Oligonucleotide analogues for the preparation of nucleic acids

Abstract

Nucleic acid technology is disclosed. Several embodiments include a modified nucleotide 12 having an oligonucleotide adaptor 24 attached via a cleavable linker 20. Incorporation of the modified nucleotide 12 at the 3' terminus allows for end adaptation of the free 5' end of the oligonucleotide adaptor 24 via nucleic acid linkage to the 3' reactive group of the modified nucleotide 12, and cleavage by the cleavable linker 20 to release the free 3' end.

Description

[Technical field]

[0001] The techniques of this disclosure generally relate to techniques for preparing nucleic acids, such as sequencing library preparation, using oligo-modified nucleotide analogs. Oligo-modified nucleotide analogs, including oligonucleotide adaptors, can be used to directly incorporate adaptors into nucleic acids as part of sample preparation for downstream processing steps.

[0002] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, it should not be assumed that the problems mentioned in this section, or associated with the subject matter provided as background, have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which as such may also correspond to implementations of the claimed technology. [Background technology]

[0003] Molecular biology is currently making intensive use of nucleic acid analysis. Various nucleic acid analysis techniques involve sample preparation steps that manipulate the sample to generate an end product that is compatible with the desired analysis platform. For example, certain sequencing platforms are matched with sequencing libraries that contain specific adapter sequences that allow for strand capture and synthesis. These adapter sequences may include universal adapters for high throughput parallel processing of large numbers of nucleic acids.

[0004] Addition of a universal adaptor for sequencing can be accomplished by a variety of methods. In one example, an adaptor containing a universal priming sequence can be ligated to the end of a template nucleic acid. A single adaptor or two different adaptors may be used in the ligation reaction. If the template nucleic acid is engineered such that its ends are the same (i.e., both are blunt or both have the same overhang), then ligation of a single compatible adaptor will generate a template with that adaptor at both ends. However, if two differently compatible adaptors (e.g., adaptor A and adaptor B) are used, three sequences of ligation products are formed: a template with adaptor A at both ends, a template with adaptor B at both ends, and a template with adaptor A at one end and adaptor B at the other end. This final product may, under some circumstances, be the only desired product from the ligation reaction, and therefore an additional purification step is required after the ligation reaction to purify it from the undesired ligation products that have the same adaptors at both ends. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, certain techniques for adding universal adaptors to samples involve inherent losses of sample as well as additional purification steps when generating and subsequently selecting suitably modified fragments. Thus, more efficient techniques for adding adaptors to nucleic acids are of interest. [Means for solving the problem]

[0006] In one embodiment, the present disclosure provides an oligo-modified nucleic acid analogue composition. The composition comprises a modified nucleotide comprising a ribose (e.g., deoxyribose, ribonucleic acid, dideoxyribose), a 5' phosphate attached to the deoxyribose, a 3' reactive group attached to the ribose, and an oligonucleotide adaptor attached to the ribose by a linker, e.g., a cleavable linker, and terminating at a reactive 5' oligonucleotide end. In one embodiment, the oligonucleotide can be attached to the linker at the terminal 3' end or the reactive 3' end, depending on the desired subsequent reaction after reaction with the modified nucleotide.

[0007] In one embodiment, the disclosure provides a method of modifying a nucleic acid. The method includes providing a nucleic acid and contacting the nucleic acid with a modified nucleotide. The modified nucleotide includes a deoxyribose, a 5' phosphate group attached to the deoxyribose, a 3' reactive group attached to the deoxyribose, and an oligonucleotide adaptor attached to the deoxyribose by a cleavable linker and terminating at a 5' oligonucleotide end. The method includes incorporating the modified nucleotide at the 3' end of the nucleic acid to generate an extended nucleic acid, and reacting the 5' oligonucleotide end of the oligonucleotide adaptor on the extended nucleic acid with the 3' reactive group to attach the 5' oligonucleotide end to the deoxyribose such that the oligonucleotide adaptor forms a loop. In one embodiment, the method includes cleaving the linker to release the 3' end of the oligonucleotide adaptor.

[0008] In one embodiment, the disclosure provides a method of modifying a nucleic acid. The method includes contacting a single-stranded nucleic acid with a modified nucleotide, the modified nucleotide including a deoxyribose, a 5' phosphate group attached to the deoxyribose, and a single-stranded oligonucleotide adaptor attached to the deoxyribose and terminating at a 5' oligonucleotide end. The method also includes incorporating the modified nucleotide at the 3' end of the single-stranded nucleic acid using a polymerase to generate an extended single-stranded nucleic acid, annealing a primer including a recognition site for the 5' region of the single-stranded oligonucleotide adaptor, and extending the primer to synthesize a complementary strand of the single-stranded nucleic acid.

[0009] In one embodiment, the disclosure provides a method for preparing a sequencing library. The method includes providing a double-stranded nucleic acid sample, and fragmenting the double-stranded nucleic acid sample using a transposome homodimer to incorporate a first adaptor at the 5' end of the double-stranded fragments generated from the double-stranded nucleic acid sample. The method also includes contacting the double-stranded fragments with a modified nucleic acid, the modified nucleic acid including a deoxyribose, a 3' reactive group attached to the deoxyribose, and an oligonucleotide adaptor attached to the deoxyribose by a cleavable linker and terminating at a 5' oligonucleotide end. The method also includes incorporating a modified nucleotide at the 3' end of the double-stranded fragment to generate an extended nucleic acid, reacting the 5' oligonucleotide end of each of the modified nucleotides with a corresponding 3' reactive group to bind the 5' oligonucleotide end to the deoxyribose such that the oligonucleotide adaptor of the modified nucleotide forms a loop, and cleaving the cleavable linker of the modified nucleotide to release the 3' end of the oligonucleotide adaptor to generate the adapted double-stranded nucleic acid fragments of the sequencing library.

[0010] In one embodiment, the disclosure provides a method for preparing a sequencing library. The method includes providing a double-stranded nucleic acid sample. The method also includes contacting the double-stranded fragments with modified adenosines, each modified adenosine includes a deoxyribose, a 5' phosphate group attached to the deoxyribose, a 3' reactive group attached to the deoxyribose, an adenine nucleobase attached to the deoxyribose, and an oligonucleotide adaptor attached to the deoxyribose or the adenine nucleobase by a linker, the oligonucleotide adaptor includes a fork adaptor including a first fork, a second fork, and a double-stranded portion, the double-stranded portion includes a 3' thymine overhang, and the linker is attached to the first fork. The method also includes incorporating a modified adenosine via the 5' phosphate group at the 3' end of the double-stranded nucleic acid to generate an extended nucleic acid comprising a 3' modified adenosine end; ligating the double-stranded portion of a fork adaptor to the 3' modified adenosine end of the double-stranded nucleic acid via the 3' thymine overhang; and cleaving the linker from the first fork after ligation to generate a double-stranded nucleic acid having fork adaptors at both ends.

[0011] In one embodiment, the disclosure provides a method for preparing a sequencing library. The method includes providing a double-stranded nucleic acid sample. The method also includes contacting the double-stranded fragments with modified, double-stranded nucleic acid with modified adenosines, each modified adenosine comprising a deoxyribose, a 5' phosphate group attached to the deoxyribose, a 3' reactive group attached to the deoxyribose, an adenine nucleobase attached to the deoxyribose, and a first oligonucleotide adaptor attached to the deoxyribose or the adenine nucleobase by a first linker. The method also includes incorporating a modified adenosine via the 5' phosphate group at a 3' end of the double stranded nucleic acid to generate an extended nucleic acid comprising the 3' modified adenosine end; contacting the extended nucleic acid comprising the 3' modified adenosine end with a fork adaptor comprising a first fork, a second fork, a double stranded portion comprising a 3' thymine overhang, and a second oligonucleotide adaptor complementary to the first oligonucleotide adaptor, thereby allowing the second oligonucleotide adaptor to extend from the first fork or the second fork via a second linker and hybridize the first oligonucleotide adaptor to the second oligonucleotide adaptor; ligating the double stranded portion of the fork adaptor to the 3' modified adenosine end of the double stranded nucleic acid via the 3' thymine overhang; and cleaving the first linker and the second linker to generate a double stranded nucleic acid having a fork adaptor at both ends.

