Preparation of nucleic acid fragments of controlled size

Abstract

Described herein are transposon complexes capable of producing nucleic acid fragments of controlled size. In some embodiments, the transposon complexes comprise a plurality of inactive transposons, with active transposons on both ends of the plurality of inactive transposons. Applications, uses, and variants of the disclosed transposon complexes include, but are not limited to, library preparation of nucleic acids and tuning of the length of the transposon complexes to produce nucleic acid fragments of a predetermined or desired length.

Description

Background Art

[0001] The disclosed technology relates to nucleic acid sequencing. In particular, the disclosed technology relates to transposome complexes that can be used to generate size-controlled nucleic acid fragments, such as as part of generating a sequencing library for nucleic acid sequencing.

[0002] The subject matter discussed in this section should not be considered to be prior art simply because it is mentioned in this section. Similarly, problems mentioned in this section or associated with the subject matter provided as background technology should not be considered to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which themselves may also correspond to specific implementations of the technology protected by the claims.

[0003] Sample preparation (e.g., library preparation) for next generation sequencing can involve fragmenting nucleic acids (such as genomic DNA or double-stranded cDNA (prepared from RNA)) into smaller fragments, followed by the addition of functionalized tag sequences ("tags") to the strands of the fragments. Such tags can include priming sites for DNA polymerases used in sequencing reactions, restriction sites, and domains for capture, amplification, detection, addressing, and transcription promoters. Previous methods for generating DNA fragment libraries can involve mechanically fragmenting target DNA using a sonicator, a nebulizer, or by nucleases, and then joining (e.g., by ligation) oligonucleotides containing these tags to the ends of these fragments.

[0004] The use of transpososomes, protein-DNA complexes of transposases, and transposon sequences that tag and fragment DNA by transposition ("tagment") allows for simultaneous genomic fragmentation and adapter incorporation into the fragments, thereby simplifying library preparation. Methods for rapidly performing these steps using transpososomes to generate fragments from any double-stranded DNA (e.g., genomes, amplicons, viruses, phages, cDNA derived from RNA, etc.) are disclosed in US 2010 / 0120098 to Grunenwald, which is incorporated herein by reference for all purposes. Transposon systems include the hyperactive Tn5 transposon system described in U.S. Pat. Nos. 5,965,443 and 6,437,109 to Reznikoff, and the Mu transposon system described in U.S. Pat. No. 6,593,113 to Tenkanen, all of which are incorporated herein by reference. Reznikoff described a 19-base transposase end sequence, which is often referred to as "ME", a mosaic end sequence. Transposon end tagging is used to label nucleic acid fragments generated from biological samples. This article describes techniques for improving the process of transposon-mediated nucleic acid fragment generation, and thus improving subsequent nucleic acid sequencing from such fragments. Summary of the invention

[0005] In one embodiment, the present disclosure relates to a transposome complex. The transposome complex comprises a plurality of inactive transposomes coupled to each other. Each of the plurality of inactive transposomes comprises a transposase and an oligonucleotide adaptor. The transposome complex also comprises a first active transposome coupled to a first end of the plurality of inactive transposomes. Further, the transposome complex comprises a second active transposome, which is coupled to a second end of the plurality of inactive transposomes, such that the plurality of inactive transposomes are located between the first active transposome and the second active transposome.

[0006] In another embodiment, the present disclosure relates to a method for preparing a transposome complex. The method includes providing a starting transposome. The starting transposome comprises a transposome dimer, a first at least partially double-stranded oligonucleotide adapter coupled to the transposome dimer, and a second at least partially double-stranded oligonucleotide adapter coupled to the transposome dimer. The method also includes hybridizing at least one ligating transposome with the starting transposome via the at least partially double-stranded ligating adapter of the at least one ligating transposome, wherein the at least partially double-stranded ligating adapter is complementary to the first at least partially double-stranded oligonucleotide adapter, the second at least partially double-stranded oligonucleotide adapter, or both. Further, the method includes coupling at least one terminal transposome with the at least one ligating transposome via the at least partially double-stranded terminal adapter of the terminal transposome, the at least partially double-stranded terminal adapter being complementary to the at least partially double-stranded ligating adapter or a different ligating adapter of the at least one ligating transposome, wherein the terminal transposome is catalytically active and wherein the at least one ligating transposome is catalytically inactive.

[0007] In another embodiment, the present disclosure relates to a method for preparing a nucleic acid library. The method includes contacting a target nucleic acid with a plurality of transposome complexes. Each transposome complex in the plurality of transposome complexes comprises a first active transposome coupled to a second active transposome via a plurality of intervening inactive transposomes to allow the plurality of transposome complexes to bind to the target nucleic acid. The method also includes tag-fragmenting the target nucleic acid to generate nucleic acid fragments. The size of the generated nucleic acid fragments is a function of the size of the individual transposome complexes in the plurality of transposome complexes.

[0008] In another embodiment, the present disclosure relates to a surface-attached transposome complex. The surface-attached transposome complex comprises a surface and a plurality of transposomes coupled to the solid surface. Each transposome in the plurality of transposomes comprises a transposase and an oligonucleotide adaptor. Based on modification of the oligonucleotide adaptor, each transposome in the plurality of transposomes is inactive.

[0009] In another embodiment, the present disclosure relates to a method for isolating nucleic acids. The method includes contacting a plurality of transposome complexes with a mixed nucleic acid sample in a solution. The mixed nucleic acid sample comprises double-stranded DNA and RNA, such that the double-stranded DNA selectively binds to the plurality of transposome complexes relative to the RNA. Each of the plurality of transposome complexes comprises a plurality of transposomes coupled to a surface. Further, based on modification of an oligonucleotide adaptor, each of the plurality of transposomes is inactive. The method also includes separating the double-stranded DNA from the RNA by removing the plurality of transposome complexes with bound double-stranded DNA from the solution, the solution comprising the RNA.

[0010] In another embodiment, the present disclosure relates to a method for normalizing the amount of nucleic acid of multiple samples. The method includes contacting a first plurality of double-stranded nucleic acids of a first sample with a first plurality of transposome complexes. Each transposome complex in the first plurality of transposome complexes contains a predetermined amount or range of transposomes coupled to a bead surface. Further, based on the modification of an oligonucleotide adapter, each transposome of the first plurality of transposome complexes is inactive. Still further, the contact is performed under conditions such that a portion of the first plurality of double-stranded nucleic acids binds to the first plurality of transposome complexes. The method also includes contacting a second plurality of double-stranded nucleic acids of a second sample with a second plurality of transposome complexes. Each transposome complex in the second plurality of transposome complexes contains a predetermined amount or range of transposomes coupled to a bead surface. Based on the modification of an oligonucleotide adapter, each transposome of the second plurality of transposome complexes is inactive. Further, the contact is performed under conditions such that a portion of the second plurality of double-stranded nucleic acids binds to the second plurality of transposome complexes. Still further, the method includes sequencing the bound portions of the first plurality of double-stranded nucleic acids and the bound portions of the second plurality of double-stranded nucleic acids.

[0011] In another embodiment, the present disclosure relates to a method for performing buffer exchange. The method includes contacting a plurality of nucleic acids suspended in a first buffer solution with a plurality of transposome complexes. Each transposome complex in the plurality of transposome complexes comprises a plurality of transposomes coupled to a surface. Further, based on modification of an oligonucleotide adaptor, each transposome in the plurality of transposomes is inactive. The method also includes generating a precipitate comprising a plurality of nucleic acids bound to the plurality of transposome complexes. Further, the method includes separating the precipitate from the first buffer solution. Still further, the method includes suspending the precipitate in a second buffer solution.

[0012] The foregoing description is presented to enable making and using the disclosed technology. Various modifications to the disclosed implementations will be apparent, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but to conform to the widest scope consistent with the principles and features disclosed herein. The scope of the disclosed technology is defined by the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] These and other features, aspects and advantages of the present technology will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters refer to like parts throughout, and in which:

[0014] Figure 1 is a schematic representation of a method for preparing a nucleic acid library according to aspects of the present disclosure;

[0015] Figure 2 is a schematic diagram of an example of a transposome complex that can be used to generate size-controlled fragments according to aspects of the present disclosure;

[0016] Figure 3 According to various aspects of the present disclosure, for use Figure 2 A schematic diagram of a method for preparing a library from a target nucleic acid using a transposome complex;

[0017] Figure 4A According to various aspects of the present disclosure, it can be used to generate Figure 2 Schematic diagram of the inactive transposome and the active transposome of the transposome complex;

[0018] Figure 4B is a schematic diagram of hybridization of a first inactive transposome with a second inactive transposome via a corresponding adaptor according to various aspects of the present disclosure;

[0019] Figure 4C is a schematic diagram of hybridization of a second inactive transposome with a third inactive transposome via a corresponding adaptor according to various aspects of the present disclosure;

[0020] Figure 4D is a schematic diagram of hybridization of a third inactive transposome with an active transposome via a corresponding adaptor according to various aspects of the present disclosure;

[0021] Figure 5 According to various aspects of the present disclosure, for generating Figure 2 A schematic diagram of a method for producing a transposome complex;

[0022] Figure 6 is a perspective view of an asymmetric transposome complex attached to a substrate according to aspects of the present disclosure;

[0023] Figure 7 shows a graph of template insert sizes resulting from sequencing a DNA library according to aspects of the present disclosure;

[0024] Fig. 8A shows a schematic diagram of a surface-linked transposome complex (SLTC) including an inactive transposome according to aspects of the present disclosure;

[0025] Figure 8B shows a schematic diagram of a SLTC including an inactive transposome and an active transposome according to aspects of the present disclosure;

[0026] FIG. 9 shows a graph illustrating fragment size regulation based on the addition of inactive transposomes to SLTCs, according to aspects of the present disclosure;

[0027] Fig.10 shows gel electrophoresis results illustrating transposome formation according to aspects of the present disclosure;

[0028] Fig.11 shows a first graph illustrating the tag fragmentation activity of a bead-linked transposome complex (BLT) comprising an active transposome and the tag fragmentation activity of a bead-linked transposome complex comprising an inactive transposome (zBLT) according to aspects of the present disclosure;

[0029] Fig.12 shows a second graph illustrating products of tag fragmentation treatment of BLT containing active transposomes and products of BLT containing only inactive transposomes according to aspects of the present disclosure;

[0030] Fig.13 shows a graph illustrating the amount of nucleic acid bound to BLT and zBLT according to aspects of the present disclosure;

[0031] Fig.14 shows a first graph illustrating the tag fragmentation activity of BLT and the tag fragmentation activity of zBLT for different ratios of active transposomes and inactive transposomes according to aspects of the present disclosure;

[0032] Fig.15A showing a first graph illustrating tag fragmentation activity of zBLT having a first amount of inactive transposomes according to aspects of the present disclosure;

[0033] Fig. 15B showing a second graph illustrating tag fragmentation activity of zBLT having a second amount of inactive transposomes according to aspects of the present disclosure;

[0034] Fig. 15Cshowing a third graph illustrating tag fragmentation activity of zBLT having a third amount of inactive transposomes according to aspects of the present disclosure;

[0035] Fig.16 showing graphs illustrating transformation efficiency, sensitivity, and average insert length of control bead-linked transposomes and bead-linked transposomes comprising inactive transposomes, according to an embodiment;

[0036] Fig.17 According to various aspects of the present disclosure, for use Fig. 8A A schematic diagram of the SLTC method for isolating nucleic acids;

[0037] Fig.18 According to various aspects of the present disclosure, for use Fig. 8A A graphical representation of the method of normalizing the amount of nucleic acid by SLTC;

[0038] Fig.19A is a diagrammatic representation of a method for transferring nucleic acid from a first solution to a second solution according to aspects of the present disclosure;

[0039] Fig.19B According to various aspects of the present disclosure, for use Fig. 8A A schematic diagram of a first method of transferring nucleic acid from a first solution to a second solution by SLTC;

[0040] Fig.19C According to various aspects of the present disclosure, for use Fig. 8A A schematic diagram of a second method of transferring nucleic acid from a first solution to a second solution using SLTC;

[0041] Fig. 20 is a schematic diagram showing inactivation of a transposome according to aspects of the present disclosure;

[0042] Fig.21A shows a graph illustrating the distribution of fragment sizes before being subjected to zSLTC in accordance with aspects of the present disclosure;

[0043] Fig. 21B shows a graph illustrating the distribution of fragment sizes bound to inactive surface-linked transposome complexes (zSLTCs) having a first density of inactive transposomes bound to the zSLTCs according to aspects of the present disclosure;

[0044] Fig. 21C shows a graph illustrating the distribution of fragment sizes bound to zSLTCs having a second density of inactive transposomes bound to the zSLTCs according to aspects of the present disclosure;

[0045] Fig.21Dshows a graph illustrating the distribution of fragment sizes bound to zSLTCs having a third density of inactive transposomes bound to the zSLTCs according to aspects of the present disclosure;

[0046] Fig.22A shows the results of gene expression analysis using a first amount of Universal Human Reference (UHR) RNA according to aspects of the present disclosure;

[0047] Fig. 22B shows the results of gene expression analysis using a second amount of UHR RNA according to aspects of the present disclosure;

[0048] Fig.23A shows the results of gene expression analysis using a first amount of human brain reference (HBR) RNA according to aspects of the present disclosure;

[0049] Fig. 23B shows the results of gene expression analysis using a second amount of HBR RNA according to aspects of the present disclosure;

[0050] Fig.24A shows the distribution of nucleic acids obtained using a normalization technique without zBLT according to aspects of the present disclosure;

[0051] Fig. 24B shows the distribution of nucleic acids obtained using a normalization technique with zBLT according to aspects of the present disclosure;

[0052] Fig.25A shows the distribution of genes with different normalizations obtained using manual normalization; and

[0053] Fig.25B The distribution of genes with different normalizations obtained with zBLT is shown. DETAILED DESCRIPTION

[0054] The following discussion is presented to enable any person skilled in the art to make and use the disclosed technology, and is provided in the context of a specific application and its requirements. Various modifications to the disclosed implementations will be apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0055] Library preparation for downstream processing and analysis (such as for nucleic acid sequencing) generally involves nucleic acid (e.g., genomic DNA) fragmentation to generate fragments (e.g., nucleic acid fragments) that are subsequently amplified and sequenced. Depending on the fragment preparation technology, the generated fragments can have a relatively wide size range, such as between 10 base pairs to 1000 base pairs. At least in some cases, the instrument for sequencing the generated fragments can only operate on fragments within a specific fragment size range, and therefore, not all of these fragments can be sequenced by the instrument. Therefore, fragments outside the operable size range are not used to generate sequencing data and are wasted. For low-concentration samples, this waste may result in a decline in low sequencing coverage and sequencing data quality.

