Tagmentation Workflow

Abstract

In one example of the method, the deoxyribonucleic acid sample is exposed to tagmentation in the presence of a tagmentation buffer containing a divalent cation cofactor and a transposase enzyme to generate a tagmented DNA fragment complex. A chelating agent mixture is added to the tagmented DNA fragment complex. The chelating agent mixture contains a chelating agent for the divalent cation cofactor in a weight ratio of at least 1:1 with the divalent cation cofactor and has a pH in the range of 8 to 9. The tagmented DNA fragment complex is incubated in the chelating agent mixture at a temperature of at least 55° C. for at least about 60 seconds such that the transposase enzyme dissociates from the tagmented DNA fragment of the tagmented DNA fragment complex.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 392,729, filed Jul. 27, 2022, the content of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Double - stranded deoxyribonucleic acid (dsDNA) target molecules can be fragmented and tagged to generate a library of smaller double - stranded DNA molecules. The smaller double - stranded DNA molecules can be denatured to produce small single - stranded DNA molecules (ssDNA). These small single - stranded DNA molecules can be used as templates in DNA sequencing reactions. The templates can allow for obtaining short read lengths, and then, during data analysis, overlapping short sequence reads can be aligned to reconstruct longer nucleic acid sequences. Some methods for fragmentation and tagging of double - stranded DNA produce excessive waste, involve expensive equipment for fragmentation, and are time - consuming.

Summary of the Invention

[0003] The methods disclosed herein involve tagmentation, followed by dissociation and removal of the transposase enzyme used for tagmentation. The transposase enzyme is dissociated with a chelating agent for the divalent cation cofactor used during tagmentation. Thus, the methods are carried out without chaotropic agents, and the presence of chaotropic agents can denature enzymes (e.g., ligase, polymerase) used downstream of the tagmentation process.

Brief Description of the Drawings

[0004] The features of the examples of the present disclosure will become apparent by referring to the following detailed description and drawings. In the drawings, like reference numerals correspond to components that are similar, if not identical. For the sake of brevity, reference numerals or features having the aforementioned functions may or may not be described in connection with other drawings in which they appear.

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Mode for Carrying Out the Invention

[0005] Tagmentation is the process by which a deoxyribonucleic acid (DNA) sample is cleaved / fragmented and tagged (e.g., with an adapter) for analysis. Tagmentation is an in vitro transposition reaction.

[0006] In the example shown in FIG. 1, tagmentation relies on a transposase enzyme 12 (e.g., Tn5) that fragments and simultaneously adds an adapter sequence to the 5' end of a double-stranded DNA fragment. The transposase enzyme 12 is part of a transpososome complex 10. The transpososome complex 10 includes the transposase enzyme 12 non-covalently bound to the transposon ends. Each transposon end is a double-stranded nucleic acid strand, one of which is part of the transfer strand 14 and the other of which is part of the non-transfer strand 16 (FIGS. 1 and 2) or 16' (FIGS. 3A and 3B). In other words, the transposon end includes a portion of the transfer strand 14 that hybridizes to a portion of the non-transfer strands 16, 16'. By way of example, the transposon end can be the relevant but non-identical 19 base pair (bp) outer and inner end sequences that function as substrates for the activity of the Tn5 transposase 12, or a mosaic end recognized by wild-type or mutant Tn5 transposase, or the R1 and R2 ends recognized by the MuA transposase.

[0007] The tagmentation process generates a tagmentation complex (also referred to herein as a "tagmented DNA fragment complex") that includes the transposase enzyme 12 bound to the tagmented DNA.

[0008] After the tagmentation process, one or more additional steps independent of the transposase enzyme 12 follow. In this step or these steps, an adapter (which may be similar to or different from the adapter added to the 5' end during tagmentation) is added to the 3' end of the fragment. The 3' end adapter can be added by either of the examples shown in Figure 2 or Figure 3B (described in more detail below). In the example shown in Figure 2, the adapter added includes adapters for clustering (e.g., P5, P7), indexing (i5, i7), and sequencing primer binding sites (A14, B15). Thus, the library template 18 generated via the example shown in Figure 2 is in a form ready for flow cell clustering and sequencing. In the example shown in Figure 3B, the sequencing primer binding sites (A14, B15) are added first, and the resulting library template 18' can be exposed to additional processing to add adapter sequences for clustering and sequencing and indexing of the sequences (e.g., barcode sequences). Alternatively, the fork-shaped adapters shown in Figures 3A and 3B can instead include all of the desired sequences (e.g., P5-i5-A14 and P7-i7-B15) as part of the non-transferred strand 16' of the transpososome complex 10'.

[0009] As mentioned, an example of the addition of the 3' end adapter is shown in FIG. 2. In this example, the free 3' end of the fragment is extended in the presence of polymerase and deoxyribonucleoside triphosphate (dNTP), heated to remove non-transferred strand 16, then the complement of the 5' adapter (i.e., transferred strand 14) can be copied, and finally, a polymerase chain reaction (PCR) reaction using two separate primers can be used to enrich the primary tagged molecules, such that they have a P5-based adapter at one end and a P7-based adapter at the other end. In the example shown in FIG. 3, the two separate primers are P5-i5-A14 and P7-i7-B15, each of which contains an adapter sequence (P5, P7), an index sequence (i5, i7), and a sequencing primer binding site (A14, B15) for clustering and sequencing.