[0012] In one embodiment, the disclosure provides a nucleic acid fragment comprising a single-stranded or double-stranded nucleic acid fragment and a modified nucleotide attached to a 3' end of the nucleic acid fragment, wherein the modified nucleic acid comprises an oligonucleotide adaptor attached to a ribose by a linker at a first end and terminating at a 5' oligonucleotide end or a 3' oligonucleotide end at a second end.

[0013] In one embodiment, the disclosure provides a method of modifying a nucleic acid. The method includes contacting a double-stranded nucleic acid with a modified nucleotide, the modified nucleotide comprising a deoxyribose, a 5' phosphate group attached to the deoxyribose, and a single-stranded oligonucleotide adaptor attached to the deoxyribose via a linker at a first end and terminating at a 3' oligonucleotide end at a second end. The method also includes incorporating the modified nucleotide at the 3' end of the first strand of the double-stranded nucleic acid via the 5' phosphate group to generate an extended first strand, annealing a primer comprising a recognition site for the 3' region of the single-stranded oligonucleotide adaptor, and extending the primer using a polymerase having 5' to 3' exonuclease activity to synthesize a complementary strand of the single-stranded nucleic acid while degrading the 5' portion of the second strand of the double-stranded nucleic acid.

[0014] In one embodiment, the disclosure provides a method of modifying a nucleic acid. The method includes contacting a double-stranded nucleic acid with a modified nucleotide, the modified nucleotide comprising a deoxyribose, a 5' phosphate group attached to the deoxyribose, and a single-stranded oligonucleotide adaptor attached to the deoxyribose. The method also includes incorporating the modified nucleotide into a recessed 3' end of a first strand of the double-stranded nucleic acid via the 5' phosphate group to generate an extended first strand, extending the first strand from the 3' end of the modified nucleotide, annealing the single-stranded portion of the fork adaptor to the single-stranded oligonucleotide adaptor, and ligating the double-stranded portion of the fork adaptor to an end of the double-stranded nucleic acid.

[0015] In one embodiment, the disclosure provides a method for modifying a nucleic acid. The method includes contacting a single-stranded RNA with a plurality of single-stranded oligonucleotides comprising a 3' random portion and a 5' fixed sequence portion, such that a 3' random portion of one of the plurality of single-stranded oligonucleotides anneals to the 3' end of the single-stranded RNA and a 5' fixed sequence portion does not anneal to the single-stranded RNA. The method also includes incorporating a modified nucleotide onto the 3' end of the single-stranded RNA using the fixed sequence portion as a template, the modified nucleotide comprising a deoxyribose, a 5' phosphate group attached to the deoxyribose, and a single-stranded oligonucleotide adaptor attached to the deoxyribose and terminating at a free 3' end.

[0016] The foregoing description is presented to enable making and using the disclosed technology. Various modifications to the disclosed embodiments will be apparent, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosed technology. Thus, the disclosed technology is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. The scope of the disclosed technology is defined by the appended claims. These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read in conjunction with the accompanying drawings, in which like features represent like parts throughout the drawings. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of modified nucleotides, e.g., oligo-modified nucleotide analogs, according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram of a process for incorporating modified nucleotides at the 3′ end of a double-stranded nucleic acid according to an embodiment of the present disclosure. [Diagram 3]FIG. 1 is a schematic diagram of a process for incorporating modified nucleotides containing hybridized adaptors into the 3′ end of a double-stranded nucleic acid according to an embodiment of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram of a process for incorporating modified nucleotides, including a fork adaptor, at the 3′ end of a double-stranded nucleic acid according to an embodiment of the present disclosure. [Diagram 5] FIG. 1 is a schematic diagram of a process for incorporating modified nucleotides at the 3′ end of a double-stranded nucleic acid to enhance ligation of a fork adaptor according to an embodiment of the present disclosure. [Figure 6A] 1 illustrates a typical prior art fragmentation workflow. [Figure 6B] 1 illustrates a typical prior art fragmentation workflow. [Figure 6C] 1 illustrates an exemplary fragmentation workflow using modified nucleotides according to an embodiment of the present disclosure. [Figure 7] 1 shows adapter primer extension over a modified nucleotide according to an embodiment of the present disclosure. [Figure 8] 1 shows Lure-based adaptor ligation using modified nucleotides according to an embodiment of the present disclosure. [Figure 9] An embodiment is shown in which modified nucleotide 12 is incorporated at the 3′ end of the RNA molecule 220. [Figure 10] FIG. 1 is a schematic diagram of primer extension as part of cDNA synthesis using modified nucleotides according to an embodiment of the present disclosure. [Figure 11] FIG. 1 is a schematic diagram of a process for priming from an oligonucleotide adaptor of a modified nucleotide incorporated at the 3′ end of a nucleic acid according to an embodiment of the present disclosure. [Figure 12] FIG. 1 is a schematic diagram of the process of chain extension from an oligonucleotide adaptor of a modified nucleotide incorporated at the 3′ end of a nucleic acid, according to an embodiment of the present disclosure. [Figure 13] FIG. 1 is a schematic diagram of the process of chain extension from a loop of an oligonucleotide adaptor, according to an embodiment of the present disclosure. [Figure 14]FIG. 13. Diagram of the incorporation of modified nucleotides into blunt and recessed duplex DNA and the size of the resulting products. [Figure 15] 1 shows the results of incorporation of modified nucleotides into blunt and recessed duplex DNA. [Figure 16] FIG. 1 is a diagram of modified nucleotide primed extension into blunt and recessed duplex DNA. [Figure 17] 1 shows the results of modified nucleotide primed extension into blunt and recessed duplex DNA. [Figure 18] FIG. 1 is a diagram of adaptor nucleic acid ligation to blunt double-stranded DNA containing modified nucleotides. [Figure 19] 4 shows the results of Lure-based adaptor ligation. [Figure 20] 4 shows the results of Lure-based adaptor ligation. [Figure 21] Exonuclease inhibition based on scar (Sequence Characterized Amplified Region) size of modified nucleotides is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The following discussion is presented to enable those skilled in the art to make and use the disclosed technology and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosed technology. Thus, the disclosed technology is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0019] The disclosed technology relates to oligo-modified nucleotide analogs (e.g., modified nucleotides, as provided herein) and techniques for using the same. Oligo-modified nucleotide analogs exploit the ability of polymerases to catalyze the incorporation of nucleotides attached to oligonucleotide adaptors (or other functional sequences). Thus, in some embodiments, a desired sequence can be added to the 3' end of a nucleic acid via a polymerase-mediated reaction rather than a nucleic acid ligation reaction, but the use of a polymerase allows for a higher yield of the desired end product compared to nucleic acid ligation. In one embodiment, the disclosed oligo-modified nucleotide analogs include adaptor sequences that are used to incorporate adaptors during sequencing library preparation. Compared to ligase-mediated adaptation of nucleic acid samples, direct incorporation of modified nucleotides conjugated to sequencing adaptors increases the efficiency of library preparation and simplifies the user's workflow. The implementation also facilitates, for example, asymmetric adaptation of libraries with different 5' and 3' adaptors, allowing for strand library generation and paired end sequencing.