[0056] Some techniques, such as using electrophoresis gel, using coated magnetic beads that can be re-formulated to enable size selection, etc., can be used to select nucleic acid fragments with a specific fragment size range suitable for the instrument. However, such techniques may still result in the discarding of a significant portion of the nucleic acid sample composed of fragments in a specific size range that is not suitable for the instrument. Some techniques, such as Nextera Flex and the bead-connected transposome method of Nextera Flex for enrichment, give greater control of the amount and reproducibility of the generated fragment size. However, for some applications, the distribution of fragment size may still be relatively wide, and may involve additional size selection to be performed, which may result in discarding fragments that are too large in size and fragments that are insufficient in size. In addition, separation (for example, during size selection) of relatively wide fragment sizes may be time-consuming. Therefore, it is beneficial to generate nucleic acid fragments or generate nucleic acid fragments with a relatively narrow size range in a size-controlled manner, while also limiting the amount of nucleic acid fragments that are not in a specific size range that are discarded.

[0057] Thus, aspects of the present disclosure relate to methods, compositions, and kits, and in particular to methods, compositions, and kits for fragmenting nucleic acids to generate fragments of a specific size or size range. Certain techniques for fragmenting nucleic acids include tag fragmentation or using transposomes to perform tag fragmentation reactions.

[0058] Transposome is a protein-DNA complex comprising a transposase (e.g., Tn5 enzyme) and a transposon. Transposome can carry out tag fragmentation to a target nucleic acid sample via a transposition reaction. In general, "tag fragmentation" or performing a "tag fragmentation reaction" involves joining the transposon end sequence to a nucleic acid, thereby labeling the nucleic acid (that is, transferring the chain to join), and simultaneously cutting the nucleic acid to produce a fragment. As discussed in further detail herein, transposomes are inserted as dimers so that transposomes carry out tag fragmentation (e.g., labeling and fragmentation) to two chains of nucleic acid. More specifically, two transposases in a transposome dimer (that is, each transposome has a transposase in two transposases) are inserted into different chains of a double-stranded nucleic acid. Each transposase of a transposome dimer cuts its corresponding nucleic acid chain, and the transfer chain of the transposome (e.g., of a transposome dimer) is connected to the cut end of the nucleic acid. The non-transferred chain of the transposome can hybridize with the transferred chain, but is not connected by a transposase. Using multiple transposomes (that is, each transposome is a dimer) to tag fragment the target nucleic acid involves joining the transposon end sequence of each transposome in the transposome to different positions along the target nucleic acid and cutting the target nucleic acid at different positions. Therefore, the target fragment is formed between two adjacent positions along the corresponding chain (for example, two positions without an intervening transposome), at which the transposon end sequences of the two transposomes are joined, and the target fragment has a length corresponding to the distance between the two adjacent positions. In addition, the target fragment is tag-fragmented and therefore contains two transposon end sequences at the opposite ends of the target fragment. That is, the target fragment contains a first transposon end sequence derived from the first transposome of the two transposomes at the first end. In addition, the tag-fragmented target fragment contains a second transposon end sequence derived from the second transposome of the two transposomes at the second end (for example, different from the first end). It should be noted that although the above disclosure describes that both transposases of the dimer tag-fragment the nucleic acid, it should be noted that, at least in some cases, only one of the transposases can be tag-fragmenting (that is, one of the transposases can be inactivated, as described in further detail herein).

[0059] At least in some cases, the ends of the target fragments are single-stranded (e.g., have single-stranded gaps) along a portion of the target fragments after being fragmented by the tag. For example, the target fragments may include single-stranded gaps extending along a portion of the target fragments adjacent to the transposon end sequence (e.g., at the first end and / or the second end). It should be noted that a gap-filling reaction can be performed to add additional nucleic acid along the single-stranded gaps so that the target nucleic acid is double-stranded along the portion of the target fragments adjacent to the transposon end sequence.

[0060] As discussed in more detail herein, the disclosed technology includes the use of a transposome complex (e.g., a Cascade complex) formed by a plurality of enzymes (e.g., transpososomes) that can each bind to a region of a target nucleic acid. As discussed in more detail herein, a transposome complex can include a combination of active transpososomes and inactive transpososomes. In some embodiments, the transposome complex can include inactive transpososomes that are all coupled to each other, and a first active transposome coupled to a first end of the transposome complex, and a second active transposome coupled to a second end of the transposome complex. Typically, an active transposome is catalytically active (e.g., the transposome is not inactivated by chemical modification or heat), and is therefore capable of inserting into a target nucleic acid. An "inactive" transposome refers to a catalytically inactive transposome that cannot be joined (e.g., inserted via ligation) to a target nucleic acid and / or that prevents or removes the ability of a transposase to cut a nucleic acid chain. In certain embodiments, a transposome can be inactivated via selective mutation to remove or reduce enzyme activity. At least in some cases, the inactive transposome can still bind to the target nucleic acid. At least in some cases, the inactive transposome can be inactive due to the inactivation of the transposase of the transposome, such as by modifying the amino acid sequence of the transposase. In some embodiments, the transposome can be inactive due to the modification of the oligonucleotides of the adapter forming the transposome rendering the transposome inactive, while the transposase can still be active. Therefore, when each transposome of the transposome complex (e.g., the inactive transposome, the first active transposome, and the second active transposome) binds to the corresponding region of the target nucleic acid, the target nucleic acid can be fragmented only at the region where the first active transposome binds to the second active transposome, thereby generating fragments whose length is proportional to the length of the transposome complex or the footprint of the bound transposome complex on the target nucleic acid. Therefore, a tag fragmentation reaction using the disclosed transposome complex can generate multiple fragments each having approximately the same length. In addition, by tuning (e.g., increasing or decreasing) the number of inactive transposomes, the length of fragments generated by tag fragmentation using the disclosed transposome complexes can be controlled. Thus, the disclosed technology can reduce the amount of unused nucleic acid, which can be beneficial for applications where the amount of nucleic acid is limited. Further, the disclosed technology can increase the speed of generating fragments by reducing the number of additional steps (such as size selection) to be performed on the fragments.

[0061] Considering the foregoing, Figure 1 A schematic flow diagram illustrating the transposase-catalyzed insertion of a transposome end sequence into a nucleic acid to generate a nucleic acid fragment is shown. Fig.10, which can be performed in combination with the size-controlled fragment generation technology provided herein. In the illustrated embodiment, a plurality of transpososomes 12 comprising at least one transposase 14 and a transposon end sequence 16 are provided to a target nucleic acid 18. Typically, the transposon end sequence 16 can be a part of a transposome complex or a transposome composition, which can insert or transpose the transposon end sequence 16 into the target nucleic acid, such as when the transposase 14 is incubated with the target nucleic acid 18 in an in vitro transposition reaction. Typically, the transposase 14 (e.g., integrase / integration enzyme) recognizes and binds to the transposon end sequence 16 to form the transposome 12. For example, the transposon end sequence 16 can be a nucleic acid that can form a complex with a transposase 14 (such as a hyperactive Tn5 transposase). In this example, the transposon end sequence 16 typically comprises a transferred transposon end sequence (e.g., a transferred strand) and an untransferred transposon end sequence (e.g., a non-transferred strand). In the in vitro transposition reaction, the 3' end of the transferred strand is joined or transferred to the target nucleic acid 18. In the in vitro transposition reaction, the non-transferred strand, which exhibits a transposon end sequence complementary to the transferred transposon end sequence, is not joined or transferred to the target DNA.

[0062] Other examples of transposon end sequences 16 include, but are not limited to, wild-type, derivative, or mutant transposon end sequences that form a complex with a transposase 14 selected from a wild-type, derivative, or mutant transposase. For example, the transposon end sequence can be a wild-type or mutant form of Tn5 transposase and MuA transposase. In some embodiments, the transposon end sequence 16 that binds to the transposase 14 has a suitable size to provide selectivity for binding between the transposon end sequence 16 and the transposase 14. For example, Tn5 derived EZ-Tn5 TM The transposon end sequence of the Aβ transposon comprises only 19 nucleotides, whereas some other transposases require much larger end sequences for transposition (e.g., MuA transposase which utilizes a transposon end sequence of approximately 51 nucleotides).

[0063] In some embodiments, one or more additional nucleotide sequences may be attached to the 5' end of the transferred strand or the 3' end of the non-transferred strand. For example, the one or more additional nucleotide sequences may comprise a barcode, a universal molecular identifier (UMI), or other adapter sequences that can facilitate sequencing of the target nucleic acid 18 by enabling identification of the relative ordering of the fragments.

[0064] Return to reference Figure 1, the transposon end sequence 16 of each transposome 12 is joined to the corresponding region 20 of the target nucleic acid 18. In the depicted embodiment, three transposomes 12 are shown: a first transposome 12a, a second transposome 12b, and a third transposome 12c. The transposon end sequence 16 of the first transposome 12a is joined to regions 20a and 20b of the strand of the target nucleic acid 18. The transposon end sequence 16 of the second transposome 12b is joined to regions 20c and 20d of the strand of the target nucleic acid 18. The transposon end sequence 16 of the third transposome 12c is joined to regions 20e and 20f of the strand of the target nucleic acid 18. Thus, when the transposome 12 (e.g., using the transposase 14) fragments the target nucleic acid 18, the transposome 12 generates target fragments 22 (e.g., nucleic acid fragments) each having a length 24 (that is, the length 24 of the fragment 22 is shown) that is proportional to the distance between the two regions 20 (in which the two flanking transposon end sequences 16 join the target nucleic acid 18) and represents the nucleotide base or base pair length of the fragment 22. For example, the length 24 shown in the illustrated embodiment can be proportional to the length between region 20a and region 20c. It should be noted that the transposon end sequence 16 can be double-stranded, and the joining of the transposon end sequence 16 to the strands of the target nucleic acid 18 can generate target fragments 22 that each contain a single-stranded gap (e.g., about 9 base pairs) extending along the end of the target fragment 18 to the transposon end sequence.

[0065] The sizes (e.g., lengths 24) of the fragments 22 generated by the transposome 12 can have a relatively large size distribution, and therefore, at least a portion of the fragments 22 may be discarded because they are too large or too small for certain applications (such as for sequencing by a particular instrument). In order to generate fragments with a controllable size distribution, a transposome complex formed by a plurality of inactive enzymes (e.g., transposase 14) and a plurality of active transpososomes can be used to fragment the target nucleic acid. To illustrate this, Figure 2 A schematic diagram of a transposome complex 26 is shown, which can provide size-controlled DNA strands for sequencing.

[0066] In the illustrated embodiment, individual transposome complexes 26 include a plurality of inactive transposomes 28 and active transposomes 30, each of which has an associated transposase 14. As shown, the inactive transposomes 28 include 13 inactive transposomes 28. However, the transposome complexes 26 may have any suitable number of inactive transposomes 28. In one example, a transposome complex 26 as provided herein includes a first active transposome 30 separated from a second active transposome 30 by one or more inactive transposomes 28. The active transposome 30 and the one or more inactive transposomes 28 are coupled to each other (e.g., connected, bound, hybridized to each other via complementary sequences). In one embodiment, an intervening inactive transposome 28 or an inactive transposome 28 (located between the first active transposome 30 and the second active transposome 30) is connected to an adjacent transposome that may be active or inactive, depending on the specific arrangement of the transposome complex 26. The active transposomes 30 form the ends (first end 31, second end 33) of the transposome complex 26, such that each active transposome 30 at the ends 31, 33 is linked to only one adjacent transposome (e.g., an inactive transposome 28). In embodiments, there is a single intervening inactive transposome 28 that is linked to both terminal active transposomes 30 at the ends 31, 33 of the transposome complex. In embodiments, the ratio of active transposomes 30 to inactive transposomes 28 in the transposome complex 26 is 2:1, 2:2, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 2:9, 2:10, 2:15, 2:20, 2:25, 2:30, 2:40, or 2:N.

[0067] However, as discussed herein, the number, arrangement and / or type of intervening inactive transposomes 28 between terminal active transposomes 30 may be selected to provide desired length control or to facilitate a particular sequencing technique. In the depicted embodiment, the transposome complex 26 comprises an inactive transposome 28. For example, the depicted embodiment includes a first inactive transposome, a second inactive transposome, and a third inactive transposome, etc. In an embodiment, each of the inactive transposomes 28 within the transposome complex 26 may be structurally different from an adjacent or neighboring transposome due to different terminal sequences and / or connection sequences that couple the inactive transposomes 28 together (i.e., to adjacent transposomes). It should be noted that providing a different adapter for each of the inactive transposomes 28 may enable the transposome complex 26 to grow in a controlled manner. That is, each adapter of each of the inactive transposomes may provide selectivity for adjacent binding, as discussed in more detail with respect to FIG. 4 . Thus, as illustrated, the inactive transposome 28 may include a starting transposome 32 that forms the seed from which the transposome complex 26 grows, and various adapter transposomes 34 , 36 that may include sequences that are complementary to each other and / or to the starting transposome 32 .

[0068] In some embodiments, the adapter can be an adapter sequence (e.g., an oligonucleotide adapter) specific to each type of inactive transposome. For example, the starting transposome 32 can include a first adapter (e.g., a first adapter sequence) whose nucleic acid sequence is configured to hybridize (via complementary binding) with a second adapter (e.g., a second adapter sequence) of the adapter transposome 34. In addition, the adapter transposome 36 can include a third adapter (e.g., a third adapter sequence) that is configured to hybridize with the second adapter but not with the first adapter. Thus, when assembling the inactive transposome 28, such as by sequentially adding each of the inactive transposomes in the inactive transposome to a solution to assemble the inactive transposome (this will be with respect to Figure 5 By sequentially adding each different type of inactive transposome 28, the growth and therefore the length of the transposome complex 26 can be controlled (as discussed in more detail herein).