[0010] Another example of the addition of the 3' end adapter is shown in FIG. 3B. The transposome complex 10' used in this exemplary method contains a single double-stranded fork-type adapter shown in FIG. 3A. In the method of FIG. 3B, non-transferred polymerase is used at a temperature below the primer melting temperature (Tm) of non-transferred strand 16' to extend the free 3' end of the fragment until it reaches the 5' end of non-transferred adapter strand 16', and then ligase covalently attaches non-transferred strand 16' to the fragment.

[0011] In any example of tagmentation, transposase enzyme 12 is removed from the tagmentation complex (including the transposase enzyme 12 bound to the tagmented DNA fragment) before it can be used to add an adapter to the 3’ end of the DNA that has been tagmented as shown in FIGS. 2 and 3B. This is because the transposase enzyme 12, particularly Tn5, remains tightly bound to the tagmented DNA and inhibits the enzyme used to complete the addition of the 3’ adapter from accessing the DNA. Heating the tagmentation complex, such as in PCR designed to add an adapter sequence to the 3’ end, is only partially effective as demonstrated by the reduced yield and larger insert sizes in the prepared library (see FIG. 4, line A, described in more detail in the Examples section). Complete Tn5 displacement can be achieved by adding a strong denaturing or chaotropic agent such as sodium dodecyl sulfate (SDS) to the tagmentation complex to denature and release the Tn5 protein, completely dissociating it from the tagmented DNA. This method is more effective than heating alone as demonstrated by the data shown in line B of FIG. 4. However, strong denaturing agents can denature enzymes (e.g., ligase, polymerase) used in downstream processes such as extension and amplification reactions. Therefore, the denaturing agent should be thoroughly washed away to prevent inhibition of subsequent enzyme steps.

[0012] The examples described herein provide an alternative method for removing a transposase enzyme (e.g., Tn5 protein) from its tagmentation complex without a denaturing agent and thus compatible with downstream enzyme steps. This method simplifies the library preparation workflow, such as Tn5 library preparation, by eliminating the need for strong denaturing agents and washes.

[0013] More specifically, the method described herein uses a transposase removal fluid and employs a chelating agent molecule under conditions where the chelating agent molecule binds to the active site of the transposase enzyme 12 and sequesters the divalent cation cofactor therefrom, thereby weakening the interaction between the transposase enzyme 12 and the tagged DNA. By doing so, the transposase removal fluid dissociates the transposase enzyme 12 from the tagged DNA and makes the DNA accessible for downstream enzyme processing.

[0014] Examples of the methods disclosed herein include exposing a deoxyribonucleic acid sample to tagging in the presence of a tagging buffer containing a divalent cation cofactor and the transposase enzyme 12, thereby generating a tagged DNA fragment complex, and adding a transposase removal fluid to the tagged DNA fragment complex, wherein the transposase removal fluid contains a chelating agent for the divalent cation cofactor in a weight ratio of at least 1:1 with the divalent cation cofactor and has a pH in the range of 8 - 9, incubating the tagged DNA fragment complex in the transposase removal fluid at a temperature of at least 55°C for at least about 60 seconds, whereby the transposase enzyme 12 dissociates from the tagged DNA fragment of the tagged DNA fragment complex. Some examples of the method further include washing the transposase removal fluid and the dissociated transposase enzyme 12 from the tagged DNA fragment. Other examples of the method further include adding a reagent to add an adapter to the 3' end of the tagged DNA fragment.

[0015] The transposase removal fluid contains a chelating agent for the divalent cation cofactor (used in tagging), a buffer, and water. In some examples, the transposase removal fluid also contains a salt.

[0016] Chelating agents are reagents that sequester cations used in tagmentation, such as Mg 2+ , Co 2+ , etc. Therefore, the chelating agent in the transposase removal fluid will depend on the divalent cation cofactor used in tagmentation. In one example, the divalent cation cofactor is Mg 2+ , and the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and diketo acid antiviral compounds. In another example, the divalent cation cofactor is Co 2+ , and the chelating agent is selected from the group consisting of aza-crown-ether compounds, N-acetyl-cysteine, porphyrin, and crown ethers. When the chelating agent is an aza-crown-ether compound, the aza-crown-ether compound can be 1,4,7,10-tetraazacyclododecane tetrahydrochloride. When the chelating agent is a crown ether, the crown ether can be selected from the group consisting of 15-crown-5 and 18-crown-6. When the chelating agent is a diketo acid antiviral compound, the diketo acid antiviral compound can be selected from the group consisting of 2-hydroxy-4-oxo-4-thiophen-2-yl-but-2-enoic acid, 4-thiophen-2-yl-2,4-dioxobutanoic acid, and 2,4-dioxo-4-phenylbutanoic acid, which are used as integrase inhibitors. Other viral integrase inhibitors may be used, such as those having a common structural motif where the transposase has been removed. When the chelating agent is porphyrin, the porphyrin can be cobalt-free vitamin B12.

