given nucleic acid fragment, and p is a number of number of nucleotides in the strand of the given nucleic acid fragment spanned by a given nucleic acid binding compound when bound to the given nucleic acid fragment.

62. The system of claim 43, wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

63. The system of claim 43, wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

64. The system of claim 43, wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

65. The system of claim 43, wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

66. The system of claim 43, wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

67. The system of claim 66, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

68. The system of claim 66, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

69. The system of claim 66, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

70. The system of claim 66, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.71 . The system of claim 43, wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

72. The system of claim 43, wherein the nucleic acid molecules compriseDNA molecules.

73. The system of claim 43, wherein the nucleic acid molecules comprise RNA molecules.

74. The system of claim 43, wherein the nucleic acid molecules are double-stranded.

75. The system of claim 43, wherein the nucleic acid molecules are singlestranded.

76. The system of claim 43, wherein the sample is disposed in a chamber of the sample container that comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber is configured to comprise a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: joining two or more nucleic acid fragments in the sample having less than the minimum desired length together under conditions sufficient to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber and using the fluid handling subassembly; producing a nucleic acid fragment-bound compound in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length and using the fluid handling subassembly; and, producing a captured nucleic acid fragment-bound compound in the first section of the chamber using the nucleic acid fragment-bound compound and using the fluid handling subassembly.

77. The system of claim 76, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:flowing the concatenated nucleic acid fragment having the minimum desired length from the second section of the chamber into the first section of the chamber through the membrane prior to producing a nucleic acid fragment-bound compound in the first section of the chamber using the fluid handling subassembly.

78. The system of claim 76, wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: concatenating one or more adapter or marker molecules to at least one of the nucleic acid fragments in the sample having less than the minimum desired length and / or to the concatenated nucleic acid fragment having the minimum desired length using the fluid handling subassembly.

79. The system of claim 43, wherein the sample is disposed in a chamber comprising a membrane that divides the chamber into at least two sections, wherein a first section of the chamber is configured to comprise the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber is further configured to comprise set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising: flowing the sample that comprises the nucleic acid fragments over the first and second solid supports in the first section of the chamber under conditions sufficient for at least some of the nucleic acid fragments to be cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length using the fluid handling subassembly; and flowing at least some of the cleaved nucleic acid fragments that are below the minimum desired length from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane using the fluid handling subassembly.

80. The system of claim 79, wherein one or more of the nucleic acid fragments to be cleaved are above a maximum desired length.81 . The system of claim 79, wherein the conditions comprise mixing conditions.

82. A device, comprising at least one chamber that comprises: a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

83. The device of claim 82, wherein the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein a second section of the chamber comprises a set of ligase enzymes attached to one or more second solid supports, wherein the membrane is impermeable to the first andsecond solid supports, wherein the membrane is permeable to reagents in solution, and wherein conditions in the chamber are such that: two or more nucleic acid fragments in the sample having less than the minimum desired length are joined together to produce a concatenated nucleic acid fragment having the minimum desired length using the set of ligase enzymes attached to the one or more second solid supports in the second section of the chamber, a nucleic acid fragment-bound compound is produced in the first section of the chamber using the concatenated nucleic acid fragment having the minimum desired length, and a captured nucleic acid fragment-bound compound is produced in the first section of the chamber using the nucleic acid fragment-bound compound.

84. The device of claim 83, wherein the chamber comprises a membrane that divides the chamber into at least two sections, wherein a first section of the chamber comprises the mixture of nucleic acid binding compounds and the set of capture reagents that are attached to the one or more first solid supports, wherein the first section of the chamber further comprises a set of nuclease and / or transposase enzymes attached to one or more second solid supports, wherein a second section of the chamber comprises an outlet, wherein the membrane is impermeable to the first and second solid supports, wherein the membrane is permeable to reagents in solution, and wherein conditions in the chamber are such that: the sample that comprises the nucleic acid fragments flows over the first and second solid supports in the first section of the chamber so that at least some of the nucleic acid fragments are cleaved by the nuclease and / or transposase enzymes to produce cleaved nucleic acid fragments that are below the minimum desired length; and at least some of the cleaved nucleic acid fragments that are below the minimum desired length flow from the first section of the chamber and out of the outlet of the second section of the chamber through the membrane.