[0069] For example, the transposome complex 26 may include any number of inactive transposomes 28 such that the resulting length of the fragments generated using the transposome complex 26 is greater than 50 base pairs, 100 base pairs, 500 base pairs, or greater than 1000 base pairs. The length of the nucleic acid fragments generated may be between 50 and 150 base pairs, 50 and 500 base pairs, 150 and 500 base pairs, 500 and 1000 base pairs. As described above, the active transposome 30 is capable of inserting into the target nucleic acid and is therefore catalytically active. For example, the active transposome 30 may have a catalytically active end that can insert a sequence into the nucleic acid. As also discussed herein, an "inactive" transposome, such as inactive transposome 28, refers to a catalytically inactive transposome (e.g., due to modification of the transposome 28 or the adaptor of the transposase) that is unable to join (e.g., insert) into a target nucleic acid (e.g., target nucleic acid 18), but the inactive transposome 28 still binds to the target nucleic acid 18.

[0070] Typically, the inactive transposome 28 can be inactivated using a suitable chemical or thermal inactivation technique, such as via chemical modification or by blocking the terminal sequence of the transposase of the transposome. For example, such techniques for inactivating the transposome to generate the inactive transposome 28 include, but are not limited to, heating the transposase, dephosphorylating the 5' end of the transposase, and blocking the 3' end with a chemical modification. Although the active transposome 30 and the inactive transposome 28 are described as being different (that is, active or inactive), it should be noted that in some embodiments, the active transposome 30 and the inactive transposome 28 may contain the same type of integrase (e.g., transposase).

[0071] Thus, the transposome complex 26 incubated with the target nucleic acid 18 will use the active transposomes to join to the target nucleic acid 18, while the inactive transposomes will each bind to the corresponding portion of the target nucleic acid 18. That is, the 3' end of the transposon end of the active transposome 30 will join to the 5' end of the target nucleic acid 18. It should be noted that controlling the number of inactive transposomes 28 can be used to control the length of the final fragmented DNA chain, as described in relation to Figure 5 discussed in more detail.

[0072] In the illustrated embodiment, the transposome (e.g., active transposome 30 and inactive transposome 28) is a dimer. That is, each transposome comprises a dimer, the monomers of which have a transposase (e.g., Tn5 transposase) coupled to a transposon or other adapter sequence. For example, active transposome 30 includes an active transposome dimer, and inactive transposome 28 includes an inactive transposome dimer. In some embodiments, a portion of the transposome (e.g., a full transposome) may be a homodimer. In some embodiments, the transposome may be a connected dimer. That is, the monomers of the dimer may be connected, such as by post-translational addition of a linker, or the protein of the transposome may be expressed as a tandem fusion when manufactured. For example, the transposome may be a genetic fusion of the Tn5 transposase, producing a single transposase protein backbone with two identical domains (e.g., both are Tn5 transposases). In some embodiments, a portion of the transposome may be a heterodimer. It should be noted that transposomes may generally include other types of integrants capable of binding to DNA. For example, DNA binding enzymes may include, but are not limited to, Crispr / Cas proteins.

[0073] Thus, the transposome complex 26 can be used to bind to DNA and generate size-controlled fragments. It should be noted that each of the enzymes of the transposome complex 26 can be capable of binding to nucleic acids, regardless of whether the enzymes are catalytically active. That is, although the transposase of the inactive transposome 28 is catalytically inactive, the inactive transposome 28 can still be allowed to bind to the target nucleic acid 18. At least in some cases, the initial binding of one or more enzymes in the complex to the nucleic acid can cause a synergistic effect, so that the remaining enzymes of the complex 26 bind to the same nucleic acid molecule. As a result, the transposome complex 26 can position the active transposomes 30 (e.g., active end transposomes) of the complex on the same DNA molecule in "cis". It should be noted that because each of the transposomes of the transposome complex 26 is capable of binding to nucleic acids, the transposome complex 26 can bind to nucleic acids synergistically and in "cis". By binding in "cis", the size of the fragmented nucleic acid can be proportional to the number of transposomes of the transposome complex. That is, these active transposomes 30 subsequently cut the DNA at a fixed distance (e.g., size) determined by the length or spatial separation distance between the pairs of active end transposomes 30 in the transposome complex 26. In addition, tuning the ratio of the transposome complex 26 to the nucleic acid (e.g., DNA) substrate or target nucleic acid 18 of the sample of interest can facilitate fragmentation of the nucleic acid to a uniform size. For example, the ratio of the transposome complex 26 to the nucleic acid (where the transposome complex 26 is in excess) can facilitate fragmentation of the nucleic acid to a uniform size. In addition, the amount of nucleic acid that is not covered or not used can be reduced by increasing the ratio of the transposome complex 26, thereby minimizing or reducing the amount of nucleic acid that is not used or discarded. In another embodiment, the ratio of nucleic acid to the transposome complex 26 is in excess, which can generate two populations of fragments with different size distributions. For example, a first population of fragments corresponding to regions of the nucleic acid bound by the transposome complex 26 can have a uniform size distribution, and a second population of fragments corresponding to unbound regions of the nucleic acid can have a random size distribution. Since the second population is not bound by the transposome complex 26, the second population can be digested, such as by a nuclease that cleaves accessible double-stranded nucleic acids.

[0074] At least in some cases, binding of the transposome complex 26 to the target nucleic acid and cleavage of the target nucleic acid by the active transposome 30 of the transposome complex 26 (e.g., a cleavage step) can be separated by a duration. For example, the active transposome 30 can be activated to cleave the target nucleic acid. That is, the transposome complex 26 can be provided to the target nucleic acid, and after a duration that can correspond to the period during which the transposome of the transposome complex 26 binds to the nucleic acid, the active transposome can be activated, such as by providing a salt (e.g., a magnesium-containing salt) to a solution containing the transposome complex 26 and the target nucleic acid or increasing the temperature of the solution. Providing a duration between binding and cleavage of the target nucleic acid can increase the likelihood that the transposomes of the transposome complex 26 bind cooperatively.

[0075] Figure 3 A flow chart for preparing a nucleic acid library using a transposome complex 26 that binds to a target nucleic acid 18 and generates fragments 22 of controlled length is shown. As shown in the illustrated embodiment, the transposome complex 26 binds to the nucleic acid 18 along the length 40 of the nucleic acid 18. It should be noted that although the transposome complex 26 includes an inactive transposome 28, at least a portion or all of the transposomes of the complex 26 are bound to the nucleic acid 18. As discussed herein, regardless of whether the transposome (e.g., active transposome 30 or inactive transposome 28) is active, both the active transposome 30 and the inactive transposome 28 are bound to the nucleic acid 18. In addition, the initial binding of one or more transposomes in the transposome complex 26 to the nucleic acid 18 can cause a synergistic binding effect, which can cause each of the remaining unbound transposomes to bind to the same nucleic acid 18.

[0076] Thus, the individual transposome complexes 26 each bind along a corresponding length 40 of the nucleic acid. That is, the first transposome complex 26a binds along a first length 40a of the target nucleic acid 18, the transposome complex 26b binds along a second length 40b of the target nucleic acid 18, and the third transposome complex 26c binds along a third length 40c of the target nucleic acid 18. Binding to the nucleic acid 18 results in the transposome complex 26 mediating a tag fragmentation reaction of the nucleic acid 18. As discussed herein, "tag fragmentation" or performing a "tag fragmentation reaction" involves the ligation of the transposon end sequence to the nucleic acid 18 at the binding site, thereby tagging the nucleic acid 18 (that is, transferring the chain ligation) and simultaneously cleaving the nucleic acid 18 to generate fragments 22, which can together form a nucleic acid library 39. For example, after the transposome end sequence of each active transposome 30 is ligated to the target nucleic acid 18, the fragments 22 are generated. When the 3' ends of the transposon end sequences of active transposomes 30a and 30b are joined to the nucleic acid 18 along the length 40a, a fragment 22a is formed. When the 3' ends of the transposon end sequences of active transposomes 30c and 30d are joined to the nucleic acid 18 along the length 40b, a fragment 22b is formed. When the 3' ends of the transposon end sequences of active transposomes 30e and 30f are joined to the nucleic acid 18 along the length 40c, a fragment 22c is formed. Since the inactive transposomes between the active transposomes 30 are not joined to the target nucleic acid 18, the length 42 of the fragment 22 is a function of the binding length covered by the transposome complex 26. Therefore, the length 42 of the fragment 22 is based on the number of transposomes (e.g., active transposomes 26 and inactive transposomes 28) of the transposome complex 26.

[0077] In embodiments where a plurality of transposome complexes 26 are provided, each transposome complex 26 has approximately the same length 40 relative to one another (that is, the same number of total transposomes or the same transposome arrangement and / or the same number of inactive transposomes 28 and active transposomes 30), such that each resulting fragment will have approximately the same length 40. In embodiments where a plurality of transposome complexes 26 of different sizes are used, the resulting cleaved nucleic acid fragments 22 will have correspondingly different lengths 40.

[0078] At least in some cases, a portion of the nucleic acid 18 may be uncovered or not associated with the transposome complex 26, and therefore may not be of a suitable length (e.g., for measurement by an instrument). Therefore, the uncovered portion of the nucleic acid 18 can be solubilized or digested by suitable means known to those of ordinary skill in the art. In this way, the foreign nucleic acid 18 can be substantially removed from the solution or substrate from which the library preparation is performed. The digestion can occur in conjunction with the binding of the transposome complex, such that only the uncovered portion is digested, while the covered portion of the nucleic acid 18 is protected by the presence of the associated transposome complex 26. Alternatively, a size exclusion method can be used to filter out the uncovered portion having a first size from the covered portion having a second size.

[0079] As described above, each of the transposomes (that is, the inactive transposome 28 and the active transposome 30) may be capable of binding only to one or more transposomes of a specific type. To illustrate this, 4A to 4D (For example, Figure 4A , Figure 4B , Figure 4C ,and Figure 4D ) shows an inactive transposome and an active transposome each comprising an adaptor sequence that provides selective binding to the inactive transposome and the active transposome.

[0080] Figure 4A The depicted embodiment shows a first inactive transposome 32, a second inactive transposome 34, and a third inactive transposome 36. Each of the transposomes (e.g., the first inactive transposome 32, the second inactive transposome 34, the third inactive transposome 36, and the active transposome 30) comprises a corresponding adapter pair. As discussed herein, the transposomes can be dimer complexes, and therefore, each transposome comprises two adapters. As shown, the first inactive transposome 32 comprises a first adapter 44a and a second adapter 44b, the second inactive transposome 34 comprises a first adapter 46a and a second adapter 46b, the third inactive transposome 36 comprises a first adapter 48a and a second adapter 48b, and the active transposome 30 comprises a first adapter 50a and a second adapter 50b.

[0081] Typically, adapters 44, 46, 48, and 50 can be at least partially double-stranded oligonucleotides. In the illustrated embodiment, adapters 44, 46, 48, and 50 include a single-stranded overhang on the 3' end. However, in some embodiments, adapters 44, 46, 48, and 50 can include a single-stranded overhang on the 5' end. In some embodiments, adapters 44, 46, 48, and 50 can be coupled to corresponding transpososome monomers. For example, the first adapter 44a can be coupled to the first transposase 45a of the first inactive transpososome 32 via the first monomer, and the second adapter 44b can be coupled to the second transposase 45b of the second inactive transpososome 32 via the second monomer. Similarly, the first adapter 46a can be coupled to the first transposase 47a of the inactive transpososome 34 via the first monomer, and the second adapter 46b can be coupled to the second transposase 47b of the second inactive transpososome 34 via the second monomer. The first adaptor 48a can be coupled to the first transposase 49a of the inactive transposome 36 via the first monomer, and the second adaptor 48b can be coupled to the second transposase 49b of the second inactive transposome 36 via the second monomer. In some embodiments, the adaptors 44, 46, 48, 50 can be different for the corresponding transposomes. The adaptors 44a and 44b can comprise the same nucleotide sequence as part of the homodimer.

[0082] In the depicted embodiment, the active transposome 30 comprises a double-stranded adaptor on each active transposase 51 of the transposome dimer. It should be noted that the first adaptor 50a (e.g., a first oligonucleotide adaptor) of the first active transposome 30 and the second adaptor 50b (e.g., a second oligonucleotide adaptor) of the second active transposome 30 can each comprise a double-stranded transposon end sequence and a single-stranded adaptor sequence on each monomer of the corresponding transposome dimer.

[0083] As a non-limiting example of how an adapter oligonucleotide sequence can be used to form a transpososome complex (e.g., a Cascade complex), the Tn5 transposase adapter is a double-stranded oligonucleotide of a fixed sequence, called a mosaic end (ME) sequence. The chain that is connected to the target nucleic acid (e.g., target substrate DNA) during tag fragmentation is called the "transferred chain". It should be noted that the 3'OH-end of this chain is transferred and connected to the target nucleic acid during tag fragmentation. The complementary chain in the Tn transposome can be called the "non-transferred chain". In active transpososome enzymes, the 5'OH-end is phosphorylated; phosphorylation is required for the transposome to be active. This lack of phosphate renders the transposome catalytically inactive, but still able to bind to the substrate DNA. The ME duplex can be approximately 19 bp long. For example, the ME duplex can be short at one or both of the 5' end of the transferred chain of the ME or the 3' end of the non-transferred chain of the ME. Additional sequences can be appended to the 5' end of the transferred chain and the 3' end of the non-transferred chain. These additional bases can be of any length and sequence.

[0084] In a specific embodiment, the first inactive transposome 32 (eg, the starting transposome) comprises a non-transferred strand having an additional sequence appended to its 3' end. Figure 4B As shown, these additional sequences can be complementary to the additional sequences attached to the 3' end of the non-transferred strand of the second inactive transposome 34 (e.g., the first ligated transposome). Thus, at least a portion (e.g., at least 5 bases, at least 10 bases) of the adapter sequences 44, 46 are complementary. In addition, the additional sequences can be the same sequence and polarity as the additional sequences attached to the 3' end of the non-transferred strand of the third inactive transposome 36 (e.g., the second ligated transposome). As shown Figure 4C As shown, by definition, the additional sequence attached to the 3' end of the non-transferred strand of the second inactive transposome 34 is complementary to the additional sequence attached to the 3' end of the non-transferred strand of the third inactive transposome 36. Furthermore, the additional sequence attached to the 3' end of the non-transferred strand of the first inactive transposome 32 and the additional sequence attached to the 3' end of the non-transferred strand of the third inactive transposome 36 are the same sequence, complementary to the additional sequence attached to the 3' end of the non-transferred strand of the active transposome 30 (e.g., the terminal transposome), as shown. Figure 4D It should be noted that although 4A to 4D The assembly of the transposome complex 26 via hybridization of the 3′ end of the non-transferred strand of the transposome is exemplified, but assembly can also be achieved via hybridization of the 5′ end of the non-transferred strand of the transposome.