[0017] The amount of chelating agent in the transposase removal fluid will depend on the amount of divalent cation cofactor used in the tagging and whether salt is included in the transposase removal fluid. The chelating agent is present in a weight ratio of at least 1:1 with the divalent cation cofactor used in the tagging. When no salt is included in the transposase removal fluid, transposase removal can be achieved by including an excess of chelating agent relative to the divalent cation cofactor. The excess chelating agent is effective in promoting the chelation of divalent cation cofactors away from the active site of the protein. In these examples, the transposase removal fluid includes the chelating agent in a weight ratio ranging from about 3:1 to about 10:1 with the divalent cation cofactor used in the tagging. When salt is included in the transposase removal fluid, transposase removal can be achieved with a smaller amount of chelating agent (compared to when no salt is included). Salt can bind to the phosphate backbone of the tagged DNA, preventing it from acting like a ligand for the divalent cation cofactor and making the divalent cation cofactor more sensitive to chelation. In these examples, the transposase removal fluid includes the chelating agent in a weight ratio ranging from 1:1 to about 3:1 with the divalent cation cofactor used in the tagging. In one example of a transposase removal fluid containing salt, the weight ratio of the chelating agent in the transposase removal fluid to the divalent cation cofactor used in the tagging is 1.2:1.

[0018] When included, the salt in the transposase removal fluid can be an inorganic salt selected from the group consisting of sodium salts, potassium salts, and lithium salts. Exemplary sodium salts include sodium chloride, sodium sulfate, and sodium carbonate, exemplary potassium salts include potassium chloride, and exemplary lithium salts include lithium chloride.

[0019] When included, the salt in the transposase removal fluid is present at a concentration of at least 75 mM. In one example, the salt is present at a concentration of 150 mM.

[0020] Any suitable buffer such as tris(hydroxymethyl)aminomethane (Tris buffer), tris hydrochloride (Tris-HCl), tris acetate, etc. can be used in the transposase removal fluid.

[0021] The concentration of the buffer in the transposase removal fluid ranges from about 5 mM to about 100 mM.

[0022] The pH of the transposase removal fluid ranges from 8 to 9.

[0023] The transposase removal fluid can be part of a kit that also includes a tagging buffer. In one example, the kit includes a tagging buffer containing water, an optional co-solvent (e.g., dimethylformamide), a divalent cation cofactor for transposase 12, and a buffer, and a transposase removal fluid containing water, a chelating agent for the divalent cation cofactor in the tagging buffer in a weight ratio of at least 1:1 with the divalent cation cofactor in the tagging buffer, and a buffer. In some examples, the transposase removal fluid of the kit also includes a salt at a concentration of at least 75 mM. In one example of the tagging buffer, the optional co-solvent may be present in an amount of up to about 11%, the metal cofactor may be present at a concentration in the range of about 1.5 mM to about 5.5 mM, and the buffer may be present at a concentration in the range of about 5 mM to about 12 mM. The remainder of the tagging buffer is water (e.g., deionized water).

[0024] In one example of the method, the divalent cation cofactor (in the tagging buffer) is Mg 2+ and the chelating agent (in the transposase removal fluid) is EDTA in a weight ratio of at least 1:1, e.g., 1.2:1, to Mg in a buffer containing a salt, e.g., NaCl, at a concentration of at least 75 mM, e.g., 150 mM, and at a pH in the range of at least 8.0, e.g., 8.6 to 9. This example of the transposase removal fluid can be incubated with the tagged DNA complex at a temperature of at least 55°C, e.g., 60°C, for at least 60 seconds. 2+ ​

[0025] In another example of the method, the divalent cation cofactor (in the tagging buffer) is Mg 2+ and the chelating agent (in the transposase removal fluid) is in a buffer containing a salt, such as NaCl, at a concentration of at least 75 mM, for example 150 mM, and at a pH in the range of at least 8.0, for example 8.6 - 9, and is a diketo acid anti-retroviral compound at a weight ratio of at least 1:1 with respect to Mg 2+ This example of the transposase removal fluid is incubated with the tagged DNA complex at a temperature of at least 55 °C, for example 60 °C, for at least 60 seconds.

[0026] In yet another example of the method, the divalent cation cofactor (in the tagging buffer) is Co 2+ and the chelating agent (in the transposase removal fluid) is Cyclen (i.e., 1,4,7,10-tetraazacyclododecane tetrahydrochloride). In this example, Co 2+ is used at a concentration of at least 1.5 mM, for example 5 mM, and the Cyclen chelating agent is present at a weight ratio of at least 1:1 with respect to Co 2+ (e.g., 1.5 mM or 7.5 mM). Cyclen is present in a buffer containing a salt, such as NaCl, at a concentration of at least 75 mM, for example 150 mM, and at a pH in the range of at least 8.0, for example 8.6 - 9. This example of the transposase removal fluid is incubated with the tagged DNA complex at a temperature of at least 55 °C, for example 60 °C, for at least 60 seconds.

[0027] The time for incubating the tagged DNA complex in the transposase removal fluid can range from about 1 minute to about 5 minutes, and the temperature during this incubation period is about 60 °C. It should be understood that a temperature gradient that rises to 60 °C and is maintained at 60 °C for at least 1 minute may be used throughout the incubation period.

[0028] After tagmentation and transposase removal as described herein, and prior to downstream processing to add the 3' end of the fragment, the method uses enzymes, such as ligase and polymerase, for adding a 3' adapter to the tagmented DNA, and cations (e.g., Mg 2+ ) that support their activity may be further included. The addition of these cations compensates for the prior sequestration of the tagmentation cations to enable transposase removal.

[0029] To further illustrate the disclosure, examples are provided herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the disclosure.

[0030] Non-limiting examples Example 1 This example illustrates the results from two comparative methods (Experiment A and Experiment B) regarding the removal of the Tn5 transposase enzyme after tagmentation. For each of these experiments, the tagmentation buffer contained water, 10% dimethyl formamide (DMF), 5 mM magnesium acetate, and 10 mM Tris acetate, pH 7.6. Thus, Mg 2+ was the divalent cation cofactor.