[0020] Converting a nucleic acid sample into a sequencing-ready library involves a series of enzymatic manipulations to add oligonucleotide adaptors containing flow cell complementary sequences, primer binding sites, and indices. Depending on the particular sample preparation workflow, traditional adaptations can be inefficient. Thus, new and more efficient sample preparations that limit sample loss would improve sequencing results, especially for samples with limited amounts. Thus, the problem of complex and low-yield sample preparations involving multiple DNA manipulations (end repair, A-tailing, and inefficient ligation), each with different inefficiencies, is addressed by the direct incorporation of oligo-modified nucleotide analogs provided herein. The modified nucleotides disclosed herein can perform highly efficient intramolecular proximity ligation (either by reactive chemistry or other methods) that is more efficient than intermolecular classical ligation of adaptors to sample DNA. Proximity is mediated by the oligonucleotide adaptors of the modified nucleotides, which can mobilize or hold ligation reaction components in close proximity to each other to improve reaction efficiency. In some workflows provided herein, certain biochemical steps in traditional workflows, such as ligation, can be completely eliminated. In one embodiment, the problem of sample loss and loss of stranded information due to incorrect matching of adapters is solved by sequential addition of 1) a first adapter via fragmentation followed by 2) a second adapter via polymerase-mediated adapter addition to obtain fragments with asymmetric adapters.

[0021] The disclosed technique provided increased yield during sample preparation compared to ligation-based processes. In certain embodiments, direct incorporation of oligo-modified nucleotide analogs using polymerase reduces the number of workflow steps or sample manipulations, limiting sample loss and increasing ease of use. Additional advantages include the ability to add asymmetric adaptors sequentially (without sample loss), allowing for PCR- or PCR-free sample preparation, and retaining stranded information. The disclosed method for 3'-adaptation of nucleic acids offers the advantage of more granular control of individual incorporation of 3'-adaptors by different techniques for adding 5'-adaptors, as opposed to other methods in which both 3'-adaptors and 5'-adaptors are added in the same way (i.e., dsDNA ligation).

[0022] FIG. 1 is a representative oligo-modified nucleotide analog, also referred to herein as modified nucleotide 12. In one embodiment, modified nucleotide 12 may be referred to as an oligo-modified nucleotide analog (oNTP). Modified nucleotide 12 includes a pentose sugar, e.g., deoxyribose 14, or modified deoxyribose, and a 5' phosphate group 15 (e.g., triphosphate). Extending from the 1' carbon position of deoxyribose 14 is linker 20, which in some embodiments may be a cleavable linker that can be cleaved from deoxyribose 14. The linker may include a nucleotide base group that aids in base pairing during incorporation of the modified nucleotide. The base group may have a cleavable group between it and the oligo group. Linker 20 may include a carbon or carbon chain including one or more intervening carbons, nitrogens, oxygens, or combinations thereof, located before the cleavage position indicated by X. Linker 20 may include a benzyl functional group or a PEG spacer. Linker 20 may be chemically cleaved using tris(2-carboxyethyl)phosphine (TCEP) cleavage of the double bond or a tetrahydropyranyl (THP) cleavable moiety.

[0023] The linker 20 may be photocleavable or enzymatically or chemically cleavable. In one embodiment, the carbon-carbon bond of the linker 20 is cleaved by palladium-catalyzed cleavage. In one embodiment, the linker 20 includes a uracil at cleavage position X, which is excised via uracil DNA glycosylase (USER) to leave an abasic site at cleavage position X that can be cleaved by endonuclease VIII. In some embodiments, the linker 20 includes 1, 2, 3, 4, 5, 6, or more carbons separating the deoxyribose 14 from the oligonucleotide adaptor 24. A shorter linker 20 may leave a smaller "scar" upon incorporation of the modified nucleotide 12 into the nucleotide backbone provided herein. Table 1 shows representative cleavage chemistries that may be used in conjunction with the disclosed linkers 20.

[0024] [Table 1]

[0025] The oligonucleotide adaptor 24 is attached to and extends from the linker 20 and may comprise a single stranded oligonucleotide or a partially double stranded oligonucleotide as disclosed herein. The oligonucleotide adaptor 24 terminates at a 5' oligonucleotide end 30, designated Y. Cleavage at cleavage position X releases the 3' end of the oligonucleotide adaptor 24 (see FIG. 2). The oligonucleotide adaptor 24 may be between 10 and 1000 nucleotides in length. In one embodiment, the oligonucleotide adaptor 24 is between 10 and 100, 10 and 30, 30 and 50, or 30 and 100 nucleotides in length. The size of the adaptor varies depending on the library preparation and can be very large, for example, for PCR-released libraries, so polymerase incorporation of modified nucleotides may be reduced with large (e.g., greater than 1000 nucleotides) oligonucleotide adaptors. However, incorporation of modified nucleotides 12 into 36-mer oligonucleotide adaptors 24 has been demonstrated to have high yields.

[0026] In one embodiment, oligonucleotide adaptor 24 may comprise one or more of a barcode, an adaptor sequence, a tag sequence, a primer binding sequence, a primer recognition sequence, a mosaic end sequence, a transposome recognition sequence, a capture site or capture sequence, a sequence complementary to a capture sequence, a molecular barcode (Unique Molecular Identifier, UMI) sequence, a restriction sequence, or an index sequence (e.g., a sample index sequence). The sequence of oligonucleotide adaptor 24 may be functional in the 5' to 3' direction in several embodiments of 3' adaptations provided herein.

[0027] In one embodiment, the oligonucleotide adaptor 24 may include or be attached to an affinity binder, shown as A, which serves as a handle to enable pull-down or isolation. For example, the affinity binder may be part of a binding pair, such as a biotin / streptavidin or antibody / antigen binding pair. The affinity binder A may be a first member of the binding pair, while a second member 34 of the binding pair may be attached to a surface 36 (e.g., a substrate surface, a bead surface), allowing isolation of the modified nucleotide 12, either before or after incorporation. In one embodiment, the modified nucleotide 12 is provided on the surface 36, and the adaptation or other incorporation steps disclosed occur on the surface 36. In one embodiment, the products of the adaptation workflow step are first generated and then separated on the surface 36. Although the affinity binder is shown as being generally centrally located within the oligonucleotide adaptor 24, it should be understood that the affinity binder may be attached at other locations on the oligonucleotide adaptor 24. Further, in embodiments, the oligonucleotide adaptor 24 does not include an affinity binder and / or the workflow steps disclosed herein are performed in solution or are not bound to a surface to incorporate modified nucleotides 12.

[0028] The modified nucleotide 12 includes a reactive 5' end, shown as Y in FIG. 1. In some embodiments, the reactive 5' end is a phosphate (e.g., tri-phosphate) or alkyne group reactive end. The modified nucleotide 12 also includes a reactive 3' group, shown as Z, which may be a hydroxyl group or an azide. The reactive 3' group allows for the attachment of the 5' oligonucleotide end Y as generally disclosed herein, for example, via ligation or click reaction. One or more of the X, Y, or Z reactive groups may be selectively blocked and deblocked. In one example, the Z reactive group may be reversibly blocked with a 3'-O-azidomethyl cap, which is removable by tris(2-carboxyethyl)phosphine (TCEP), to regenerate the 3'-OH. The 5' reactive group Y may be reversibly blocked by dephosphorylation or regenerated via a phosphorylation step. Table 2 shows representative ZY ligation strategies. The disclosed strategies may include ligation without deblocking, where due to stereochemistry a single extension occurs. Other strategies may use deblocking to generate reactive 3' and / or reactive 5' groups.