[0085] Each of the transpososomes (e.g., active transpososome 30, first inactive transpososome 32, second inactive transpososome 34, and third inactive transpososome 36) can have additional sequences attached to the 5' end of the transferred strand of these transpososomes. In particular, active transpososome 30 can have additional sequences attached to the 5' end of the transferred strand, which play a role in the preparation of the library later, such as attaching additional functionality, such as sequences for amplifying the library or attaching the library to a sequencing flow cell. Such sequences may include universal adapter sequences, sequencing primers, capture sequences, etc. In one embodiment, the 5' end of the non-transferred strand of active transpososome 30 is phosphorylated. Any transpososome in the transpososome may contain a portion for attaching the transpososome to a surface. For example, the 5' end of the transferred strand of the first inactive transpososome may be biotinylated so that it binds to magnetic beads coated with streptavidin. In some embodiments, additional sequences may be attached to the 3' end of the non-transferred strand. For example, the 3' end of the non-transferred strand may contain sequences that can be recognized and bound by certain enzymes, such as polymerases used in gap-filling reactions. Thus, after the tags have been fragmented from the active transposome 30 and the transferred strand has been ligated to the DNA substrate, the non-transferred strand may also be ligated to the DNA substrate, such as by using a non-strand-displacing polymerase and a ligase. It should be noted that either the transferred strand or the non-transferred strand may contain additional sequences that may facilitate the addition of additional adapter sequences (e.g., by primer extension, ligation).

[0086] Figure 5 is a flow chart of a method 52 for preparing a transposome complex 26. At box 54, a first inactive transposome 32 (e.g., a starting transposome) is provided. In some embodiments, providing a transposome complex may include providing a substrate 56, shown here as a magnetic bead. At box 58, the first inactive transposome 32 is attached to the substrate 56 (e.g., via the 5' end (that is, "Bio5'") of the first inactive transposome 32). For example, the starting transposome 32 is attached to streptavidin magnetic beads. In other embodiments, the transposome complex 26 is prepared in solution. In embodiments using a support surface substrate 56, the substrate 56 may be washed to remove any first inactive transposome 32 that remains in solution or is not bound to the substrate.

[0087] At block 60, one or more second inactive transposomes 34 (eg, ligated transposomes) are added and hybridized to the starting transposome via their complementary sequences, followed by washing to remove unbound transposomes. 4A to 4DAs discussed, the second inactive transposome 34 may include an adaptor 46 that is complementary to the adaptor 44 of the first inactive transposome 32. Thus, the adaptor 44 of the first inactive transposome 32 may be coupled, bound, or hybridized to the adaptor 46 of the second inactive transposome 34. In such embodiments where the two adaptors 44 of the first inactive transposome 32 are complementary to the two adaptors 46 of the second inactive transposome 34, the second inactive transposome 34 may bind to both sides (e.g., the two adaptors 44) of the first inactive transposome 32. In any case, once one or more second inactive transposomes 34 have hybridized to one or more first inactive transposomes 32, the substrate 56 may be washed to remove any second inactive transposome 34 remaining in the solution (that is, not bound to the first inactive transposome 34). Additionally or alternatively, the second inactive transposome 34 and the first inactive transposome may be cross-linked. At least in some cases, cross-linking the second inactive transposome 34 and the first inactive transposome together can improve the rigidity or robustness of the transposome complex 26. Further, cross-linking can improve the size control of the transposome complex 26. At least in some cases, cross-linking can improve the stability of the transposome complex 26 by preventing or substantially reducing monomer exchange between the transposomes of the transposome complex 26. In some embodiments, the transposomes of the transposome complex 26 can include stabilizers, such as locked nucleic acids (LNA), which can provide additional stability to the transposomes of the transposome complex 26.

[0088] At block 62, a third inactive transposome 36 (e.g., a second ligating transposome) is added and hybridized to the second transposome (e.g., the second inactive transposome 34) via its complementary sequence in a manner substantially similar to that described for the hybridization of the second inactive transposome 34 to the first inactive transposome 32. In some embodiments, blocks 60 and 62 may be repeated multiple times to add additional inactive transposomes (e.g., a ligating transposome, a second inactive transposome 34, a third inactive transposome 36) to the transposome complex 26, thereby increasing the length of the transposome, which increases the size of the fragments generated using the transposome complex 26. When the transposome complex 26 reaches a predetermined length, at block 64, an active transposome 30 (e.g., a terminal transposome) may hybridize to the third inactive transposome 36, thereby providing a catalytically active end to the transposome complex 26 (e.g., a Cascade complex). The inactive transposomes 28 of the transposome complex 26 can be provided as individual transposomes that have been inactivated, or can be inactivated en masse after being ligated together but before adding the active transposome 30. It should be noted that by providing a different adaptor for each of the first inactive transposome 32, the second inactive transposome 34, and the third inactive transposome 36, uncontrolled growth of the transposome complex 26 can be prevented. For example, during blocks 60 and 62, having different adaptors can prevent multiple inactive transposomes from binding to a specific end of the transposome complex 26.

[0089] As discussed herein, the disclosed transposome complexes can be used to prepare nucleic acid libraries, such as sequencing libraries, to generate DNA fragments of controlled length. The DNA is cut with the transposome complex 26. For example, the transposon end sequence can include a transferred DNA strand and a non-transferred DNA strand, which can contain a 19 base pair (bp) mosaic end sequence or a truncated DNA sequence. The non-transferred strand (e.g., with or without nuclease protection and / or chain termination groups, such as phosphorothioate and / or dideoxy) is then dissociated from the transferred strand, and a replacement oligomer (which can contain additional DNA sequences, such as sequencing tags) is annealed to a complementary transferred strand sequence with or without a nuclease protection group (e.g., phosphorothioate). The DNA can be prepared using a DNA polymerase (e.g., a thermostable polymerase, or a non-thermostable polymerase such as DNA polymerase I or Klenow fragment exo - ) and a DNA ligase. DNA polymerase and ligase are used to fill and ligate the gap between the singly tagged DNA and the replacement oligonucleotide, thereby generating a dsDNA fragment with covalently attached 5' and 3' tags. Alternatively, oligonucleotides can be provided to fill the gap and then ligated.

[0090] As generally discussed above, the second inactive transposome 34 can hybridize to both sides of the first inactive transposome 32. Thus, the transposome complex 26 can be symmetrical because there are inactive transposomes growing from opposite sides of the first inactive transposome 32. At least in some cases, the transposome complex 26 can grow asymmetrically around the first inactive transposome 32. To illustrate this, Figure 6 A schematic diagram of a transposome complex 26 that has grown asymmetrically is shown. In the depicted embodiment, the first inactive transposome 32 is coupled to a substrate 56 (e.g., a magnetic bead) via a bond 66. The bond 66 can be a hybridization between two single-stranded nucleic acids that are respectively bound to the first inactive transposome 32 and the substrate 56. In any case, by binding one side of the first inactive transposome 32 to the substrate 56, the second inactive transposome 34 can hybridize only to the opposite side of the first inactive transposome. Although the depicted embodiment shows that the transposome complex 26 has grown asymmetrically, it should be understood that the transposome complex 26 can also grow symmetrically around the first inactive transposome 32.

[0091] After being formed by the disclosed techniques, the transposome complexes 26 can be purified or otherwise subjected to a selection step (e.g., selection based on molecular weight) to form a composition enriched in transposome complexes 26, which can be of the same size and have the same number of inactive transposomes and active transposomes. At least in some cases, the transposome complexes 26 can remain bound to the substrate 56 for library preparation reactions. For example, during a library preparation reaction, a plurality of target nucleic acids can be provided to a solution containing a plurality of transposome complexes 26, and each transposome complex 26 can be bound to a corresponding substrate 56.

[0092] Figure 7 A graph of template insert sizes resulting from sequencing a DNA library is shown according to various aspects of the present disclosure. More specifically, Figure 7A graph representing the size of the insert fragment obtained by a paired end sequencing experiment. The read pairs (that is, the ends of the templates derived from the sequencing) are mapped to a reference genome and used to determine the size of the template insert fragment. This experimental group illustrates a comparison between two embodiments of the transposome. In one case (e.g., the embodiment illustrated in box 57), a cascade transposome complex is created, which comprises a single inactive "anchor" transposome 28 coupled to the surface of the beads and has two active transposomes 30 bound via complementary adapters. In another embodiment (e.g., illustrated in box 59), free active transposomes 30 are used. In both cases, equal amounts of active transposomes are used in tag fragmentation experiments. The results show that when the transposomes are cascaded, the modal distribution of the insert fragment size is greater than when they are not cascaded, illustrating that the transposome complex of the present disclosure with cascaded transposomes can regulate and expand the insert fragment size.

[0093] Thus, aspects of the present disclosure relate to preparing transposome complexes that enable the generation of size-controlled nucleic acids, such as during library preparation. In general, the disclosed transposome complexes have a plurality of inactive transposomes, each of which is coupled to an adjacent inactive transposome via an adapter (e.g., an oligonucleotide adapter sequence). In addition, the disclosed transposome complexes contain active transposomes coupled to inactive transposomes at the ends of a plurality of inactive transposomes. As discussed herein, "activity" or "inactivity" refers to the ability of a transposome or a transposase of a transposome to not be able to perform tag fragmentation on a nucleic acid. For example, an active transposome may have an available transfer strand. However, although the disclosed transposome complexes may contain inactive transposomes, the inactive transposomes may still bind to a target nucleic acid. Therefore, when the disclosed transposome complexes are provided to a nucleic acid, at least a portion of the transposomes (e.g., active transposomes and inactive transposomes) may bind to the nucleic acid. After binding to the nucleic acid, the active transposome can perform tag fragmentation on the nucleic acid, thereby inserting the transposon end sequence into the nucleic acid and fragmenting the nucleic acid, thereby generating portions of the nucleic acid that are bound to multiple transposomes. These portions of the nucleic acid that are bound to the transposomes are fragments of a size approximately equal to the length of the transposome complex after fragmentation. It should be noted that by modifying the number of transposomes (e.g., inactive transposomes) provided to the transposome complex, the length of the transposome complex and thus the size of the fragments ultimately produced by the transposome complex can be tuned. Therefore, the disclosed transposome complex can reduce the amount of fragments that are discarded due to inappropriate size for certain instruments by generating such a transposome complex having a number of inactive transposomes corresponding to the length (e.g., number of base pairs) of an instrument suitable for a specific size range.

[0094] As provided herein, "transposase" can refer to an enzyme that is capable of forming a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and, in an in vitro transposition reaction, catalyzes the insertion or transposition of the transposon end-containing composition into a double-stranded target DNA incubated therewith.

[0095] The term "transposon end" may refer to a DNA that contains a nucleotide sequence ("transposon end sequence") necessary to form a complex with a transposase or integrase that functions in an in vitro transposition reaction. The transposon end forms a "complex" or "transposome complex" or "transposome composition" with a transposase or integrase that recognizes and binds to the transposon end, and in an in vitro transposition reaction, the complex is able to insert or transpose the transposon end into a target DNA incubated therewith. The transposon end exhibits two complementary sequences consisting of a "transferred transposon end sequence" or "transferred strand" and a "non-transferred transposon end sequence" or "non-transferred strand." For example, with a hyperactive Tn5 transposase (e.g., EZ-Tn5 TM One of the transposon ends of the complex formed by the transposase (EPICENTRE Biotechnologies, Madison, Wis., USA) comprises a transferred strand exhibiting the following "transferred transposon end sequence":

[0096] 5'AGATGTGTATAAGAGACAG 3'(SEQ ID NO:1)

[0097] and a non-transferred strand exhibiting a "non-transferred transposon end sequence" as follows:

[0098] 5'CTGTCT CTTATACACATCT 3'. (SEQ ID NO:2).

[0099] In an in vitro transposition reaction, the 3' end of the transferred strand in the active transposome (that is, the active transposome 30) is joined or transferred to the target DNA. In an in vitro transposition reaction, the non-transferred strand that exhibits a transposon end sequence complementary to the transferred transposon end sequence is not joined or transferred to the target DNA. As provided herein, the inactive transposome 28 may comprise all or part of the transposon end sequence or a modified transposon end sequence that results in inactivation or inactivation of the transposase.

[0100] In some embodiments, the transfer strand and the non-transfer strand are covalently joined. For example, in some embodiments, the transfer strand sequence and the non-transfer strand sequence are provided on a single oligonucleotide, for example, in a hairpin configuration. Therefore, although the free end of the non-transfer strand is not directly joined to the target DNA by the transposition reaction, the non-transfer strand is indirectly attached to the DNA fragment because the non-transfer strand is connected to the transfer strand by the loop of the hairpin structure.

[0101] "Transposon end composition" means a composition comprising a transposon end (that is, a minimal double-stranded DNA segment capable of being acted upon by a transposase to undergo a transposition reaction) optionally plus an additional sequence or sequences. 5' of the transferred transposon end sequence and / or 3' of the non-transferred transposon end sequence. For example, a transposon end attached to a tag is a "transposon end composition". In some embodiments, a transposon end composition comprises or consists of two transposon end oligonucleotides, the two transposon end oligonucleotides consisting of a "transferred transposon end oligonucleotide" or "transferred strand" and a "non-transferred strand end oligonucleotide" or "non-transferred strand", the "transferred transposon end oligonucleotide" or "transferred strand" and the "non-transferred strand end oligonucleotide" or "non-transferred strand" in combination exhibiting the sequence of a transposon end, and one or both strands contain additional sequences in these sequences.

[0102] However, in some embodiments, the transposon end composition comprises or consists of at least one transposon end oligonucleotide that exhibits one or more other nucleotide sequences in addition to the transposon end sequence. Thus, in some embodiments, the transposon end composition comprises a transferred strand that exhibits one or more other nucleotide sequences 5' to the transferred transposon end sequence that are also represented by the tag. Thus, in addition to the transferred transposon end sequence, the tag may also have one or more other tag portions or tag domains.