[0031] In the workflow of Experiment A, DNA was first tagmented, the transfer strand of the adapter was added to the 5' end of the tagmented DNA, and then treated with water (not sodium dodecyl sulfate (SDS)), thus maintaining the Tn5 bound to the tagmented DNA. The tagmented DNA complex was washed and subjected to PCR (including high-temperature heating that can dissociate Tn5) to add the adapter sequence to the 3' end of the tagmented DNA.

[0032] In the workflow of Experiment B, DNA was first tagged, an adapter transfer strand was added to the 5’ end of the tagged DNA, and then treated with SDS to denature the Tn5 enzyme and thus dissociate it from the tagged DNA complex. The SDS was washed away, and the tagged DNA fragments were exposed to PCR to add an adapter sequence to the 3’ end of the tagged DNA.

[0033] As a non-size selection cleanup process, the tagged DNA fragments from Experiments A and B were exposed to solid phase reversible immobilization (SPRI) technology to reversibly bind the tagged DNA. In this process, SPRI beads were added to each sample at 1.8 times the volume of the PCR buffer and incubated at room temperature for approximately 5 minutes. The tagged DNA bound to the SPRI beads was pelleted, washed with ethanol, the ethanol was removed, resuspended in buffer, and pelleted again. A portion of the supernatant was analyzed using a TapeStation. The size profiles and yields of the tagged DNA fragments from Experiments A and B were measured using a TapeStation. The results for both Experiments A and B are shown in Figure 4. For libraries prepared using completely removed Tn5, the expected size profile (partially based on the transpososome complex concentration used) included fragments in the range of 100 base pairs (bp) to 4000 bp. At higher or lower transpososome complex concentrations, the expected size range could shift. The results in Figure 4 illustrate that SDS (line B) is more effective than the high-temperature heating used in PCR (line A) in removing Tn5 from the tagged product complex. In contrast to the results for SDS Tn5 removal (line B), the data for Tn5 removal without SDS (line A) showed a significantly reduced yield and distortion of the insert size profile.

[0034] Example 2 This example illustrates the results from one comparative method (Experiment A) and one example method (Experiment B) regarding the removal of the Tn5 transposase enzyme after tagmentation. For each of these experiments, the tagmentation buffer contained water, 10% DMF, 5 mM magnesium acetate, and 10 mM Tris acetate, pH 7.6. Thus, Mg 2+ was the divalent cation cofactor. Incubation for tagmentation was performed at 55 °C for 5 minutes.

[0035] In the workflow of Experiment A, DNA was first tagmented and the adapter transfer strand was added to the 5’ end of the tagmented DNA. Tn5 was removed by adding 1% SDS and incubating at 25 °C for 5 minutes. The SDS was washed away in the wash buffer and the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer bases.

[0036] In the workflow of Experiment B, DNA was first tagmented and the adapter transfer strand was added to the 5’ end of the tagmented DNA. The supernatant was discarded and Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiment B, the transposase removal fluid contained 100 mM Tris-HCl, pH 8.6, 100 mM NaCl, and 0.6 mM EDTA. The tagmented DNA complex was incubated in the transposase removal fluid at 60 °C for 1 minute. After washing in the wash buffer, the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer bases.

[0037] Except for measuring the size profiles and yields of the tagged DNA fragments from Experiments A and B in this example using a Bioanalyzer, the tagged DNA fragments from Experiments A and B were exposed to the same SPRI technology as described in Example 1 to reversibly bind the tagged DNA as a non-size-selective cleanup process. The results (labeled A and B, respectively) are shown in Figure 5. The data for Experiments A and B demonstrate equivalent yields and insert size profiles between the conventional SDS approach (line A) and the cation chelation approach (line B) regarding removing Tn5 from its product complex. Thus, the data in Figure 5, in combination with heat, demonstrate that a transposase removal fluid containing EDTA and salts in a high-pH buffer is sufficient to replace SDS as a reagent for dissociating the Tn5 enzyme from the tagged DNA complex.

[0038] Example 3 This example illustrates the results from replicated comparative methods (Experiments A and B) and replicated example methods (Experiments C and D) regarding removing the Tn5 transposase enzyme after tagging. For each of these experiments, the tagging buffer contained water, 10% DMF, 5 mM magnesium acetate, and 10 mM tris acetate, pH 7.6. Thus, Mg 2+ was the divalent cation cofactor. Incubation for tagging was performed at 55 °C for 5 minutes.

[0039] In the workflows of Experiments A and B, the DNA was first tagged and the adapter transfer strand was added to the 5' end of the tagged DNA. Tn5 was removed by adding 1% SDS and incubating at 25 °C for 5 minutes. The SDS was washed away in the wash buffer and the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0040] In the workflows of Experiments C and D, DNA was first tagged, and the adapter transfer strand was added to the 5' end of the tagged DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiments C and D, the transposase removal fluid contained 100 mM Tris-HCl, pH 8.6, 100 mM NaCl, and 6 mM EDTA. The tagged DNA complex was incubated in the transposase removal fluid at 60 °C for 1 minute. After washing in the wash buffer, the samples were processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0041] The tagged DNA fragments from Experiments A - D were exposed to the same SPRI technology as described in Example 1 to reversibly bind the tagged DNA as a non-size selected cleanup process. The size profile and yield results are shown in Figure 6. The results in Figure 6 illustrate that, in combination with heat, a transposase removal fluid containing EDTA and salts in a high pH buffer can be applied directly to the tagged reaction without removing the supernatant (performed in Example 2) and can effectively dissociate Tn5 from the tagged DNA complex. Thus, the transposase removal fluid can be introduced without performing a first wash to exchange the tagging buffer.