[0029] [Table 2]

[0030] The modified nucleotide 12 may be a modified purine or pyrimidine nucleotide. The modified oligonucleotide may be uracil, thymine, cytosine, adenine, or guanine. In one embodiment, the nucleobase and the linker 20 may both be attached to the 1' carbon position. In one embodiment, the nucleobase may be attached to a carbon of the linker 20. In one embodiment, the linker 20 may extend directly from the nucleobase such that the nucleobase 38 is between the linker 20 and the deoxyribose 14. In one embodiment, the linker 20 of the modified nucleotide 12 may be a linker (e.g., the linker 20 may have a chemical structure and may be positioned relative to and extend from the nucleobase 38) as described in U.S. Pat. No. 9,127,314, which is incorporated herein by reference in this application.

[0031] The disclosed embodiments include compositions of modified nucleotides 12. Modified nucleotides 12 may be provided as a single modified nucleotide type (e.g., only one of uracil, thymine, cytosine, adenine, or guanine) or a mixture of different nucleotides. For a particular reaction, all of the modified nucleotides 12 may have the same oligonucleotide adaptor sequence or may have distinct oligonucleotide adaptor sequences. Furthermore, modified nucleotides 12 may be provided in a reaction mixture along with unmodified nucleotides. It should be understood that certain features of modified nucleotides 12 (e.g., pentose sugar 14, nucleobase 38) may be simplified for purposes of illustration in the disclosed embodiments.

[0032] 2 is an exemplary process for incorporating modified nucleotide 12 at the 3' end of a sample DNA molecule 50, shown as double stranded DNA. The sample DNA molecule 50 may be DNA fragments generated by a suitable fragmentation technique (enzymatic fragmentation, sonication, natural fragmentation of cell-free DNA). In one embodiment, the fragments may be generated by transposome-mediated 5' adaption (see FIG. 6). Thus, the sample DNA may include a 5' adaptor in some embodiments.

[0033] In a first step, the sample DNA 50 is contacted with a modified nucleotide 12 in the presence of a polymerase to extend the 3' end via incorporation of the modified nucleotide 12 at the 5' phosphate group 15. The polymerase may be a permissive polymerase (e.g., a sequencing-by-synthesis polymerase capable of 3' addition of dye-conjugated nucleotides). Additionally, the polymerase may lack 3' to 5' exonuclease activity to prevent proofreading excision of the modified nucleotide 12. For extension, the 5' triphosphate group 15 of the modified nucleotide 12 is reacted with the 3' end of the sample DNA 50 as shown in FIG. 2. A single-stranded oligonucleotide adaptor 24 of the incorporated modified nucleotide 12 is extended from the 3' end of the sample DNA 50. The 5' oligonucleotide end Y is ligated to a reactive group Z, for example, via enzymatic or chemical ligation, which causes the oligonucleotide adaptor 24 to form a loop 52. In one embodiment, enzymatic ligation is mediated by a DNA ligase, such as the template-independent T4 ligase reaction, or a specialized single-stranded ligase, such as Circ ligase. In one embodiment, chemical ligation is mediated by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) of the Click reaction.

[0034] Cleavage at cleavage site X liberates the 3' end of the oligonucleotide adaptor 24, but leaves behind a "scar". Although incorporation of a modified nucleotide 12 at a single 3' end is shown, it should be understood that the illustrated reaction can occur in parallel at both 3' ends of the double-stranded sample DNA 50. It has been demonstrated that a polymerase can read through a non-natural backbone connection created using an azide-alkyne click reaction. Thus, the scar and potentially any non-natural 3' linkages (i.e., click chemistry) can be retained without significantly affecting the subsequent polymerase / amplification reaction.

[0035] Polymerase-mediated incorporation of the modified nucleotide 12 results in the formation of a 3'-adapted DNA 56 through the addition of a nucleotide in the oligonucleotide adaptor 24 at the 3' end of the sample DNA 50. Thus, a relatively simple and efficient reaction allows for batch addition of up to several hundred nucleotides to the 3' end of a nucleic acid through a single polymerase incorporation. As discussed herein, selective 3' addition of nucleotides separated from 5'-adaptation facilitates paired end sequencing workflows that sequence forward and reverse strands or sequencing workflows that use asymmetric adaptors such as fragmentation where the 5' end is added by a transposase. Furthermore, although relatively long oligonucleotides may be ligated to the strand ends, such ligation is relatively inefficient compared to polymerase reactions. Thus, using the disclosed techniques, more efficient addition of adaptors to the 3' end is achieved.

[0036] In the illustrated embodiment, the sample DNA 50 includes a 5' overhang. However, the DNA 50 may be provided as a blunt end or with a 3' overhang. The polymerase extends the 3' end to pair with the 5' overhang using a nucleotide complementary to the nucleotide of the 5' overhang. The 5' overhang may be a single base overhang. However, as discussed herein, the 5' overhang may represent a previously incorporated 5' adaptor. In one embodiment, the modified nucleotide 12 is a reversible extension terminator prior to cleavage and release of the 3' end. That is, the structure of the modified nucleotide 12 acts to prevent the subsequent addition of modified nucleotide 12 or unmodified nucleotides via reaction of the 5' end with the reactive group Z. In one embodiment, to prevent undesired extension, the reactive group Z may be reversibly blocked as provided herein.

[0037] 3 is a schematic diagram of a modified nucleotide 12 that includes a hybridized oligonucleotide adaptor 24. The oligonucleotide adaptor 24 includes a first strand 60 that is directly attached (e.g., covalently attached) to a linker 20. The first strand 60 hybridizes to a partially complementary second strand 62, such that the oligonucleotide adaptor 24 includes a partially double-stranded portion 63 as well as a single-stranded portion 64. The single-stranded portion 64 includes a 5' reactive end Y.

[0038] The formation of the oligonucleotide adaptor 24 as a construct may occur after incorporation of the modified nucleotide 12, which comprises only the first strand 60. That is, the polymerase-mediated incorporation may be followed by an annealing step at a temperature selected to allow specific hybridization and to be high enough to prevent non-specific binding. In other embodiments, the modified nucleotide 12 may be provided with the oligonucleotide adaptor 24, which is preformed and comprises the first strand 60 and the second strand 62. The 3' end of the oligonucleotide 63 may be blocked so that no extension occurs during incorporation of the modified nucleotide 12. The reactive 3'-Z may be reversibly blocked so that incorporation terminates the sample DNA 50 and / or prevents undesired side reactions of the single-stranded portion 64 prior to incorporation.

[0039] The nucleic acid linkage between the 5' oligonucleotide end Y and the active (or deblocked) 3' reactive group Z forms a loop 65 that is mostly single stranded but includes a 3' double stranded portion. The 3' end of the second strand 62 can be released by either or both of a cleavage or denaturation step. Cleavage results in a modified nucleotide 12 with a double stranded end 66, which can be removed in a subsequent denaturation step (e.g., for amplification). Denaturation without cleavage results in a modified nucleotide 12 with a single stranded end 68 and a single stranded scar.

[0040] Accordingly, certain embodiments of the present disclosure include the incorporation of a modified nucleotide 12 having an oligonucleotide adaptor 24 linked to the 1' position via a linker 20 and having a terminal 5' end. The oligonucleotide adaptor 24 is attached to an available reactive Z group via its 5' end and may be cleaved to release the 3' end. Thus, the disclosed technology includes allowing the oligonucleotide adaptor to freely transition between a terminal 5' end when attached to the 1' position of deoxyribose 14 and a terminal 3' end when attached to the 3' position of deoxyribose 14. However, in certain embodiments, the oligonucleotide adaptor 24 may be linked at the 1' position and have a terminal 3' end that does not react with a Z group and / or is not subject to cleavage.