[0103] As used herein, "tag portion" or "tag domain" means a portion or domain of a tag that exhibits a sequence for a desired intended purpose or application. One tag portion or tag domain is a "transposon end domain", which exhibits the transferred transposon end sequence. In some embodiments, where the transferred strand also exhibits one or more other nucleotide sequences 5' to the transferred transposon end sequence, the tag also has one or more other "tag domains" in the 5'-portion, each of which is provided for any desired purpose. For example, some embodiments comprise or consist of a transposon end composition comprising or consisting of: (i) a transferred strand exhibiting one or more sequences 5' of the transferred transposon end sequence, the one or more sequences comprising or consisting of a tag domain selected from one or more of a restriction site tag domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain; and (ii) a non-transferred strand exhibiting a non-transferred transposon end sequence. Certain embodiments of the method may use any one or more of the transposon end compositions described herein.

[0104] In some embodiments, the disclosed technology is used to generate a nucleic acid library (e.g., library 39) or a DNA fragment library, wherein the DNA fragment library comprises a target DNA fragment having a 5' end, the target DNA fragment having a 5' end comprising a sequence from a transferred strand derived from a transposon end or a transposon end composition. In a preferred embodiment, the sequence from the transferred strand comprises a 5' tag domain, and in a still more preferred embodiment, the DNA fragment library comprises a target DNA fragment containing a 3' tag, the target DNA fragment containing a 3' tag is complementary to the transferred strand from the transposon end or the transposon end composition. In some embodiments, the DNA fragment library comprises double-stranded fragments of the target DNA. The generated library can be used in a sequencing reaction as provided herein.

[0105] The generated nucleic acid library can be sequenced according to any sequencing technology, such as those incorporating sequencing by synthesis methods described in U.S. Patent Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2005 / 0100900; U.S. Patent No. 7,057,026; WO 05 / 065814; WO 06 / 064199; WO 07 / 010,251, the disclosures of which are incorporated herein by reference in their entirety. Alternatively, sequencing by ligation technology can be used in a sequencing device. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides, and are described in U.S. Pat. No. 6,969,488; U.S. Pat. No. 6,172,218; and U.S. Pat. No. 6,306,597; the disclosures of which are incorporated herein by reference in their entirety. Some embodiments may utilize nanopore sequencing, whereby a target nucleic acid strand or nucleotides removed from a target nucleic acid exotomy pass through a nanopore. As the target nucleic acid or nucleotide passes through the nanopore, each type of base can be identified by measuring the fluctuations in the conductivity of the pore (U.S. Pat. No. 7,001,792; Soni & Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); and Cockroft et al., J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entirety). Some other embodiments include detecting the proton released when the nucleotide is incorporated into the extension product. For example, sequencing based on the detection of releasing protons can use the electric detector and related technology commercially available from Ion Torrent Company (Guilford, CT, Life Technologies subsidiary) or the sequencing method and system described in US 2009 / 0026082 A1, US 2009 / 0127589 A1, US 2010 / 0137143A1 or US 2010 / 0282617 A1, each of which is incorporated herein by reference in its entirety. Specific embodiments can utilize the method related to the real-time monitoring of DNA polymerase activity.Nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide or by utilizing a zero-mode waveguide, as described, for example, in Levene et al., Science 299, 682-686 (2003); Lundquist et al., Opt. Lett. 33, 1026-1028 (2008); Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entirety. Other suitable alternative techniques include, for example, fluorescence in situ sequencing (FISSEQ) and massively parallel signature sequencing (MPSS). In a particular embodiment, sequencing can be performed via HiSeq, MiSeq, or HiScanSQ from Illumina (La Jolla, CA).

[0106] The transposome complex 26 can be provided as a pre-prepared composition in which the active transposome and the inactive transposome are already coupled to each other. In one embodiment, the transposome complex 26 is provided as part of a library preparation kit, which can include additional elements, such as appropriate primers for use in conjunction with a desired sequencing platform. The kit can include a transposome complex composition that contains only transposome complexes 26 that are all estimated to be within a specific size or weight tolerance. The sample preparation kit may also include one or more sample preparation enzymes, buffers, and / or reagents. The sample preparation kit can be provided as a pre-packaged kit for preparing a library from a single sample, or in certain embodiments, can be provided as a multi-sample kit with a plurality of different available transposome complexes 26 of different sizes that can produce different library fragment lengths. The end user can select a transposome complex 26 of the desired length and perform the library preparation step using the transposome complex 26 of the selected size. In another embodiment, a library preparation kit may allow a user to construct or make transposome complexes 26 from transposome monomers or dimers or from individual active and inactive transposomes according to the disclosed techniques.

[0107] The disclosed techniques can be used to prepare nucleic acid libraries from target nucleic acids (e.g., target nucleic acid 18). "Target nucleic acids" can be derived from any in vivo or in vitro source, including from one or more cells, tissues, organs, or organisms (whether living or non-living), or from any biological or environmental source (e.g., water, air, soil). For example, in some embodiments, the target nucleic acid comprises or consists of eukaryotic and / or prokaryotic dsDNA, which is derived from or is derived from humans, animals, plants, fungi (e.g., mold or yeast), bacteria, viruses, viroids, mycoplasmas, or other microorganisms. In some embodiments, the target nucleic acid comprises or consists of genomic DNA, subgenomic DNA, chromosomal DNA (e.g., from an isolated chromosome or part of a chromosome, e.g., from one or more genes or loci of a chromosome), mitochondrial DNA, chloroplast DNA, plasmid or other episomal derived DNA (or recombinant DNA contained therein), or double-stranded cDNA, which is prepared by reverse transcribing RNA using an RNA-dependent DNA polymerase or reverse transcriptase to generate a first-strand cDNA, and then extending a primer annealed to the first-strand cDNA to generate dsDNA. In some embodiments, the target nucleic acid comprises a plurality of dsDNA molecules in or prepared from a nucleic acid molecule (e.g., a plurality of dsDNA molecules in or prepared from a genomic DNA or cDNA, which is prepared from RNA in or from a biological source (e.g., a cell, tissue, organ, organism) or an environmental source (e.g., water, air, soil, saliva, sputum, urine, feces)). In some embodiments, the target nucleic acid is from an in vitro source. For example, in some embodiments, the target nucleic acid comprises or consists of dsDNA, which is prepared in vitro from single-stranded DNA (ssDNA) or from single-stranded or double-stranded RNA (e.g., using methods known in the art, such as primer extension using a suitable DNA-dependent and / or RNA-dependent DNA polymerase (reverse transcriptase)). In some embodiments, the target nucleic acid comprises or consists of dsDNA, which is prepared from all or a portion of one or more double-stranded or single-stranded DNA or RNA molecules using any method known in the art, including methods for the following operations: DNA or RNA amplification (e.g., PCR or reverse transcriptase-PCR (RT-PCR), transcription-mediated amplification methods, in which all or a portion of one or more nucleic acid molecules are amplified); molecular cloning of all or a portion of one or more nucleic acid molecules into a plasmid, fosmid, BAC or other vector that is subsequently replicated in a suitable host cell; or capturing one or more nucleic acid molecules by hybridization, such as by hybridization with a DNA probe on an array or microarray.

[0108] In alternative embodiments, the transposome complex may comprise a plurality of transposomes coupled or linked to a solid surface or substrate, thereby forming a surface-linked transposome complex (also referred to herein as a SLTC). For example, the substrate may be a magnetic bead, and thus a plurality of transposomes coupled to the magnetic bead may be referred to as a bead-linked transposome complex (BLT). However, it should be understood that while certain embodiments are discussed in the context of beads, other substrates are also contemplated, including flat surfaces, patterned surfaces, shaped surfaces, and the like. Further, in certain embodiments, the disclosed linked transposome complexes may be used in solution-based reactions. The surface-linked transposome complex may comprise a transposome as generally discussed herein.

[0109] The transposome of the surface-attached transposome complex may include one or more inactive transposomes, one or more active transposomes, or a combination of inactive transposomes and active transposomes. For example, each of the transposomes of the surface-attached transposome complex may be an inactive transposome. As another non-limiting example, each of the transposomes of the surface-attached transposome complex may be an active transposome. As discussed herein, an active transposome is catalytically active (e.g., the transposome is not inactivated by chemical modification or heat) and is therefore capable of inserting into a target nucleic acid. An "inactive" transposome or "zombie transposome" refers to a catalytically inactive transposome that cannot be joined (e.g., inserted via a connection) into a target nucleic acid, but the inactive transposome still binds to the target nucleic acid. That is, an inactive transposome or zombie transposome is capable of binding to a target DNA, but cannot catalyze the joining of a target DNA and a transposon binding sequence. Inactive transposomes may be inactive, at least in some cases, due to inactivation of a transposase of the transposome, such as by modifying the amino acid sequence of the transposase. It is currently recognized that surface-attached transposome complexes comprising inactive transposomes may enable tuning of insert size distribution of sequencing libraries generated using surface-attached transposome complexes. Further, in embodiments in which the surface-attached transposome complexes comprise both inactive transposomes and active transposomes, the inactive transposomes may modulate the location of tag fragmentation events. Furthermore, surface-attached transposome complexes comprising inactive transposomes may facilitate isolation and / or purification of nucleic acids, such as with respect to Figure 17 to Figure 1 9 is described in further detail.

[0110] To illustrate a first example of a surface-attached transposome complex, Fig. 8A A schematic diagram of a surface-bound transposome complex 68 is shown, wherein each transposome is an inactive transposome 36 coupled (e.g., directly or indirectly) to a substrate 56 (e.g., a magnetic bead). Fig. 8AThe surface-bound transposome complex 68 may be referred to as an inactive bead-bound transposome complex (zBLT) 70a or an inactive surface-bound transposome complex (zSLTC). That is, zBLT or zSLTC refers to a transposome complex comprising one or more catalytically inactive transposomes. Fig. 8A In this illustrated embodiment of zBLT70a, all transposomes are inactive transposomes 28. However, as described below with respect to Figure 8B As discussed, the first portion of the transposome may be an inactive transposome 28 , while the second portion may comprise an active transposome 30 .

[0111] refer to Fig. 8A Each inactive transposome 28 of the SLTC 68 can be coupled to a linker (not shown) so that each inactive transposome 28 can be individually attached to the surface of a magnetic bead (e.g., substrate 56). Figure 8B As discussed in, the linker between the magnetic beads and the inactive transposome 28 and / or the active transposome 30 may be due to a functional group on the surface of the beads (such as a carboxyl group, a hydroxyl group, or a carbonyl group), and the binding may be promoted based on the concentration of salt present in the solution. In some embodiments, the inactive transposomes 28 may be coupled to each other (e.g., via an adaptor). In some embodiments, a portion of the inactive transposome 28 may include a linker, while the remaining portion of the inactive transposome 28 may not include the linker. For example, the substrate 56 may be directly coupled to the first portion of the inactive transposome 28 and indirectly coupled to the second portion of the inactive transposome 28. That is, the first portion of the inactive transposome 28 may be directly coupled to the substrate 56 via a linker of each of the inactive transposomes 28 in the first portion. The second portion of the inactive transposome 28 (e.g., different from the first portion) may be directly coupled to the first portion of the inactive transposome 28 via an adaptor. Thus, the second portion of the inactive transposome 28 is indirectly coupled to the substrate 56 via the first portion and the linker of the inactive transposome 28. Fig. 8AThe embodiment of substrate 56 illustrated in the example shows magnetic beads (e.g., beads), but other suitable solid surfaces may also be used. For example, substrate 56 may include an array of wells, and transposomes (e.g., inactive transposomes 28, active transposomes 30, or both) may be coupled to the surface of the wells. Further, it should be noted that one or more of the inactive transposomes 28 of SLTC 68 or zBLT may be structurally different from adjacent or neighboring transposomes due to different terminal sequences and / or linker sequences that couple the inactive transposomes 28 together (i.e., to adjacent transposomes). That is, SLTC 68 or zBLT 70a may include inactive transposomes 32, 34, 36, or other types of inactive transposomes.

[0112] In some embodiments, a mixture of both active transposomes 30 and inactive transposomes 28 may be coupled to substrate 56. To illustrate a second example of SLTC 68, Figure 8B A schematic diagram of a zBLT 70b (e.g., SLTC 68) is shown, which comprises both an inactive transposome 28 and an active transposome 30 coupled to magnetic beads (e.g., a bead-linked transposome complex). Figure 8B SLTC 68 of the present invention may be referred to as an active zBLT 70b. As used herein, an "active" SLTC, BLT, zBLT, or zSLTC refers to a SLTC, BLT, zBLT, or zSLTC comprising an active transposome 30. As used herein, SLTC 68 may generally refer to an active or inactive surface-bound transposome complex. Further, zBLT refers to a bead-bound transposome complex in which at least one transposome is an inactive transposome 28.

[0113] In accordance with the above Fig. 8AIn a generally similar manner as described for SLTC 68 or zBLT 70a of zBLT 70b, each of the active transposome 30 and / or the inactive transposome 28 of zBLT 70b can have its own linker (not shown) so that each of the active transposome 30 and / or the inactive transposome 28 can be individually attached to the surface of the bead. In some embodiments, the active transposome 30 and / or the inactive transposome 28 can be coupled to each other (e.g., via an adaptor). In some embodiments, a portion of the transposome (e.g., an active transposome, an inactive transposome, or a portion of both) can include a linker, while the remainder of the transposome may not include a linker. In addition, while the illustrated embodiments include an inactive transposome 28, it should be noted that one or more of the inactive transposomes 28 of zBLT 70b can be structurally different from adjacent or neighboring transposomes due to different terminal sequences and / or linker sequences that couple the inactive transposomes 28 together (i.e., to neighboring transposomes). That is, zBLT 70b may include inactive transposomes 32, 34, 36, or other types of inactive transposomes. For example, surface-bound transposome complex 68 may include starting transposome 32, which forms a seed from which transposome complex 26 grows, and various adapter transposomes 34, 36, which may include sequences that are complementary to each other and / or to starting transposome 32.