[0042] Example 4 This example illustrates the results from a comparative method (Experiment A) and the method of the example (Experiment B) regarding the removal of the Tn5 transposase enzyme after tagging. For each of these experiments, the tagging buffer contained water, 4% DMF, 2 mM magnesium acetate, and 4 mM Tris acetate, pH 7.6. Thus, Mg 2+ was the divalent cation cofactor. Incubation for tagging was performed at 55 °C for 5 minutes.

[0043] In the workflow of Experiment A, DNA was first tagmented, and the adapter transfer strand was added to the 5' end of the tagmented DNA. Tn5 was removed by adding 1% SDS and incubating at 25°C for 5 minutes. The SDS was washed away in the wash buffer, and the sample was processed under extension-ligation and PCR cycle conditions using ligase, non-strand-displacing polymerase, and Q5 polymerase in buffer base.

[0044] In the workflow of Experiment B, DNA was first tagmented, and the adapter transfer strand was added to the 5' end of the tagmented DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiment B, the transposase removal fluid contained 140 mM Tris, pH 8.8, 100 mM KCl, and 1.8 mM EDTA. The tagmented DNA complex was incubated in the transposase removal fluid at 60°C for 1 minute. The sample was processed under extension-ligation and PCR cycle conditions using ligase, non-strand-displacing polymerase, and Q5 polymerase in buffer base containing 2.4 mM magnesium.

[0045] The tagmented DNA fragments from Experiments A and B were exposed to the same SPRI technology as described in Example 1, except that a portion of the supernatant was analyzed using a Bioanalyzer, and the tagmented DNA was reversibly bound as a non-size-selected cleanup process. The size profile and yield results are shown in Figure 7. These results, in combination with heat, illustrate that a transposase removal fluid containing EDTA and salts in a high-pH buffer can be applied directly to the tagmentation reaction and can dissociate Tn5 from the tagmented DNA complex. These results also show that following dissociation of Tn5, a composition containing ligase, non-displacing polymerase, and Q5 polymerase in a magnesium-containing base buffer can be added directly to the reaction without removing the supernatant (e.g., without changing the buffer), and the enzymes can function.

[0046] Example 5 This example illustrates the results from a positive control method (Experiment A), a first comparative method (Experiment B), the method of the example (Experiment C), and a second comparative method (Experiment D) regarding the removal of the Tn5 transposase enzyme after tagmentation. For Experiments A and D, the tagmentation buffer contained water, 10% DMF, 5 mM magnesium acetate, and 10 mM Tris-acetate, pH 7.6. In these experiments, Mg 2+ served as the divalent cation cofactor. For Experiments B and C, the tagmentation buffer contained water, 10% dimethyl formamide (DMF), 2.5 mM cobalt, and 25 mM Tris-HCl, pH 7.6. In these experiments, Co 2+ served as the divalent cation cofactor. For all of the experiments, the incubation for tagmentation was carried out at 55 °C for 5 minutes.

[0047] In the workflow of Experiment A, DNA was first tagmented using the Mg 2+ tagmentation buffer described in this example, and the adapter transfer strand was added to the 5'-end of the tagmented DNA. Tn5 was removed by adding 1% SDS and incubating at 25 °C for 5 minutes. The SDS was washed away in the wash buffer, and the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer bases.

[0048] In the workflow of Experiment B, DNA was first tagmented using the Co 2+ tagmentation buffer described in this example, and the adapter transfer strand was added to the 5'-end of the tagmented DNA. Tn5 was removed by adding 1% SDS and incubating at 25 °C for 5 minutes. The SDS was washed away in the wash buffer, and the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer bases.

[0049] In the workflow of Experiment C, the Co described in this example 2+ First, DNA was tagged using the tagmentation buffer, and the transfer strand of the adapter was added to the 5' end of the tagged DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiment C, the transposase removal fluid contained 100 mM Tris, pH 8.6, 150 mM KCl, and 5 mM Cyclen. The tagged DNA complex was incubated in the transposase removal fluid at 60 °C for 1 minute. After washing in the wash buffer, the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0050] In the workflow of Experiment D, the Mg described in this example 2+ First, DNA was tagged using the tagmentation buffer, and the transfer strand of the adapter was added to the 5' end of the tagged DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiment D, the transposase removal fluid contained 100 mM Tris, pH 8.6, 150 mM KCl, and 5 mM Cyclen. The tagged DNA complex was incubated in the transposase removal fluid at 60 °C for 1 minute. After washing in the wash buffer, the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0051] The tagged DNA fragments from Experiments A - D were exposed to the same SPRI technology as described in Example 1 to reversibly bind the tagged DNA in a non - size - selected cleanup process. The size profile and yield results are shown in Figure 8. Line B in Figure 8 illustrates that 2+ the cation can replace Mg 2+ in the catalytic mechanism of Tn5. Line C in Figure 8 illustrates that 2+ the cation can replace Mg 2+ in the catalytic mechanism of Tn5, and 2+Compositions containing Cyclen that chelate can act to dissociate Tn5 from tagged DNA complexes, exemplifying a suitable library template size profile and post-PCR yield. Line D in Figure 8 exemplifies that Cyclen is ineffective in chelating Mg 2+ from the active site of the enzyme. Overall, the results in Figure 8 demonstrate that when Co 2+ cations are used as cofactors, in combination with heat, a transposase removal fluid containing Cyclen and salts in a high-pH buffer is sufficient to replace SDS as a reagent for dissociating the Tn5 enzyme from tagged DNA complexes.