85. A composition, comprising:a sample that comprises nucleic acid fragments and a mixture of nucleic acid binding compounds under conditions sufficient to produce a set of nucleic acid fragment-bound compounds, wherein the mixture comprises a set of affinity tagged nucleic acid binding compounds and a set of untagged nucleic acid binding compounds, and wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of the nucleic acid fragments in the sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds; and a set of capture reagents that are attached to one or more first solid supports and / or to one or more charge modulation compounds under conditions sufficient to produce captured nucleic acid fragment-bound compounds, wherein the set of capture reagents selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds, and wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound.

86. A kit, comprising: a set of affinity tagged nucleic acid binding compounds; a set of untagged nucleic acid binding compounds; and, a set of capture reagents that selectively bind to affinity tags of the affinity tagged nucleic acid binding compounds.

87. The kit of claim 86, wherein one or more components of the kit are disposed in one or more containers.

88. The kit of claim 86, wherein the capture reagents are selected from the group consisting of: a biotin molecule, a streptavidin molecule, an avidin molecule, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

89. The kit of claim 86, wherein the affinity tags are selected from the group consisting of: a histone-tag, a streptavidin-tag, an avidin-tag, an aptamer, an oligonucleotide, a peptide, an antibody or an antigen binding portion thereof, and an antigen.

90. The kit of claim 86, further comprising one or more first solid supports and / or to one or more charge modulation compounds that are attached, or attachable, to the set of capture reagents.91 . The kit of claim 90, wherein the one or more first solid supports are selected from the group consisting of: a resin, a bead, a substrate, and a well of a microwell plate.

92. The kit of claim 90, further comprising a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of ligase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution.

93. The kit of claim 90, further comprising a device that comprises a chamber comprising a membrane that divides the chamber into at least two sections, one or more second solid supports, and a set of nuclease and / or transposase enzymes attached, or attachable, to the one or more second solid supports, wherein the membrane is impermeable to the first and second solid supports, and wherein the membrane is permeable to reagents in solution.

94. The kit of claim 86, wherein a mixture comprises the set of affinity tagged nucleic acid binding compounds and the set of untagged nucleic acid binding compounds.

95. The kit of claim 94, wherein a ratio of the affinity tagged nucleic acid binding compounds to the untagged nucleic acid binding compounds in the mixture is selected such that a plurality of nucleic acid fragments in a sample having a minimum desired length each bind to a nucleic acid binding compound of more than one of the affinity tagged nucleic acid binding compounds when the sample is contacted with the mixture under conditions sufficient to produce a set of nucleic acid fragment-bound compounds.

96. The kit of claim 95, wherein an affinity level between the set of capture reagents and the affinity tags is selected such that multiple affinity tags of a given nucleic acid fragment-bound compound comprising a nucleic acid fragment having the minimum desired length binds to a corresponding capture reagent in the set of capture reagents in order to capture the given nucleic acid fragment-bound compound when the set of nucleic acid fragment-bound compounds is contacted with the set of capture reagents under conditions sufficient to produce captured nucleic acid fragment-bound compounds.

97. The kit of claim 86, further comprising one or more reagents for eluting nucleic acid fragments having a minimum desired length from other components of captured nucleic acid fragment-bound compounds.

98. The kit of claim 86, further comprising one or more reagents for eluting nucleic acid fragments having less than a minimum desired length from other components of captured nucleic acid fragment-bound compounds.

99. The kit of claim 86, further comprising one or more of the affinity tags that are free in solution.

100. The kit of claim 86, further comprising one or more additional reagents selected from the group consisting of: a molecular crowder, a chaotropic salt, a monovalent salt, a divalent salt, an alcohol, a polyamine, and a denaturing agent.101 . The kit of claim 86, wherein the nucleic acid binding compounds bind to the nucleic acid fragments in a non-sequence-specific manner.

102. The kit of claim 86, wherein the nucleic acid binding compounds comprise one or more synthetic compounds.

103. The kit of claim 86, wherein the nucleic acid binding compounds comprise nucleic acid binding proteins or peptides.

104. The kit of claim 103, wherein the nucleic acid binding proteins comprise RNA-binding proteins.

105. The kit of claim 103, wherein the nucleic acid binding proteins comprise double-stranded DNA-binding proteins.

106. The kit of claim 103, wherein the nucleic acid binding proteins comprise single-stranded DNA-binding proteins.

107. The kit of claim 103, wherein the nucleic acid binding proteins are selected from the group consisting of: a histone protein, an sso7d protein, and a DNA stabilizing stress response protein.

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