[0114] It is currently recognized that a SLTC 68 (e.g., Figure 8B The size of the fragment or insert generated by zBLT 70b) shown in FIG can be tuned based on the relative number of inactive and active transposomes. To illustrate this, 9A to 9C (Right now, Fig.9A , Fig. 9B ,and Fig. 9C ) shows a schematic diagram of two examples of surface-attached transposomes comprising active transposomes 30.

[0115] More specifically, Fig.9A A schematic diagram of a first surface-attached transposome complex 68 (e.g., BLT 72) comprising an active transposome 30 bound to a target nucleic acid 18 is shown. That is, Fig.9AAll of the transpososomes of the illustrated BLT 72 are active transpososomes. As shown in the illustrated embodiment, the active transpososomes 30 are radially distributed around the substrate 56. In some embodiments, each of the active transpososomes 30 can be evenly distributed around the substrate 56 (e.g., magnetic beads) so that the active transpososomes 30 are substantially equidistant from adjacent active transpososomes 30. In other contemplated arrangements, each of the active transpososomes 30 can be distributed on the substrate 56 in a patterned arrangement that is not necessarily equidistant. In the illustrated embodiment, the substrate 56 does not include any surface-attached inactive transpososomes 28. Therefore, the fragment length of the bound nucleic acid 18 is a function of the location and spacing of the active transpososomes 30 on the substrate 56. This spacing and / or positioning can be based on printing, patterning, or other techniques for activating the substrate 56.

[0116] Fig. 9B A schematic diagram of a second surface-bound transposome complex 68 (e.g., zBLT 70b) is shown, which contains both active transposomes 30 and inactive transposomes 28 bound to a target nucleic acid 18. As shown in the illustrated embodiment, the active transposomes 30 are radially distributed around a substrate 56 (e.g., magnetic beads). In some embodiments, each of the active transposomes 30 can be evenly distributed around the substrate 56 (e.g., magnetic beads) such that the active transposomes 30 are substantially equidistant from adjacent active transposomes 30. Further, as shown in the illustrated embodiment, the number of inactive transposomes 28 between two adjacent active transposomes 30 is substantially the same for each pair of active transposomes 30. Providing an even distribution of active transposomes 30 and inactive transposomes 28 can facilitate the generation of more uniform fragments from the target nucleic acid 18.

[0117] Fig. 9C A graph 80 illustrating the size distribution of fragments of a target nucleic acid 18 generated by BLT 72 and zBLT 70b is shown. Curve 82 corresponds to the size distribution of fragments generated using BLT 72. Curve 84 corresponds to the size distribution of fragments generated using zBLT 70b. As shown in graph 80, the size distribution of fragments generated from a target nucleic acid 18 using zBLT 70b (e.g., as described with respect to FIG. 1 ) is greater than the size distribution of fragments generated from a target nucleic acid 18 using BLT 72. Figure 1 The size distribution of the fragment 22) is relatively long.

[0118] At least in some cases, it may be desirable to control the fragments generated from the target nucleic acid 18 (e.g., as described with respect to Figure 1 It is currently recognized that by tuning the substrate 56 (e.g., Figure 8BThe number of active transposomes 30 and inactive transposomes 28 bound or linked to the zBLT 70b) and / or the ratio of the number of active transposomes 30 and inactive transposomes 28 can change the size distribution (e.g., the average size of the fragments can be increased or decreased). For example, a lower transposome load of active transposomes 30 can produce relatively larger fragments compared to an SLTC 68 with a relatively higher transposome load of active transposomes 30, and can further reduce the amount of sample input to be processed by the SLTC 68. The inactive transposomes 28 can spatially block a portion of the nucleic acid 18 (e.g., a dsDNA sample) from tag fragmentation and thereby increase the resulting fragment size. Therefore, tuning the relative ratio or number of active transposomes 30 and / or inactive transposomes 28 of the SLTC 68 composed of active transposomes 30 and inactive transposomes 28 can change the fragment size. Further, the inactive transposomes 28 can increase the DNA binding capacity (e.g., the number of positions at which the target nucleic acid 18 binds to the SLTC 68) of each SLTC 68. Thus, tuning the number of inactive transposomes 28 can provide an additional mechanism to prevent nucleic acids (e.g., DNA or RNA) from dissociating from the SLTC 68 due to fluid dynamic forces occurring during library preparation. It should be noted that controlling the DNA binding capacity can be advantageous for generating relatively large fragments using the SLTC 68, since otherwise the SLTCs may have few attachment points to the magnetic beads 56 if they are mediated solely by active transposomes.

[0119] In embodiments, active transposomes 30 make up less than half of the total transposomes (e.g., active transposomes 30 and inactive transposomes 28) of the mixture distributed on a particular surface. In embodiments, active transposomes 30 are less than 20%, less than 15%, less than 10%, or less than 5% of the mixture. In embodiments, active transposomes 30 represent between 0.1% and 20%, between 1% and 5%, between 5% and 10%, between 10% and 15%, between 15% and 20%, between 20% and 30%, or between 30% and 50% of the mixture.

[0120] Fig.10 The results of gel electrophoresis illustrating transposome formation are shown. More specifically, Fig.10An image 86 of blue native protein gel results is shown, which illustrates whether the use of the altered transfer strand oligonucleotides successfully forms a dimeric transposome. The gel results show four lanes 88, 90, 92, and 94. Lane 88 shows the results 96 (i.e., bands) of the BSA marker. Lane 90 shows the results 98, 100 (i.e., bands) of a transposome mixture consisting of two transposomes with different transposon DNA lengths. Lane 92 shows the results 102 (i.e., bands) of a bead-linked transposome with an inactive transposome 28 constructed with a 3' phosphorylated transfer strand 102. Lane 94 shows the results 104 (i.e., bands) of a transposome lacking a 3' phosphate block constructed with a transfer strand of the same size as in lane 92. Comparison of control transposomes, including a mixed preparation of active transposomes (a mixture of two active forms of transposomes with different transposon DNA lengths) and transposomes with short transposons (e.g., 19 bp), showed dimeric transposome complexes of the expected size, indicating that the 3' phosphate modification does not interfere with the formation of the dimeric transposome complex.

[0121] Fig.11 A first graph 106 illustrating the tag fragmentation activity of BLT 72 (eg, "active BLT") and the tag fragmentation activity of zBLT is shown. More specifically, Fig.11 A fluorescence resonance energy transfer (FRET) tag fragmentation activity assay is shown, illustrating undetectable tag fragmentation activity of a bead-attached transposome comprising an inactive transposome 28 (right) compared to a bead-attached transposome comprising an active transposome 30 (left).

[0122] Fig.12 108 corresponding to a tag fragmentation process of BLT 72 (eg, an active BLT) and a tag fragmentation process of BLT 70 (eg, a BLT containing an inactive transposome) are shown. Fig. 8A 70a) of zBLT corresponding to the tag fragmentation process of the second graph 109. More specifically, Fig.12 Bioanalyzer traces (e.g., curves 108 and 109) of PhiX DNA subjected to tag fragmentation conditions with BLT 72 (e.g., curve 108) containing an active transposome 30 or zBLT 70a (e.g., curve 109) containing an inactive transposome are shown. The high molecular weight fragments in the zBLT trace are consistent with unfragmented PhiX (approximately 5 kB).

[0123] Fig.13A graph 110 illustrating the amount of nucleic acid bound to BLT 70a and 72 is shown. More specifically, graph 110 illustrates the use of a control without BLT 72 (e.g., a "no bead control"), a control bead that does not include an attached transposome (e.g., a streptavidin bead) and / or a control bead that is not loaded with DNA (e.g., an "unloaded bead control"), a BLT 72 (e.g., an "active BLT", such as with respect to Fig.9A 72), and zBLT 70a (e.g., "Zombie BLT"). To test whether zBLT 70a has DNA binding activity, a DNA binding assay was performed. Control beads, BLT 72 (i.e., with bound DNA), and zBLT 70a were each incubated with lambda genomic DNA under standard tag fragmentation conditions. After tag fragmentation, the concentration of DNA remaining in the supernatant was measured via a fluorescent DNA binding dye (e.g., via a Qubit fluorometer). This measurement is used to calculate the amount of DNA bound by BLT 72 or zBLT 70a. The results in chart 110 show that zBLT 70a binds to a similar amount of DNA as BLT 72. The control beads showed little nonspecific binding, indicating that the inactive transposome 28 was responsible for this DNA binding activity.

[0124] Fig.14 A graph 112 illustrating the tag fragmentation activity of BLT 72 and the tagging activity of zBLT 70b for different mixtures or ratios of active transposome 30 and inactive transposome 28 is shown. Using inactive transposomes, zBLT 70b or SLTC having a mixture of active transposome 30 and inactive transposome 28 can be constructed using a stepwise procedure for binding transposomes to beads. For example, BLT or SLTC can be generated by providing active transposomes to substrate 56, optionally washing or removing excess unbound active transposomes, and then adding inactive transposomes. In at least some cases, inactive transposome 28 and active transposome 30 can be added to beads, as described with respect to Figure 5 described.

[0125] In with Fig.14In a corresponding example, active transposomes 30 are bound to beads (e.g., substrates 56 or magnetic beads) at a desired activity density (e.g., the amount of active transposomes 30 and inactive transposomes 28 that provide DNA binding ability). After removing excess active transposomes 30 that are not bound to the beads from the supernatant, a desired concentration of inactive transposomes 28 is added. Inactive transposomes 28 are bound to substrates 56 to produce BLTs 72 containing active transposomes 30 and inactive transposomes 28. It should be noted that the addition of active transposomes 30 and then inactive transposomes 28 during the construction process can promote non-competitive binding of active transposomes 30 to beads, which can provide the desired activity through BLTs 72. Table 1 shows an example of zBLTs generated according to the disclosed technology. In addition to zBLT 70b in Table 1, pure active BLTs (e.g., active SLTC) were prepared at 10 AU / μl, 15 AU / μl, 20 AU / μl, and 40 AU / μl as controls. FRET testing of selected BLTs showed that sequential transposome binding achieved equivalent zBLT activity compared to the pure active equivalent alone. FRET activity results for zBLT and pure active equivalent BLT alone.

[0126] Table 1 - Bead-attached transposomes generated using stepwise transposome binding .

[0127]

[0128] FIG. 15A to FIG. 15C (Right now, Fig.15A , Fig. 15B ,and Fig. 15C ) illustrates the fragment size distribution of zBLT 70b compared to a pure active BLT 72 control (e.g., BLT or SLTC that does not contain an inactive transposome 28). Electrophoretic analysis of the tag fragmentation products via a bioanalyzer showed that the addition of an inactive transposome caused the fragment size distribution to shift toward larger fragment sizes compared to the control BLT 72. For example, Fig.15A A first graph illustrating a curve 114 corresponding to the tag fragmentation activity of zBLT with 10 AU / μL of inactive transposome 28 and a curve 115 corresponding to the tag fragmentation activity of pure active BLT is shown. Fig. 15B A second graph illustrating curve 116 corresponding to the tag fragmentation activity of transposome complexes attached to beads with 15 AU / μL of inactive transposome 28 and curve 117 corresponding to the tag fragmentation activity of pure active BLT is shown. Fig. 15C A third graph illustrating curve 118 corresponding to the tag fragmentation activity of BLT with 20 AU / μL of inactive transposome 28 and curve 115 corresponding to the tag fragmentation activity of pure active BLT is shown. Fig.15A , Fig. 15B ,and Fig. 15C The graph of exemplifies that the addition of inactive transposome 28 to BLT 72 with active transposome 30 results in a shift in observed fragment sizes. Samples subjected to tag fragmentation by BLT 72 (e.g., corresponding to curves 115, 117, and 119) show a shift toward fragments having larger sizes for BLT 72 containing inactive transposome 28, compared to samples subjected to tag fragmentation by zBLT 70b (e.g., corresponding to curves 114, 116, and 118). Thus, FIG. 15A to FIG. 15C A technique for shifting fragment or insert size via the addition of an inactive transposome to a SLTC 68 with an active transposome 30 is shown.

[0129] To characterize the hybrid zBLT 70 (e.g., as described in Figure 8B As described above, zBLT 70b) was used to prepare libraries in a sequencing assay using 40 AU / μL of pure active BLT (e.g., as indicated in Table 1) and 40 AU / μL of zBLT from 10 ng of sample with 1% NA12877 in a NA12878 background for enrichment and sequencing. Fig.16 As shown, pure active BLT and zBLT provide similar library conversion efficiency and somatic variant calling sensitivity. These metrics were maintained, while an insert size shift of approximately 50 bp was seen. Fig.16 A first graph 120 illustrating conversion efficiency, a second graph 122 illustrating sensitivity, and a third graph 124 illustrating average insert lengths of BLT 72 (e.g., active BLT) and zBLT 70b are shown. More specifically, the graphs show an evaluation of the performance of zBLT 70 compared to control BLT 72 in a sequencing assay using 10 ng of NA12878 input in a NA12877 background.

[0130] As described herein, SLTC 68 can be used to separate mixtures of different types of nucleic acids. For example, a zSLTC or zBLT 70a that does not include a catalytically active transposome (e.g., an active transposome 30), such as described with respect to Fig. 8A The zBLT 70a described herein can bind to nucleic acids without tagging and fragmenting these nucleic acids. To illustrate this, Fig.17A flow chart of a method 130 for separating a mixture of different types of nucleic acids (e.g., DNA and RNA) using zBLT 70a is shown. At box 132, zBLT 70a is provided to a mixture of nucleic acids (e.g., a first nucleic acid 134 (e.g., DNA) and a second nucleic acid 136 (e.g., RNA)). In the illustrated embodiment, zBLT 70a comprises an inactive transposome 32. As discussed herein, the inactive transposome 28 may be able to bind nucleic acids, however, the inactive transposome is catalytically inactive. That is, the inactive transposome 28 does not perform tag fragmentation and cleavage on nucleic acids. In the exemplary method 130, zBLT 70 may selectively bind to the first nucleic acid 134, but not to the second nucleic acid 136. Therefore, at box 138, zBLT 70a may bind to the first nucleic acid 134 via an adaptor, while the second nucleic acid 136 remains unbound (e.g., in solution). At block 140, the second nucleic acid 136 can be removed from the solution containing the zBLT 70a bound to the first nucleic acid 134 via a washing and elution step. In this way, the zBLT 70a facilitates the separation of different types of nucleic acids present in the solution. At least in some cases, the use of zBLT 70a to separate the first nucleic acid from the second nucleic acid can improve the retention of the second nucleic acid that may be lost or otherwise damaged during certain separation techniques. In addition, it is currently recognized that separation using zBLT 70a can be relatively easier than techniques using reagents (such as trizol or chloroform) used in the extraction of DNA, RNA, or protein.