[0052] Example 6 This example exemplifies the results from the method of the example (Experiment A), the positive control method (Experiment B), and the comparative method (Experiment C) regarding removing the Tn5 transposase enzyme after tagging. For each of these experiments, the tagging buffer contained water, 10% DMF, 5 mM magnesium acetate, and 10 mM tris acetate, pH 7.6. Thus, Mg 2+ was the divalent cation cofactor. Incubation for tagging was performed at 55 °C for 5 minutes.

[0053] In the workflow of Experiment A, DNA was first tagged and the adapter transfer strand was added to the 5' end of the tagged DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiment A, the transposase removal fluid contained 500 mM tris, pH 8.6, 100 mM NaCl, and 15 mM 2-hydroxy-4-oxo-4-thiophen-2-yl-but-2-enoate integrase inhibitor. The tagged DNA complex was incubated in the transposase removal fluid at 60 °C for 1 minute. After washing in the wash buffer, the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0054] In the workflow of Experiment B, DNA was first tagged, and the transfer strand of the adapter was added to the 5' end of the tagged DNA. Tn5 was removed by adding 1% SDS and incubating at 25°C for 5 minutes. The SDS was washed away in the wash buffer, and the samples were processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0055] In the workflow of Experiment C, DNA was first tagged, the transfer strand of the adapter was added to the 5' end of the tagged DNA, and then it was treated with water (not the transposase removal fluid or sodium dodecyl sulfate (SDS)), thus maintaining Tn5 bound to the tagged DNA. The tagged DNA complex was washed and subjected to PCR (including high-temperature heating that can dissociate Tn5) to add the adapter sequence to the 3' end of the tagged DNA.

[0056] The tagged DNA fragments from Experiments A - C were exposed to the same SPRI technology as described in Example 1 to reversibly bind the tagged DNA as a non-size selection cleanup process. The size profile and yield results are shown in Figure 9. These results demonstrate that a compound designed to inhibit the integrase enzyme can chelate cations from the active site of the Tn5 transposome product complex, thus dissociating Tn5 from the DNA and enabling the generation of a sequencing library for use.

[0057] Example 7 This example illustrates the results from the positive control methods (Experiments A and B), which relate to removing the Tn5 transposase enzyme after tagging, and the methods of the examples (Experiments C and D). For Experiments A and B, the tagging buffer contained water, 2 mM magnesium acetate, and 10 mM Tris-HCl, pH 7.5. In these experiments, Mg 2+was a divalent cation cofactor. For Experiments C and D, the tagmentation buffer contained water and 1.25 mM cobalt. In these experiments, Co 2+ was a divalent cation cofactor. For all of the experiments, incubation for tagmentation was performed at 37 °C for 15 minutes.

[0058] In the workflows of Experiments A and B, Mg 2+ tagmentation buffer was used to first tagment the DNA, and the adapter transfer strand was added to the 5' end of the tagmented DNA. Tn5 was removed by adding 1% SDS and incubating at 25 °C for 5 minutes. The SDS was washed away in the wash buffer, and the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0059] In the workflows of Experiments C and D, Co 2+ tagmentation buffer was used to first tagment the DNA, and the adapter transfer strand was added to the 5' end of the tagmented DNA. Tn5 was removed by adding an example of the transposase removal fluid disclosed herein. In Experiments C and D, the transposase removal fluid contained 95 mM Tris, pH 8.8, 75 mM KCl, and 3.75 mM Cyclen. The tagmented DNA complex was incubated in the transposase removal fluid at 65 °C for 1 minute. After washing in the wash buffer, the sample was processed under PCR cycle conditions using EPM as the polymerase and buffer base.

[0060] As a size selection cleanup process, the tagged DNA fragments from Experiments A - D were exposed to solid phase reversible immobilization (SPRI) technology to reversibly bind the tagged DNA. In this process, SPRI beads were added to each sample at 0.5 times the volume of the PCR buffer and incubated at room temperature for approximately 5 minutes. The tagged DNA bound to the SPRI beads was pelleted. Next, the supernatant was transferred to a new tube and additional SPRI beads were added at a ratio of 0.12 times compared to the supernatant volume. The sample was incubated at room temperature for approximately 5 minutes. Then, the SPRI beads were pelleted, washed with ethanol, the ethanol was removed, resuspended in buffer, and then pelleted again. A portion of the supernatant was analyzed using a TapeStation. The size profile and yield results are shown in Figure 10. The peaks of the lines (A - D) in Figure 10 were marker peaks used to align these portions of the lines for the sample data and were not part of the sample data, so they are not reproduced herein. Lines A and B in Figure 10 show the size profile and yield obtained from a control workflow using Mg and SDS in the tagging reaction to dissociate Tn5. Lines C and D in Figure 10 illustrate that Co cations can replace Mg in the catalytic mechanism of Tn5, and that a composition containing Cyclen that chelates Co can act to dissociate Tn5 from the tagged DNA complex, resulting in a suitable library template size profile and yield after PCR. Overall, the results in Figure 10 demonstrate that when Co cations are used as cofactors, in combination with heat, a transposase removal fluid containing Cyclen and salts in a high - pH buffer is sufficient to replace SDS as a reagent for dissociating the Tn5 enzyme from the tagged DNA complex. 2+ and the size profile and yield obtained from a control workflow using Mg and SDS in the tagging reaction to dissociate Tn5. Lines C and D in Figure 10 show that Co 2+ cations can replace Mg in the catalytic mechanism of Tn5, and that Co 2+ can replace Mg in the catalytic mechanism of Tn5, and that Co 2+ cations can replace Mg in the catalytic mechanism of Tn5, and that a composition containing Cyclen that chelates Co can act to dissociate Tn5 from the tagged DNA complex, resulting in a suitable library template size profile and yield after PCR. Overall, the results in Figure 10 demonstrate that when Co 2+ cations are used as cofactors, in combination with heat, a transposase removal fluid containing Cyclen and salts in a high - pH buffer is sufficient to replace SDS as a reagent for dissociating the Tn5 enzyme from the tagged DNA complex.