[0041] FIG. 4 illustrates an approach in which conventional adaption via ligation is enhanced using modified nucleotides 12 that include oligonucleotide adaptors 24 that form a fork adaptor. The illustrated modified nucleotides 12 are attached to a fork adaptor 76 at a cleavage site 72. The cleavage site 72 is attached to an end 74 of a first fork 78 of the fork adaptor. The first fork 78 includes a first adaptor that is not complementary to a second adaptor on a second fork 80. Thus, in an example in which the sample DNA 50 is adapted to include different end adaptors (e.g., an A adaptor and a B adaptor), the fork adaptor 76 carries both of the different adaptors on different forks such that ligation of the fork adaptor 76 to the sample DNA 50 adds both adaptors at once, resulting in a fork end. In one embodiment, the fork adaptor 76 may be a Nextera adaptor (Illumina, Inc.) that includes a P5 sequence and an i5 sequence on the first fork 78 and a P7 sequence and an i7 sequence on the second fork 80. Fork adapter 76 may be a universal adapter, whereby all of the different sample fragments are compatible with fork adapters 76 that have a common sequence.

[0042] The fork adaptor 76 is covalently and cleavably linked to the linker 20 of the modified nucleotide 12. However, in some embodiments, the fork adaptor 76 may hybridize to a complementary oligo that extends from the cleavable linker 20 (see FIG. 2 and FIG. 5). The fork adaptor includes a double-stranded portion 84 that includes a 3′ T-overhang 86. The T-overhang 86 is ultimately ligated to the A-tail sample DNA 50 via, for example, T4 ligase. The A-tail consists of a modified nucleotide 12 that is a modified adenosine. Thus, incorporation of the adenosine modified nucleotide 12 into the 3′ end of the double-stranded sample DNA 50 modified by the fork adaptor 76 with the T-overhang 86 creates an A-tail that is used in subsequent ligation of the T-overhang 86.

[0043] In the illustrated embodiment, the double-stranded sample DNA 50 is blunt-ended, and incorporation of the adenosine modified nucleotide 12 into the double-stranded sample DNA 50 generates a 3' A-tail 90 from which the fork adaptor 76 is extended. A-tailing may be performed by a compatible A-tailing polymerase, such as taq polymerase, Klenow, or terminal transferase. The A-tail 90 generates a substrate for ligation of the 3' T 86 of the double-stranded portion 84. The fork adaptor 76 extends from the cleavable linker 20 of the incorporated adenosine, and the ligation efficiency of these elements is enhanced by coupling, e.g., by holding the elements in close proximity to each other prior to and during ligation. Thus, ligation occurs while the fork adaptor 76 is still attached to the cleavable linker 20.

[0044] After ligation, retention of the fork adaptor 76 via the cleavable linker 20 to the incorporated adenosine can be reversed leaving a scar at an internal site corresponding to the position of the incorporated modified nucleotide 12. Sample DNA 50 includes a fork adaptor 76 added via ligation. Although only one end of sample 50 is illustrated, it should be understood that the illustrated adaption can occur at both ends of fragments of sample 50 and as part of the preparation of a sequencing library.

[0045] FIG. 5 illustrates an approach using modified nucleotide 12 (here, modified adenosine) containing oligonucleotide adaptor 24 to recruit fork adaptor 100 via hybridization. Here, sample DNA 50 is shown as cell-free DNA fragments. However, other sample DNA formats are also contemplated. End repair and A-tailing using modified nucleotide 12 adds a terminal adenosine 102 containing oligonucleotide adaptor 24 to the 3' end of sample DNA 50. Fork adaptor 100 may include a T overhang to facilitate ligation of the double-stranded portion of fork adaptor 100 to A-tailed sample DNA 50. For the approaches herein (e.g., as in FIG. 4 and FIG. 5), the ligation step can be either enzymatic or chemical.

[0046] The fork adaptor 100 also includes a modified nucleotide 12 at each 3' non-complementary end of the fork adaptor 100. When the fork adaptor 100 is added to a reaction mixture containing the sample DNA 50, the respective oligonucleotide adaptors 24 extending from both the 3' A-tail 102 and the fork adaptor 100 are brought into close proximity to each other. The oligonucleotide adaptors 24 are self-complementary as shown and can hybridize to form a double-stranded paired adaptor construct 108. This construct 108 adds stability to the ligation of the fork adaptor 100 by holding the fork adaptor 100 in close proximity to the A-tail 102, facilitating ligation of the fork adaptor 100 and increasing ligation efficiency. Hybridization to form the double-stranded hybridized adaptor construct 108 can occur prior to and / or during ligation. In one embodiment, the oligonucleotide adaptor 24 of the A-tail 102 and the oligonucleotide adaptor 24 of the fork adaptor 100 can be the same sequence with high self-complementarity. In the illustrated embodiment, the 5'-most six nucleotides are self-complementary such that the 5' portion of the first oligonucleotide adaptor 24 is the reverse complement of at least the first six nucleotides of the second nucleotide adaptor 24. The self-complementary region may include at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides and may be located at the 5' end or may be an internal region. Additionally, the nucleotide adaptor 24 may include one or more self-complementary regions.

[0047] To complete preparation of adapted sample DNA for subsequent target enrichment and / or amplification steps, the oligonucleotide adaptors 24 may be cleaved at the cleavage site 110 of each of the incorporated modified nucleotides. Cleavage results in adapted double-stranded fragments 120 that include a fork adaptor 100 ligated to both ends. The fragments 120 may be part of a sequencing library for use in a sequencing reaction. The fragments 120 may be provided to a subsequent step (e.g., amplification, enrichment) or may be provided directly to a sequencing step without sequencing or amplification.

[0048] Each 3' end of the double-stranded fragment 120 includes a respective scar from the remaining portion of the linker at each modified nucleotide site. Thus, for each strand of the double-stranded fragment 120, a first scar is present at an internal site corresponding to the A-tail 102, and a second scar is present at the 3' end of the fork adaptor 100. Two scars are present on each strand. However, in some embodiments, the scars are removed in a subsequent amplification or extension step. Furthermore, only the scar at the A-tail 102 is covered by the polymerase for subsequent processing. The scar on the fork adaptor 100 may extend beyond the adaptor sequence and cannot be copied over / covered by the polymerase.

[0049] 6A-6B are schematic diagrams of the fragmentation workflow of the prior art. For 5'-adaptation, a transposome-based strategy is used. FIG. 6C shows the fragmentation workflow using modified nucleotides as disclosed herein. Compared to current transposome-based sample preparation, 3'-adaptation with modified nucleotides 12 offers several advantages.

[0050] A typical Nextera™ or Illumina DNA prep (Illumina, Inc.) sample preparation (Figure 6A) may use two different transposomes. Because the transposome is a dimeric complex, only 50% of the doubly transposed library fragments have both adapter types and are sequencing compatible. In fragmentation with fork adapters (Figure 6B), each fragmentation event introduces a 5'A-type adapter, and then 3'-adaptation is achieved via an extension ligation step. However, in practice, this extension ligation step is low-yielding, since only a portion of the product contains the desired double adapter type.