[0131] Despite the above Fig.17 The discussion of generally describes separation of DNA from RNA, but it should be noted that SLTC 68 (such as zBLT 70a) can be used to separate other components. For example, zBLT 70a can be used to separate DNA from RNA in the same manner as described above. Fig.17 The substantially similar manner described is used to separate double-stranded DNA from single-stranded RNA. Further, zBLT 70 can be used to separate double-stranded DNA from single-stranded DNA. In addition, zBLT 70a can be used to separate or extract double-stranded DNA from a mixture of other types of nucleic acids, proteins, and organic substances (lipids, carbohydrates, etc.).

[0132] In some embodiments, SLTC 68 facilitates normalizing the amount of nucleic acid in different samples. For example, zSLTC or zBLT 70a that does not contain a catalytically active transposome may bind to a certain amount of nucleic acid based on the nucleic acid binding capacity of zBLT 70a conferred by the amount of inactive transposome 28 bound to substrate 56 of zBLT 70a. To illustrate this, Fig.18A flow chart of a method 150 for normalizing the amount of nucleic acid of different samples is shown. At box 152, zBLT 70a is provided to a solution containing nucleic acid 154. In general, nucleic acid 154 may include nucleic acid or nucleic acid fragments. It should be noted that tuning the ratio of zBLT 70a or zSLTC to nucleic acid 154 can facilitate separation of uniform amounts of nucleic acid or nucleic acid fragments across multiple samples. For example, a ratio of SLTC 68 or zBLT 70a to nucleic acid 154 in which nucleic acid 154 is in excess can facilitate production of uniform amounts of nucleic acid fragments. When zBLT 70 are of the same type or all have predictable DNA binding capacity, based on generally identical or similar distributions of inactive transposomes 28 distributed on the surface relative to each other, the amount of DNA bound by each bead is predicted to be useful for normalizing the DNA between samples.

[0133] At block 156, zBLT 70a binds at least a portion of the nucleic acid or nucleic acid fragment to form a nucleic acid-surface-attached transposome complex. In the illustrated embodiment, zBLT 70a binds to a first portion 154a of nucleic acid 154, while a second portion 154b of nucleic acid 154 remains unbound in solution. Thus, first portion 154a can be separated from second portion 154b via magnetic beads (e.g., substrate 56) of zBLT 70a. Second portion 154b of nucleic acid 154 that is not coupled to substrate 56 of zBLT 70a can be washed away. After removing unbound nucleic acid, at block 158, first portion 154a can be retrieved. Thus, method 150 can be repeated for multiple samples of nucleic acid 154 to generate a normalized amount of nucleic acid for each sample. That is, method 150 can be applied to a first sample having a first amount of a first nucleic acid and a second sample having a second amount of a second nucleic acid. For each sample, the amount of nucleic acid can exceed the surface-bound transposome complex to capture a uniform amount of nucleic acid for each sample using the beads. Thus, after applying the techniques described in method 150, a first portion of a first amount of nucleic acid and a second portion of a second amount of nucleic acid can be recovered. By tuning the amount of zBLT 70a to the amount of nucleic acid, the first portion and the second portion can be substantially equal (e.g., within 1%, 5%, or 10% of each other as estimated by the DNA concentration). Additionally or alternatively, method 150 can be used to standardize the concentration of a sample to provide a desired or optimal (e.g., based on the detection limit of the device) load concentration for sequencing. For example, normalization can be used to standardize the concentration of a combined sample.

[0134] It should be noted that method 150 can be used to normalize DNA fragments. In addition, normalization with zBLT 70a can increase the speed of normalization compared to certain conventional techniques (e.g., manual normalization). For example, normalization with zBLT 70a can enable the omission of certain steps of manual normalization (e.g., quantifying individual samples, running size analysis of individual samples, or normalizing samples with different volumes). In this way, the disclosed technology can increase the speed of normalization of nucleic acid fragments.

[0135] In some embodiments, SLTC 68 can facilitate buffer exchange. For example, zBLT 70a can capture nucleic acids in a first solution, and the captured nucleic acids can be transferred and suspended in a second solution. To illustrate this, FIG. 19A to FIG. 19C (For example, Fig.19A , Fig.19B ,and Fig.19C ) generally illustrates techniques for buffer exchange.

[0136] Fig.19A A flow chart of a method 160 for buffer exchange using magnetic beads is shown. At box 162, a container 164 (e.g., a centrifuge tube) containing a nucleic acid sample 166 is provided. It is currently recognized that certain biochemical manipulation techniques (e.g., reactions involving enzymes 168 that can add or remove adapters 170 to nucleic acid samples 166) can react more efficiently under certain buffer conditions. Therefore, it may be advantageous to transfer nucleic acid samples 166 to solutions with different buffers. At box 172, magnetic beads (e.g., solid phase reversible immobilization (SPRI beads)) are added to the solution in container 164. The magnetic beads can be paramagnetic particles with carboxyl groups that reversibly bind to the nucleic acid sample in container 164. The magnetic beads can rely on charged interactions and crowding agents to drive the nucleic acids to bind non-specifically to the magnetic beads. For example, a salt-PEG solution or an 80% ethanol solution can be used to suspend the SPRI beads to maintain the interaction between the nucleic acids and the beads during purification. After the magnetic beads bind to the nucleic acids, multiple washing cycles can be implemented at box 174. After a sufficient number of wash cycles have been performed (eg, the steps at block 174 ), the nucleic acids may be eluted in a desired buffer at block 176 .

[0137] It is currently recognized that utilizing zBLT 70a with inactive transposomes 28 instead of magnetic beads as described with respect to method 160 to facilitate buffer exchange can be more efficient and less time consuming. For example, utilizing zBLT 70a can reduce the number of wash cycles. To generally illustrate this point, Fig.19B A flow chart of a method 180 for performing buffer exchange using zBLT 70a during biochemical manipulation of a nucleic acid sample 166 is shown.

[0138] At box 182, zBLT 70a bound to nucleic acid sample 166 can be provided with enzyme 168. In the illustrated embodiment, enzyme 168 is capable of adding adapter 170 (e.g., end sequence) to nucleic acid sample 166. It is currently recognized that zBLT 70a can bind to nucleic acid sample 166 so that when nucleic acid sample 166 is bound to zBLT 70a, enzyme 168 can be capable of performing biochemical manipulation (e.g., adding adapter 170). Therefore, this can reduce the amount of time by reducing the number of steps used to perform buffer exchange. At least in some cases, zBLT 70a can preferentially bind to certain nucleic acids rather than other nucleic acids. For example, zBLT 70a can have a relatively higher affinity for binding to DNA than to RNA. At least in some cases, salt can promote the formation of transposome-nucleic acid complexes. For example, the salt can contain divalent cations such as Mg. 2+ At block 184, the zBLT 70a containing the nucleic acid sample 166 may be centrifuged or subjected to a magnetic field (e.g., via magnetic separation) to form a precipitate. At block 186, the supernatant may be removed and the zBLT containing the nucleic acid sample 166 may be suspended in a desired buffer to continue the biochemical manipulation reaction. In at least some cases, the zBLT 70a bound to the nucleic acid sample 166 may be treated with a surfactant (e.g., an anionic detergent such as sodium dodecyl sulfate (SDS)) or treated with ethylenediaminetetraacetic acid (EDTA) to chelate Mg. 2+ Auxiliary factors promote the debinding of nucleic acids.

[0139] As another example of a technique for performing buffer exchange using zBLT 70a, Fig.19C A flow chart of a method 190 for performing a buffer exchange after a biochemical manipulation is shown. At block 192, a container 164 containing a nucleic acid sample 166 may be provided with an enzyme 168 and an adaptor 170 for performing a biochemical manipulation reaction. After adding the adaptor 170 to the nucleic acid sample 166, zBLT 70a may be added. Subsequently, the nucleic acid sample 166 containing the adaptor 170 and bound to the zBLT 70a may be precipitated via magnetic separation. At block 196, the supernatant may be removed and the zBLT containing the nucleic acid sample 166 containing the adaptor 170 may be suspended in a desired buffer.

[0140] As discussed herein, an inactive transposome refers to a catalytically inactive transposome that is incapable of joining (e.g., inserting via ligation) into a target nucleic acid, but the inactive transposome may still bind to the target nucleic acid. At least in some cases, an inactive transposome may be inactive due to inactivation of a transposase of the transposome, such as by modifying the amino acid sequence of the transposase. In some embodiments, a transposome may be inactive due to modification of an oligonucleotide that forms an adaptor of the transposome rendering the transposome inactive, while the transposase may still be active. To illustrate examples of inactive transpossomes, Fig. 20 A schematic diagram illustrating the binding mechanism between a transposome and a target nucleic acid is shown.

[0141] During transposition, Tn5 transposase promotes the nucleophilic attack of the 3' hydroxyl group of the mosaic end (ME) transfer strand (TS) on the phosphodiester backbone of the target DNA, resulting in the attachment of the transfer strand to the substrate DNA ( Figure 1 ). Certain methods for inactivating enzyme activity involve mutagenesis and protein engineering efforts. The technology according to the present disclosure includes blocking the 3' hydroxyl group of METs to block this chemical reactivity, while maintaining the ability to form a dimeric transposome and the ability of the resulting transposome to bind DNA. Examples of sequences of such inactive transposomes (i.e., inactive transposomes 28) or "Zombie transposomes" (specifically, as shown in the illustrated embodiments) formed by blocking the 3' hydroxyl group of the transferred chain with a phosphate group are shown in Table 2. It should be noted that alternative blocking groups can be used instead of phosphate groups, such as esters, sulfates, nitrates, carboxyl groups, or dideoxycytosine (ddC), and ddC can remove oxygen at the 3' position. In the table, / 3Phos / and / 5Phos / refer to 3' phosphate groups and 5' phosphate groups, respectively. / 3BiotinN / refers to 3' biotin used to connect the transposome to streptavidin-coated magnetic beads.

[0142] Table 2 - Oligonucleotide sequences used to prepare inactive transposomes .

[0143] Oligonucleotide name Oligonucleotide sequence (5'-3') Inactive TS AGATGTGTATAAGAGACAG / 3Phos / ME'-Biotin / 5Phos / CTGTCTCTTATACACATCT / 3BiotinN /

[0144] As about FIG. 15A to FIG. 15CAs described above, the concentration or density (e.g., AU / μL) of the tuning transposome (e.g., the inactive transposome 28) can selectively adjust the size of the SLTC 68 (e.g., the size of the nucleic acid fragment bound to the SLTC 68). In some embodiments, the density can be a predetermined amount or range, such as between 10 AU / μL and 100 AU / μL, between 10 AU / μL and 70 AU / μL, between 20 AU / μL and 60 AU / μL, between 30 AU / μL and 50 AU / μL, less than 100 AU / μL, less than 80 AU / μL, less than 70 AU / μL, less than 60 AU / μL, less than 50 AU / μL, less than 30 AU / μL, etc. Further, zSLTC 68 (e.g., BLT 70 that does not contain a catalytically active transposome) can be used to normalize nucleic acid fragments. Thus, tuning the density of inactive transposomes 28 of zSLTCs 68 used for normalization can tune the size and / or amount of the resulting normalized nucleic acid fragments. Fig.21A , Fig. 21B , Fig. 21C ,and Fig.21D It is generally illustrated that tuning the amount of inactive transposome 28 bound to zSLTC on substrate 56 can tune the fragment size selectivity of zBLT 70a. For example, Fig.21A A graph 200 showing a nucleic acid fragment size distribution of an example nucleic acid is shown. The nucleic acid fragment size distribution can be generated by a tag fragmentation reaction, as described herein. Fig. 21B A graph 210 illustrating the size distribution of nucleic acid fragments bound to beads 56 having a density of approximately 22 AU / uL is shown. Fig. 21C A graph 220 illustrating the size distribution of nucleic acid fragments bound to beads 56 having a density of approximately 44 AU / uL is shown. Fig.21D A graph 230 illustrating the size distribution of nucleic acid fragments bound to beads 56 having a density of approximately 66 AU / uL is shown. FIG. 21A to FIG. 21D In each of the zSLTCs 68, line 232 is shown at approximately 300 base pairs (bp) to illustrate the shift in the size of nucleic acid fragments bound to the zSLTCs 68. As shown, the average fragment size of nucleic acid fragments bound to the zSLTCs 68 increases as the density of inactive transposomes 28 bound to the beads 56 increases. Thus, the system may include zSLTCs 68 that are generated to preferentially bind nucleic acid fragments of a particular size or distribution by tuning the density of inactive transposomes 28 on the zSLTCs 68.

[0145] Fig.22A and Fig. 22BGraphs illustrating the results of gene expression analysis demonstrate that normalization with zSLTC 68 shows little difference in gene expression for nucleic acids compared to conventional normalization techniques (e.g., manual normalization). In particular, Fig.22A The results of gene expression analysis (e.g., R 2 =0.93) in Figure 240. Fig. 22B The gene expression analysis results (eg, R 2 =0.96). In general, both graphs 240 and 250 show fold changes between -2 and 2, indicating that normalization with zSLTC 68 produces generally similar gene expression results. However, normalization with zSLTC 68 may be significantly faster than certain conventional normalization techniques. For example, normalization with zSLTC 68 may take up to 80% less time than manual normalization.

[0146] Fig.23A and Fig. 23B Another example of a graph illustrating gene expression analysis results. Fig.23A The results of gene expression analysis (e.g., R 2 =0.92) in Figure 260. Fig. 23B The gene expression analysis results (eg, R 2 =0.96) of the graph 270. Fig.22A and Fig. 22B In a substantially similar manner as described in , the fold change indicates that normalization using zSLTC 68 produced substantially similar gene expression results compared to manual normalization. Thus, the disclosed normalization techniques can increase the speed of normalizing nucleic acid fragments with little or no reduction in the quality of the results.