[0061] Next, these comparative samples and the samples of the examples were sequenced on a NOVASEQ™ 6000 instrument according to the customer user guide, the runs were analyzed, and downsampled to 30x coverage using the Fluente:Dragen Downsample v1.0 App in Basespace. The data in Figure 11 show equivalent performance in accuracy or collability of the comparative samples and the samples of the examples across regions of the human genome having different AT and GC contents. The AT and GC contents referred to in Figure 11 are defined in Table 1 below.

[0062] [Table 1]

[0063] During sequencing, single nucleotide polymorphisms (SNPs) change a single nucleotide in the DNA sequence and indels incorporate or remove one or more nucleotides. These secondary metrics were also collected and are shown in Figures 12A - 12D. In these graphs, accuracy refers to correctness and is calculated as the following ratio, [Number of true positive calls / (Number of true positive calls + Number of false positive calls)] Recall refers to sensitivity and is calculated as the following ratio. [Number of true positive calls / (Number of true positive calls + Number of false negative calls)] The results in Figures 12A - 12D illustrate equivalent performance of the comparative method and the method of the examples for detecting and correctly calling both SNPs and indels as compared to a known true set for the NA12878 human platinum genome sample.

[0064] The results in Figures 11 and 12A - 12D demonstrate that the transposase removal fluid does not have a detrimental effect on downstream sequencing.

[0065] Representative items Item 1. A method, Exposing a deoxyribonucleic acid sample to tagmentation in the presence of a tagmentation buffer containing a divalent cation cofactor and a transposase enzyme, thereby generating a tagmented DNA fragment complex; Adding a transposase removal fluid to the tagmented DNA fragment complex, wherein the transposase removal fluid contains a chelating agent for the divalent cation cofactor in a weight ratio of at least 1:1 with the divalent cation cofactor, has a pH in the range of 8 - 9, and adding; Incubating the tagmented DNA fragment complex in the transposase removal fluid at a temperature of at least 55 °C for at least about 60 seconds, thereby dissociating the transposase enzyme from the tagmented DNA fragment of the tagmented DNA fragment complex. A method comprising incubating. Item 2. The divalent cation cofactor is Mg 2+ and The method according to item 1, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and diketo acid anti-retroviral compounds. Item 3. The divalent cation cofactor is Co 2+ and The method according to item 1, wherein the chelating agent is selected from the group consisting of azacrown ether compounds, N-acetyl-cysteine, porphyrins, and crown ethers. Item 4. The chelating agent is an azacrown ether compound, The method according to item 3, wherein the azacrown ether compound is 1,4,7,10-tetraazacyclododecane tetrahydrochloride. Item 5. The chelating agent is a crown ether, The method according to item 3, wherein the crown ether is selected from the group consisting of 15-crown-5 and 18-crown-6. Item 6. The method according to item 1, further comprising a salt at a concentration of at least 75 mM. Item 7. The method according to item 6, wherein the salt is an inorganic salt selected from the group consisting of sodium salts, potassium salts, and lithium salts. Item 8. The method according to item 1, further comprising washing the transposase removal fluid and the dissociated transposase enzyme from the tagged DNA fragment. Item 9. The method according to item 1, further comprising introducing a reagent into the tagged DNA fragment to add an adapter sequence to the 3' end of the tagged DNA fragment. Item 10. A transposase removal fluid, a chelating agent for a divalent cation cofactor, a buffer, and water, the transposase removal fluid containing the same. Item 11. The divalent cation cofactor is Mg 2+ and the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and diketo acid antiretroviral compounds, the transposase removal fluid according to item 10. Item 12. The divalent cation cofactor is Co 2+ and the chelating agent is selected from the group consisting of azacrown-ether compounds, N-acetyl-cysteine, porphyrin, and crown ethers, the transposase removal fluid according to item 10. Item 13. The chelating agent is an azacrown-ether compound, and the azacrown-ether compound is 1,4,7,10-tetraazacyclododecane tetrahydrochloride, the transposase removal fluid according to item 12. Item 14. The chelating agent is a crown ether, and the crown ether is selected from the group consisting of 15-crown-5 and 18-crown-6, the transposase removal fluid according to item 12. Item 15. The transposase removal fluid according to item 10, further comprising a salt at a concentration of at least 75 mM, wherein the salt is optionally an inorganic salt selected from the group consisting of sodium salts, potassium salts, and lithium salts. Item 16. A kit comprising: A tagmentation buffer comprising: Water; A co-solvent; A divalent cation cofactor for the transposase enzyme; A buffer; A transposase removal fluid comprising: Water; A chelating agent for the divalent cation cofactor in the tagmentation buffer, having a weight ratio of at least 1:1 with the divalent cation cofactor in the tagmentation buffer; A buffer; and Item 17. The kit according to item 16, wherein the transposase removal fluid further comprises a salt at a concentration of at least 75 mM.