[0051] Efficient incorporation of modified nucleotides 12 with oligonucleotide adaptors 24 has been demonstrated. In one embodiment, modified nucleotides 12 provided herein are used in polymerase-mediated adaption to improve the yield of sequenceable fragments in sequencing library preparation, as shown in FIG. 6C. Sample preparation using modified nucleotides 12 may use a single transposome type for 5' adaption, followed by 3' adaption with modified nucleotides 12, but in principle will only generate AB-adapted libraries. Thus, the simplified transposome design in FIG. 6C does not require fork adaptors or a strategy to selectively denature ME' and reanneal new fragments.

[0052] In the example shown in FIG. 6C, the transposome complex is a homodimer 130, with monomer 132 containing only a single adaptor type, the 5' adaptor, or A adaptor 134, in the embodiment shown. Fragmentation using homodimer 130 adds a 5' adaptor and a double-stranded ME (mosaic end) sequence to the 5' end of the insert. In the next step, modified nucleotides 12 carrying a 3' adaptor or a B adaptor can be incorporated at the 3' end. This may be done with a mixture of four different modified nucleotides 12 for each base in the template strand, or with various mixtures of natural and modified nucleotides 12.

[0053] Ligation to the 3' reactive group and linker cleavage results in an exchange of the base-paired backbone of the B adaptor such that the 3' end of the insert is extended by the B adaptor sequence. The B adaptor, in some embodiments, is non-complementary to the 5' adaptor. Subsequent steps after asymmetric adaptation may include first strand extension to remove the scar or non-natural backbone, and clustering and sequencing of the forward and reverse strands.

[0054] 1-5 and 6C show different configurations of oligonucleotide adaptors 24 used to form and / or mobilize terminal adaptors in sequencing library preparation. In certain embodiments, sequential addition of adaptors allows for the creation of asymmetric or dumbbell libraries. For example, 3' adaption via modified nucleotide 12 can be used after 5' adaptor addition. Dumbbell libraries consist of double-stranded nucleic acid fragments with hairpin adaptors at both ends. The dumbbell structure allows for sequencing around the entire dumbbell via a strand-displacing polymerase, resulting in sequences representing both the sense and antisense strands. Dumbbell-based amplification can also be rolling circle amplification, which generates multiple copies of an insert. In one example, dumbbell sequencing hairpin adaptors can be attached (via a linker) to modified nucleotides 12 that are added to double-stranded nucleic acid fragments by A-tailing (see FIG. 4). Oligonucleotide adaptors 24 can form hairpins, whereby the 5' end of the hairpin is ligated to a 3' reactive group after incorporation of the relevant nucleotide. Cleavage from the linker releases the 3' end of the hairpin. The 3' end of the hairpin can then be ligated to the free 5' end of the fragment. Ligation of the bound hairpin adaptor is improved because the hairpin adaptor is extended from the incorporated 3' modified adenosine that is added by the polymerase.

[0055] FIG. 7 shows adapter primer extension over a modified nucleotide 12 using a polymerase with 5' to 3' exonuclease activity. In the illustrated example, a double-stranded nucleic acid 150 has a modified nucleotide 12 already incorporated via addition at the 3' end 152 of a first strand 154, whereby the first strand includes an oligonucleotide adapter 156, shown here as a single-stranded oligonucleotide. Incorporation of the modified nucleotide 12 may occur as generally discussed herein. In one embodiment, the oligonucleotide adapter includes a free 3' end that may be generated via cleavage as discussed herein, or the oligonucleotide adapter may be attached to the modified nucleotide 12 such that the 3' end is free, as in the illustrated embodiment. The adapter 156 may be single-stranded, and an adapter primer 158 complementary to the adapter 156 is extended using a polymerase to generate a double-stranded adapted fragment. Instead of using a strand-displacing polymerase to extend from the adapter primer 158 across the modified nucleotide 12, a polymerase with 5'-3' exonuclease activity extends the adapter primer 158 across and past the modified nucleotide 12 toward the 5' end 160 of the second strand 162 of the nucleic acid 150. By continuing the extension, a nick 166 in the nucleic acid 150 is shifted via "nick translation", followed by ligation to seal the nick and generate a nucleic acid 168 that incorporates a double-stranded adapter 170 (e.g., adapter 156 and its complementary adapter primer 158) at at least one end. An advantage of the illustrated embodiment is that the nick 166 is separated or moved away from the position of the modified nucleotide 12, such that the structural difference of the modified nucleotide 12 relative to the unmodified nucleotide does not inhibit ligation at the nick 166. Thus, one or more adaptors, e.g., sequencing adaptors, may be incorporated onto a nucleic acid fragment end(s) via the illustrated workflow, which includes modified nucleotide addition, primer extension with exonuclease activity, and subsequent nucleic acid ligation.

[0056] FIG. 8 illustrates an embodiment in which a modified nucleotide 12 is incorporated at a recessed 3' end that includes a double-stranded DNA fragment 180. The modified nucleotide is added to pair with an oligonucleotide at a 5' overhang 182. The addition of the modified nucleotide 12 also incorporates a linked oligonucleotide adaptor 184 that includes a single-stranded lure site 186. Here, the oligonucleotide adaptor 184 is linked to the 1' position and the 3' position 188 is available for the initiation of dNTP addition. A polymerase then adds a dNTP and extends to flush out the 3' recess, using the overhang 182 as a template. The polymerase extended 3' end 190 may be blunt or may include an overhang. The oligo portion of the modified nucleotide 12 that includes the lure site 186 then serves as a "lure" to which an adaptor 200 is hybridized via a complementary sequence 192. This fixes the adaptor 200 close to the template 180 and facilitates a more efficient ligation reaction at the blunt end 210 shown, which adds the adaptor 200 to the template to generate the adapted fragment 212.

[0057] FIG. 9 illustrates an embodiment in which modified nucleotides 12 are incorporated at the 3' end of an RNA molecule 220. Typically, modified nucleotides are not incorporated at the end of a single-stranded nucleic acid. However, a "sprint" may be hybridized to the RNA 220 by contacting the single-stranded RNA 220 with a single-stranded oligo 230 having a 3' end portion 234 that is random in base composition (e.g., a hexamer end portion 234) or a mixture of a 3' end portion 234 that is blocked from extension (e.g., via a 3' end blocking on the oligo 230) and a 5' end portion 236 that has a known sequence. A set of oligos 230 with a mixture of random portions 234 of different random sequences may hybridize based on the sequence of a particular random end portion 234 at any position along the molecule. However, when a particular oligo 230 hybridizes at the 3' end of an RNA molecule 220 based on the random portion 234 being complementary to the 3' end portion of the molecule 220, an overhang of the 5' end portion 236 with a known sequence is generated. This overhang allows for the incorporation of a modified nucleotide 12 at the 3' end. An example of a splint sequence is 3'block-nnnnnn-TTTTT-5', where "n" is any base including inosine. In this particular example, the modified nucleotide 12 is derived from an adenosine base to facilitate complementary binding to the splint oligo 230.

[0058] FIG. 10 shows an example of using modified nucleotides 12 in an extension reaction. A template strand 250 is primed with a primer 252 that includes a randomer with a 5′ adaptor 254. However, in one embodiment, the primer 252 may be a target primer, for example, for a target sequencing reaction. Extension using a reaction mixture of unmodified and modified nucleotides results in a termination of the extension upon incorporation of the modified nucleotide 12. Each modified nucleotide may be attached to a 3′ adaptor 260 as provided herein. Thus, the extension products 262 have asymmetric adaptors 254, 260 at their ends. This technique eliminates the need for end repair and ligase-catalyzed adaption. In addition to eliminating the need for multiple enzymatic adaptor ligation steps, this approach also has the advantage of eliminating the need for fragmentation and the associated post-fragmentation size selection. That is, the combination of randomer-primer annealing and termination based on the incorporation of the modified nucleotide creates a separation between the extension products 262. Although priming and extension from a single strand is shown, it should be understood that the reaction can occur on both forward and reverse strands.