[0147] Thus, the disclosed techniques can enable tuning of the size selectivity of zSLTC 68 and improve the effectiveness of normalization techniques. This is further illustrated in Fig.24A and Fig. 24B More specifically, Fig.24A Graph 280 shows the distribution of nucleic acids obtained using the normalization technique without zSLTC 68 (eg, average size = 346 bp). Fig. 24B Graph 290 shows the distribution of nucleic acids (eg, average size = 554 bp) obtained using the normalization technique with zSLTC 68. In general, Fig. 24BThis indicates that large nucleic acid fragments can be obtained using zBLT. In some embodiments, the distribution of nucleic acids can more closely match the distribution of nucleic acids from the source. Therefore, normalization using zSLTC 68 can enable the user to capture more nucleic acids during normalization, thereby providing a normalization technique with reduced waste (e.g., less nucleic acid is wasted) and tunable size selectivity.

[0148] Fig.25A A graph showing the percentage of reads mapped to each indexed sample (eg, "1," "2," "3," etc. represent different samples) versus the corresponding percentage for each sample obtained using manual normalization. Fig.25B The read segments mapped to each sample are compared with z BLT 70a shows a graph of the percentages of the corresponding samples obtained. In general, Fig.25A and Fig.25B Prove that using z BLT Normalization with 70a yielded nucleic acid fragment distributions comparable to manual normalization.

[0149] Thus, additional aspects of the present disclosure relate to SLTC or BLT comprising active transposomes 30 and / or inactive transposomes 28. As described above, BLTs comprising active transposomes can produce fragments of target nucleic acids via a tag fragmentation reaction occurring between active transposomes and target nucleic acids. As discussed herein, chemical inactivation of transposomes (e.g., by adding 3' phosphate to the transfer strand) may not disrupt the binding of ME. Further, although the position of this modification is within the active site of the complex, inactivation may not inhibit dimer transposome formation or prevent binding of target DNA. In addition, by changing the number of active transposomes 30 and inactive transposomes 28 bound or connected to the substrate 56 of SLTC 68 (e.g., BLT72) and / or the ratio of the number of active transposomes 30 and inactive transposomes 28, the size of the fragments of the target nucleic acid can be tuned. Certain techniques can utilize size selection SPRI to narrow the fragment size distribution to the desired range for sequencing, and such techniques can reduce library conversion efficiency by discarding undesirable fragment sizes. The disclosed technology provides control over insert size during the tag fragmentation step, thereby reducing additional steps downstream, such as size selection. Still further, SLTC 68, such as zBLT 70a, can be used for applications including nucleic acid separation, normalizing nucleic acid amounts between different samples, and performing buffer transfers.

[0150] The technology presented and claimed herein is cited and applied to specific examples of material objects and practical nature that demonstrably improve the art and are therefore not abstract, intangible, or purely theoretical. Further, if any claim appended to the end of this specification contains one or more elements designated as "means for (performing) (function) ..." or "steps for (performing) (function) ...", such elements are intended to be interpreted in accordance with 35 U.S.C. 112(f). However, for any claim containing elements designated in any other manner, these elements are not intended to be interpreted in accordance with 35 U.S.C. 112(f).

[0151] This written description uses examples to enable any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems and performing any incorporated methods. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

1. A transposome complex, comprising: a plurality of inactive transposomes, wherein the plurality of inactive transposomes are coupled to each other; a first active transposome coupled to first ends of the plurality of inactive transposomes; a second active transposome, wherein the second active transposome is coupled to the second ends of the plurality of inactive transposomes, such that the plurality of inactive transposomes are located between the first active transposome and the second active transposome; and The first active transposome, the second active transposome, and each of the plurality of inactive transposomes comprise a transposase and an adaptor. 2 . The transposome complex of claim 1 , wherein the first active transposome and the second active transposome are on opposite ends of the transposome complex. 3 . The transposome complex of claim 1 , wherein the first active transposome and the second active transposome further comprise an insertion sequence. 4 . The transposome complex of claim 1 , wherein the first active transposome, the second active transposome, and each inactive transposome of the plurality of inactive transposomes are dimers. 5 . The transposome complex of claim 1 , wherein each inactive transposome of the plurality of inactive transposomes is configured to bind to a target nucleic acid and is inactive such that the adaptor is not inserted into the target nucleic acid when bound. The transposome complex according to claim 5 , wherein each inactive transposome is inactive based on the modification of the adaptor.

7. The transposome complex of claim 6, wherein the modification is a blocked 3' end of the adaptor to remove catalytic activity.

8. The transposome complex according to claim 6, wherein the modification is dephosphorylation of the 5' end of the adaptor to remove catalytic activity. 9 . The transposome complex according to claim 1 , wherein the first active transposome and the second active transposome form a catalytically active end of the transposome complex.

10. The transposome complex of claim 1, wherein the plurality of inactive transposomes are coupled together via complementary adaptor sequences.

11. The transposome complex of claim 1, wherein the first active transposome comprises a first active transposase and a first adaptor that is different from the adaptors of each inactive transposome. 12 . The transposome complex of claim 11 , wherein the second active transposome comprises a second active transposase and a second adaptor different from the adaptors of each inactive transposome.

13. The transposome complex of claim 1, wherein each inactive transposome is inactive based on modification of the amino acid sequence of the transposase of each inactive transposome.

14. The transposome complex of claim 1, wherein the plurality of inactive transposomes, the first active transposome, and the second active transposome are homodimers.

15. The transposome complex of claim 1, wherein the adaptor is at least partially double-stranded, and wherein each inactive transposome in the plurality of inactive transposomes comprises a second adaptor, wherein the adaptor and the second adaptor are identical.

16. The transposome complex of claim 15, wherein the adapter comprises a first adapter sequence that is at least partially double-stranded, and wherein the second adapter sequence coupled to the second transposase of the plurality of inactive transpososomes comprises a second adapter sequence that is at least partially double-stranded, wherein the first adapter sequence and the second adapter sequence on individual inactive transposomes of the plurality of inactive transposomes are identical.

17. The transposome complex of claim 1, wherein the first adaptor of the first active transposome and the second oligonucleotide adaptor of the second active transposome each comprise a double-stranded transposon end sequence and an at least partially double-stranded adaptor sequence.

18. The transposome complex of claim 1, wherein each inactive transposome is coupled to an adjacent transposome of the transposome complex via cross-linking.

19. The transposome complex of claim 1, wherein at least one inactive transposome comprises a stabilizer configured to reduce monomer exchange between at least one inactive transposome of the plurality of inactive transposomes and the first active transposome, the second active transposome, or both.

20. A kit comprising a plurality of transposome complexes according to claim 1, wherein each transposome complex in the plurality of transposome complexes has the same number of inactive transposomes between the first active transposome and the second active transposome.

21. A method for preparing a transposome complex, the method comprising: providing a starting transposome comprising a first oligonucleotide adaptor and a second oligonucleotide adaptor; as well as hybridizing at least one ligated transposome to the starting transposome via a ligated adaptor of the at least one ligated transposome, wherein the at least one ligated transposome is catalytically inactive, and wherein the ligated adaptor is catalytically inactive with the first oligonucleotide adaptor, the second oligonucleotide adaptor or both are complementary; and At least one terminal transposome is coupled to the at least one ligation transposome via a terminal adaptor of the terminal transposome that is complementary to the ligation adaptor or a different ligation adaptor of the at least one ligation transposome, wherein the terminal transposome is catalytically active.

22. The method of claim 21, wherein the starting transposome is an active transposome, such that the starting transposome is capable of ligating to a target nucleic acid.

23. The method of claim 21, wherein the starting transposome is an inactive transposome such that the starting transposome is prevented from binding to a target nucleic acid.

24. The method of claim 21, comprising attaching the starting transposome to a substrate surface.

25. The method of claim 21, comprising hybridizing at least two ligated transposomes to opposite sides of the starting transposome via corresponding ligated adaptors of the at least two ligated transposomes, wherein the corresponding ligated adaptors are complementary to the first oligonucleotide adaptor and the second oligonucleotide adaptor.

26. The method of claim 21, wherein the first oligonucleotide adaptor and the second oligonucleotide adaptor comprise different nucleic acid sequences.

27. The method of claim 21, comprising, after hybridizing the at least one ligated transposome to the starting transposome via the ligated adaptor of the at least one ligated transposome, washing the substrate containing the hybridized ligated transposome and the starting transposome.

28. The method of claim 21, wherein the at least one ligating transposome comprises a plurality of ligating transposomes, and wherein the coupling of the terminus comprises coupling to a ligating transposome different from the ligating transposome that hybridized to the starting transposome.

29. A method for preparing a nucleic acid library, the method comprising: contacting the target nucleic acid with a plurality of transposome complexes, wherein each transposome complex in the plurality of transposome complexes comprises a first active transposome coupled to a second active transposome via an intervening plurality of inactive transposomes, to allow the plurality of transposome complexes to bind to the target nucleic acid; as well as The target nucleic acid is subjected to tag fragmentation to generate nucleic acid fragments, wherein the size of the generated nucleic acid fragments is a function of the size of individual transposome complexes in the plurality of transposome complexes.

30. The method of claim 29, further comprising digesting regions of the target nucleic acid that are not bound by the plurality of transposome complexes.

31. The method of claim 29, further comprising removing the plurality of transposome complexes after generating the nucleic acid fragments.

32. The method of claim 29, further comprising sequencing the generated nucleic acid fragments.

33. The method of claim 29, wherein the plurality of transposome complexes all have approximately the same number of intervening inactive transposomes between the first active transposome and the second active transposome such that the generated nucleic acid fragments are within a range of sizes.

34. The method of claim 29, wherein each transposome complex in the plurality of transposome complexes is bound to a corresponding substrate.

35. A surface-bound transposome complex, the surface-bound transposome complex comprising: solid surfaces; and A plurality of transposomes are coupled to the solid surface, and wherein at least one transposome in the plurality of transposomes is inactive based on modification of an oligonucleotide adaptor of the at least one transposome.

36. The transposome complex of claim 35, wherein the oligonucleotide adaptor comprises a blocked 3' end to remove catalytic activity.

37. The transposome complex of claim 36, wherein the 3' end is blocked via a phosphate group, dideoxycytosine, ester, sulfate, carboxyl group, or any combination thereof.

38. The transposome complex of claim 35, wherein each transposome of the plurality of transposomes is configured to bind to a target nucleic acid and is inactive such that the oligonucleotide adaptor is not inserted into the target nucleic acid when bound.

39. The transposome complex of claim 35, wherein the solid surface is a magnetic bead.

40. The transposome complex of claim 35, wherein the solid surface is a flat substrate.

41. The transposome complex of claim 35, wherein each transposome in the plurality of transposomes is coupled to the solid surface via a linker.

42. The transposome complex of claim 35, wherein the plurality of transposomes are at regular distances from each other on the solid surface.

43. The transposome complex of claim 35, comprising a nucleic acid associated with at least a portion of the plurality of transposomes.

44. The transposome complex of claim 43, wherein the nucleic acid is a double-stranded nucleic acid.

45. The transposome complex of claim 35, wherein the solid surface is not coupled to any active transposomes.

46. ​​A method for isolating nucleic acid, the method comprising: contacting a plurality of inactive transposome complexes with a mixed nucleic acid sample in a solution, the mixed nucleic acid sample comprising double-stranded DNA and RNA, such that the double-stranded DNA selectively binds to the plurality of inactive transposome complexes relative to the RNA, wherein each inactive transposome complex in the plurality of inactive transposome complexes comprises a plurality of inactive transposomes coupled to a surface to allow the plurality of inactive transposome complexes to bind to the double-stranded DNA; as well as The double-stranded DNA is separated from the RNA by removing the plurality of inactive transposome complexes with bound double-stranded DNA from the solution, the solution comprising the RNA.

47. The method of claim 46, comprising increasing the Mg content of the solution. 2+ to promote the binding of the double-stranded DNA to the plurality of inactive transposome complexes.

48. The method of claim 46, comprising separating the bound double-stranded DNA from the plurality of inactive transposome complexes by eluting the double-stranded DNA into a second solution.

49. The method of claim 46, wherein the surface comprises magnetic beads, and wherein the separating comprises magnetic separation.

50. A method for normalizing the amount of nucleic acid in a plurality of samples, the method comprising: contacting a first plurality of double-stranded nucleic acids of a first sample with a first plurality of inactive transposome complexes, wherein each inactive transposome complex of the first plurality of transposome complexes comprises a predetermined amount of inactive transposomes coupled to a bead surface, and wherein the contacting is performed under conditions such that a portion of the first plurality of double-stranded nucleic acids binds to the first plurality of inactive transposome complexes; contacting a second plurality of double-stranded nucleic acids of a second sample with a second plurality of inactive transposome complexes, wherein each inactive transposome complex of the second plurality of inactive transposome complexes comprises a predetermined amount of inactive transposomes coupled to a bead surface, and wherein the contacting is performed under conditions such that a portion of the second plurality of double-stranded nucleic acids binds to the second plurality of inactive transposome complexes; as well as The bound portions of the first plurality of double-stranded nucleic acids and the bound portions of the second plurality of double-stranded nucleic acids are sequenced.

51. The method of claim 50, comprising separating the bound portion of the first plurality of double-stranded nucleic acids from unbound nucleic acids in the first sample prior to sequencing.

52. The method of claim 51, comprising separating the bound portion of the second plurality of double-stranded nucleic acids from unbound nucleic acids in the second sample prior to sequencing.

53. The method of claim 51, wherein the binding portion of the first plurality of double-stranded nucleic acids and the binding portion of the second plurality of double-stranded nucleic acids are approximately the same amount of nucleic acids relative to each other.

54. The method of claim 51, wherein the predetermined range of inactive transposomes coupled to the bead surface is between about 10 AU / μL and about 70 AU / μL.

55. The method of claim 51, wherein the predetermined range of inactive transposomes coupled to the bead surface is between about 20 AU / μL and about 60 AU / μL.

56. A method of performing a buffer exchange, the method comprising: contacting a plurality of nucleic acids suspended in a first buffer solution with a plurality of inactive transposome complexes, wherein each inactive transposome complex in the plurality of inactive transposome complexes comprises a plurality of inactive transposomes coupled to a surface; generating a precipitate comprising the plurality of nucleic acids bound to the plurality of inactive transposome complexes; separating the precipitate from the first buffer solution; as well as The precipitate is suspended in a second buffer solution.

57. The method of claim 56, comprising washing the precipitate.

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