[0066] Supplementary Notes It should be understood that all combinations of the foregoing concepts and further concepts, to be considered in more detail below, are intended to be part of the subject matter of the invention disclosed herein (provided such concepts are not mutually inconsistent). Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein. It should also be understood that any terms used explicitly herein and that may also appear in any disclosure incorporated by reference should be given the meaning that most closely matches the particular concepts disclosed herein.

[0067] References throughout this specification to "one example", "another example", "an example", etc. mean that a particular element (e.g., a feature, a structure, and / or a property) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it should be understood that, unless the context clearly dictates otherwise, elements described in connection with any example may be combined in any suitable manner in various examples.

[0068] Although some embodiments have been described in detail, it should be understood that the disclosed examples may be modified. Therefore, the foregoing description should be considered non-limiting.

Claims

**Claim 1** A method comprising: exposing a deoxyribonucleic acid sample to tagmentation in the presence of a tagmentation buffer containing a divalent cation cofactor and a transposase enzyme, thereby generating a tagmented DNA fragment complex; adding to the tagmented DNA fragment complex a transposase removal fluid, wherein the transposase removal fluid: contains a chelating agent for the divalent cation cofactor in a weight ratio of at least 1:1 with the divalent cation cofactor; has a pH in the range of 8 - 9; incubating the tagmented DNA fragment complex in the transposase removal fluid at a temperature of at least 55°C for at least about 60 seconds, thereby dissociating the transposase enzyme from the tagmented DNA fragments of the tagmented DNA fragment complex. **Claim 2** wherein the divalent cation cofactor is Mg ２＋ and The method according to claim 1, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and diketo acid antiviral compounds. **Claim 3** wherein the divalent cation cofactor is Co ２＋ and The method according to claim 1, wherein the chelating agent is selected from the group consisting of aza-crown-ether compounds, N-acetyl-cysteine, porphyrins, and crown ethers. **Claim 4** The method according to claim 3, wherein the chelating agent is the aza-crown-ether compound, and the aza-crown-ether compound is 1,4,7,10-tetraazacyclododecane tetrahydrochloride. **Claim 5** The method according to claim 3, wherein the chelating agent is the crown ether, and the crown ether is selected from the group consisting of 15-crown-5 and 18-crown-6. **Claim 6** The method according to any one of claims 1 - 5, wherein the transposase removal fluid further contains a salt at a concentration of at least 75 mM, and the salt is optionally an inorganic salt selected from the group consisting of sodium salts, potassium salts, and lithium salts. **Claim 7** The method according to any one of claims 1 - 6, further comprising washing the transposase removal fluid and the transposase enzyme dissociated from the tagmented DNA fragments. **Claim 8** The method according to any one of claims 1 to 7, further comprising introducing a reagent into the tagged DNA fragment to add an adapter sequence to the 3'-end of the tagged DNA fragment.

9. A transposase removal fluid, comprising: a chelating agent for a divalent cation cofactor; a buffer; water, and a transposase removal fluid.

10. The divalent cation cofactor is Mg ２＋ and The transposase removal fluid according to claim 9, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and diketo acid anti-retroviral compounds.

11. wherein the divalent cation cofactor is Co ２＋ and The transposase removal fluid according to claim 9, wherein the chelating agent is selected from the group consisting of azacrown ether compounds, N-acetyl-cysteine, porphyrin, and crown ethers.

12. i) the chelating agent is the azacrown ether compound, and the azacrown ether compound is 1,4,7,10-tetraazacyclododecane tetrahydrochloride, or ii) the chelating agent is the crown ether, and the crown ether is selected from the group consisting of 15-crown-5 and 18-crown-6. The transposase removal fluid according to claim 11.

13. The transposase removal fluid according to any one of claims 9 to 12, further comprising a salt at a concentration of at least 75 mM, wherein the salt is optionally an inorganic salt selected from the group consisting of sodium salts, potassium salts, and lithium salts.

14. A kit, comprising: a tagging buffer, comprising: water; a co-solvent; a divalent cation cofactor for a transposase enzyme; a buffer, and a tagging buffer; a transposase removal fluid, comprising: water; a chelating agent for the divalent cation cofactor in the tagging buffer at a weight ratio of at least 1:1 to the divalent cation cofactor in the tagging buffer; a buffer, and a transposase removal fluid, and a kit.

15. The kit according to claim 14, wherein the transposase removal fluid further comprises a salt at a concentration of at least 75 mM.

16. i) the divalent cation cofactor is Mg ２＋ and The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid and diketo acid anti-retroviral compounds, or ii) the divalent cation cofactor is Co ２＋ and The kit according to claim 14 or 15, wherein the chelating agent is selected from the group consisting of an aza-crown-ether compound, N-acetyl-cysteine, porphyrin, and crown ether.

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