[0059] FIG. 11 shows an example where modified nucleotides 12 are used, for example, in RNA sequencing library preparation from single-stranded RNA molecules 180. The use of modified nucleotides 12 carrying a 3′ adaptor 282 allows for direct addition of the 3′ adaptor in the first strand cDNA synthesis step. The workflow is carried out as follows: Reverse transcriptase extends from a primer 284 containing a 5′ adaptor 186, shown as a random hexamer or an A14 oligo linked to an oligo(dT) (for attachment of a polyA RNA tail). The reaction mixture contains modified nucleotides 12 supplemented at low concentration with dNTPs. Modified nucleotides 12 terminate the extension once incorporated. Thus, the concentration of modified nucleotides 12 can be adjusted to alter the length of the cDNA (e.g., higher concentrations of modified nucleotides 12 result in shorter cDNA products). The cDNA incorporating modified nucleotides 12 can then be captured using a biotin handle for purification and buffer exchange. Next, a 3' adaptor 282 (potentially B15) is ligated to the 3' end by proximity enhanced ligation and revealed through a USER cleavage step that liberates the 3' end. Once the 3' oligo is cleaved, index primer PCR amplifies the cDNA. The sequential addition of adaptors to the same strand means that this library preparation essentially allows for the determination of sense / antisense strand information in the final sequencing library. This approach also eliminates the need for complex template conversion protocols.

[0060] Variations of this approach can also be used for targeted RNA-seq applications (e.g., splice variants and gene fusion analysis, and whole exome sequencing). Compared to other approaches (e.g., RASL-seq for gene fusions) that require two probes and nucleic acid ligation events, enrichment that requires an additional step, or multiplex PCR that requires an additional step and two primers for each target, this approach combines sequence-specific binding with library adaptation. Since the adaptors are added sequentially, subsequent steps from the initial strand extension with modified nucleotide 12 can be performed on beads (e.g., streptavidin beads). This can increase the efficiency of subsequent processes due to the high concentration of adaptors that can be co-immobilized in the same space as the single-adapted library.

[0061] Depending on the polymerase used, the oligonucleotide adaptor 24 of the modified nucleotide 12 may not need to be ligated to a 3'-OH to function as an adaptor, as shown in the example of FIG. 12. In the illustrated example, the oligonucleotide adaptor 24 is partially double-stranded and includes or is hybridized to a primer 290 that can be extended in the 3' direction. In the illustrated embodiment, the addition of base 294 based on template 296 "jumps" the modified nucleotide 12. However, depending on the configuration of the modified nucleotide 12, the extension may use the base associated with the modified nucleotide 12 as a template. As shown, the 3'-OH reactive group 300 is not directly ligated to the end of the oligonucleotide adaptor 24, and in the illustrated example, there is no exchange of base pairing backbone. FIG. 13 shows an arrangement in which an exchange of base pairing backbone occurs at the reactive 3'-OH 310, forming a loop (or hairpin) that allows the oligonucleotide adaptor 24 to be extended in the 3' direction to copy the single-stranded template molecule 312.

[0062] Figure 14 is a diagram of reactions performed to incorporate modified nucleotide incorporation into blunt and recessed duplex DNA (left) and the expected resulting product sizes (right). Figure 15 shows the results of modified nucleotide incorporation into blunt and recessed duplex DNA from the reactions shown in Figure 14. Modified nucleotide oNTPs were incorporated by 1901pol in both templated and non-templated reactions with approximately 90% templated incorporation and approximately 95% non-templated incorporation in 5 minutes.

[0063] FIG. 16 is a diagram of modified nucleotide primed extension onto blunt and recessed duplex DNA, and FIG. 17 shows the results of modified nucleotide primed extension onto blunt and recessed duplex DNA. In the reactions, single-stranded oligonucleotide adaptors were incorporated into the blunt and recessed duplex fragments at the 3' end. The single-stranded oligonucleotides bind to the primers via sequence complementarity. 1901pol or Bst2.0 were used to extend the primers, and strand displacement of the duplexes occurred during primer extension. That is, the displaced strand was the strand that did not contain the incorporated modified nucleotide. Bst2.0pol exhibited the highest activity in both displacement extension of blunt and recessed duplexes with 60° C. incubation for 5 min.

[0064] FIG. 18 is a diagram of adaptor ligation to blunt double-stranded DNA containing modified nucleotides. Either modified adenosine containing lures or dATP controls were incorporated at the ends of the duplexes. FIG. 19-FIG. 20 show the results of lure-based adaptor ligation compared to ligation with other nucleotide ends. It was demonstrated that ligation occurred, although ligation containing lure tails was less preferred compared to other ends. T4 ligase showed the best ligation in the presence of oNTP lure tails.

[0065] Figure 21 shows exonuclease inhibition based on scar size of modified nucleotides. 3' to 5' exonuclease activity can vary significantly with dNTP type and base modification. Scar size can be inversely correlated with ligation efficiency.

[0066] The disclosed techniques may be used to modify a nucleic acid sample or sample nucleic acid. A "sample nucleic acid" may be derived from any in vivo or in vitro source, including one or more cells, tissues, organs, or organisms, living or dead, or any biological or environmental source (e.g., water, air, soil). For example, in some embodiments, the sample nucleic acid comprises or consists of eukaryotic and / or prokaryotic dsDNA originating or derived from humans, animals, plants, fungi (e.g., molds or yeasts), bacteria, viruses, viroids, mycoplasma, or other microorganisms. In some embodiments, the nucleic acid of the sample comprises or consists of genomic DNA, subgenomic DNA, chromosomal DNA (e.g., from an isolated chromosome or portion of a chromosome, e.g., from one or more genes or loci from a chromosome), mitochondrial DNA, chloroplast DNA, DNA from a plasmid or other episome (or recombinant DNA contained therein), or double-stranded cDNA made by reverse transcribing RNA using an RNA-dependent DNA polymerase or reverse transcriptase to generate first strand cDNA and then extending a primer annealed to the first strand cDNA to generate dsDNA. In some embodiments, the nucleic acid of the sample 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 genomic DNA or cDNA prepared from RNA in or derived from a biological (e.g., cell, tissue, organ, organism) or environmental (e.g., water, air, soil, saliva, sputum, urine, feces) source). In some embodiments, the nucleic acid of the sample is from an in vitro source. For example, in some embodiments, the nucleic acid of the sample comprises or consists of dsDNA prepared in vitro from single-stranded DNA (ssDNA) or from single- or double-stranded RNA (e.g., using methods well 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 nucleic acid of the sample comprises or consists of dsDNA 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 amplification of DNA or RNA (e.g., PCR or reverse transcriptase PCR (RT-PCR), transcription-mediated amplification methods, involving amplification of all or a portion of one or more nucleic acid molecules); molecular cloning of all or a portion of one or more nucleic acid molecules in a plasmid, fosmid, BAC (Bacterial Artificial Chromosome), or other vector that is then replicated in a suitable host cell; or methods for capture of one or more nucleic acid molecules by hybridization, such as hybridization to DNA probes on an array or microarray.

[0067] The disclosed nucleic acid techniques may be implemented as part of a sequencing workflow. Sequencing techniques may include 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 / 010251, the disclosures of which are incorporated herein by reference in their entireties. Some embodiments may utilize nanopore sequencing, whereby a sample nucleic acid strand or nucleotides are removed from the sample nucleic acid by an exonuclease and passed through a nanopore. As a sample nucleic acid or nucleotide passes through the nanopore, each base type can be identified by measuring the change in electrical conductance 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 entireties). Still other embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons may use electrical detectors and related technology available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or may use the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082(A1), 2009 / 0127589(A1), 2010 / 0137143(A1), and 2010 / 0282617(A1). Certain embodiments may utilize methods that involve real-time monitoring of DNA polymerase activity.Nucleotide incorporation can be detected via Fluorescence Resonance Energy Transfer (FRET) interactions between a fluorophore-bearing polymerase and a gamma-phosphate-labeled nucleotide, or using zero-mode waveguides, for example, as described 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, Fluorescent In Situ SEQuencing (FISSEQ) and Massively Parallel Signature Sequencing (MPSS). In certain embodiments, the sequencing device 260 may be an iSeq from Illumina (La Jolla, Calif.).

[0068] In some embodiments, the modified nucleotide 12 may include a cleavable moiety that is subject to photochemical cleavage to allow for the release of the 3' end of the oligonucleotide adaptor. Cleavage at the cleavage site disrupts the linkage of the oligonucleotide adaptor 24 to the linker 20, e.g., a carbon linker. Photochemical cleavage encompasses any method that utilizes light energy to achieve cleavage of a nucleic acid (e.g., one or both strands of a double-stranded nucleic acid molecule). Sites for photochemical cleavage may be provided by non-nucleotide chemical moieties in the nucleic acid, such as phosphoramidites [4-(4,4'-dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite) (Glen Research, Sterling, VA, USA, Catalog No. 10-4913-XX).

[0069] In some embodiments, the oligonucleotide adaptor 23 may include an affinity binder or affinity tag that serves as a handle to allow for pull-down or purification of nucleic acids incorporating modified nucleotides 12. The affinity tag may be useful for bulk isolation of target nucleic acids. As used herein, the term "affinity binder" may refer to components of a multi-component complex that specifically interact or bind to one another. For example, the affinity tag may include biotin or His (Histidine), which may bind to streptavidin or nickel, respectively. Other examples of multicomponent affinity tag complexes include ligands and their receptors, such as avidin-biotin, streptavidin-biotin, and derivatives of biotin, streptavidin, or avidin, including, but not limited to, 2-iminobiotin, desthiobiotin, NeutrAvidin (Molecular Probes, Eugene, OR), CaptAvidin (Molecular Probes), and maltose-Maltose Binding Protein (MBP), calcium-Calcium Binding Protein / Peptide (Calcium Binding Protein / Peptide), and the like. binding proteins / peptides, including CBP (Carboxylic Acid Bacterium Protein / Peptide), antigen-antibody including epitope tags and their corresponding anti-epitope antibodies, haptens, e.g., dinitrophenyl and digoxigenin and their corresponding antibodies, aptamers and their corresponding targets, poly-His tags (e.g., penta-His and hexa-His) and corresponding Immobilized Metal ion Affinity Chromatography (IMAC) materials and their binding partners, including anti-poly-His antibodies, fluorophores and anti-fluorophore antibodies, etc.

[0070] The disclosed modified nucleotides 12 (e.g., oligo-modified nucleotide analogues, oNTPS) are non-naturally occurring molecules. In some embodiments, the modified nucleotides are incorporated into naturally occurring and / or synthetic nucleic acid sequences. In some embodiments, the modified nucleotides are attached to an oligonucleotide adaptor that includes a non-naturally occurring nucleic acid sequence, such as a universal adaptor sequence suitable for sequencing or other nucleic acid manipulation workflows. For multiplex reactions, different modified nucleotides 12 may be used for each different sample that is otherwise the same, differing by the distinguishable sample index sequences of the oligonucleotide adaptors.

[0071] Embodiments of the present disclosure include compositions of modified nucleotides 12 and / or nucleic acids having one or more incorporated modified nucleotides 12. Embodiments of the present disclosure include nucleic acids generated as part of a sequencing library preparation having a 3' adaptor added via incorporation of a modified nucleotide 12 and exchange of the base paired backbone of the adaptor to the 3' end of the incorporated modified nucleotide 12 via nucleic acid ligation and cleavage at a linker that extends from the nucleic acid base and releases the 3' end of the adaptor. Embodiments of the present disclosure include sample preparation kits that include modified nucleotides 12 and associated reagents for a workflow, for example, associated reagents for sequencing library preparation. The associated reagents may include chemical reagents or enzymes (e.g., ligases and / or polymerases, as discussed herein).

[0072] This written description enables any person skilled in the art to practice the disclosed embodiments, including making and using any device or system and performing any incorporated method, using the examples as part of the disclosure. 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 include structural elements that do not differ from the literal words of the claims, or if they include equivalent structural elements that have only minor differences from the literal words of the claims.

Claims

1. An oligonucleotide-modified nucleic acid analog composition, A modified nucleotide, Ribose, A 5'-phosphate group bonded to the ribose, A 3'-reactive group bonded to the ribose, An oligonucleotide adapter bonded to the ribose by a linker and terminated at the 5'-oligonucleotide end, comprising a modified nucleotide.

2. The composition according to claim 1, wherein the oligonucleotide adapter is bonded to the 1'-position of the ribose via the linker.

3. The composition according to claim 1, wherein the modified nucleotide comprises a nucleobase bonded to the 1'-position of the ribose, and the linker extends from the nucleobase.

4. The composition according to claim 3, wherein the nucleobase is uracil, thymine, cytosine, adenine, or guanine.

5. The composition according to claim 3, wherein the composition comprises a plurality of modified nucleotides comprising a mixture of nucleotide bases.

6. The composition according to claim 5, comprising a plurality of unmodified nucleotides, wherein the unmodified nucleotides comprise one or more of uracil, thymine, cytosine, adenine, or guanine.

7. The composition according to claim 1, wherein the linker comprises a carbon chain containing two or more carbons, and the oligonucleotide adapter is directly or indirectly bonded to the carbon chain.

8. The composition according to claim 1, wherein the linker is a cleavable linker.

9. The composition according to claim 8, wherein the cleavable linker comprises an enzymatically cleavable molecule, a chemically cleavable molecule, or a photocleavable molecule.

10. The composition according to claim 1, wherein the 5'-oligonucleotide end reacts with the 3'-reactive group to bond the 5'-oligonucleotide end to the ribose at the 3'-position.

11. The composition according to claim 1, comprising a reversible blocking factor at the 5'-oligonucleotide end or the 3'-reactive group.

12. The composition according to claim 1, wherein the 5'-oligonucleotide end comprises a phosphate group or an alkyne group.

13. The composition according to claim 1, wherein the 3'-reactive group comprises a hydroxyl group or an azide.

14. The composition according to claim 1, wherein the oligonucleotide adapter comprises a primer binding site, a capture site, an index, or a combination thereof.

15. The composition according to claim 1, wherein the oligonucleotide adapter is bound to an affinity conjugate.

16. The composition according to claim 1, wherein the oligonucleotide adapter is single-stranded.

17. The composition according to claim 1, wherein the oligonucleotide adapter comprises a sequence that hybridizes to a recognition sequence extending from the linker.

18. The composition according to claim 1, wherein the oligonucleotide adapter comprises a fork adapter.

19. The composition according to claim 1, wherein the oligonucleotide adapter has a nucleotide length of 10 to 1000.

20. The composition according to claim 1, wherein the ribose is deoxyribose or dideoxyribose.

21. An oligo-modified nucleic acid analog composition, a modified nucleotide, a ribose, a 5'-phosphate group bound to the ribose, a 3'-reactive group bound to the ribose, and an oligonucleotide adapter bound to the ribose by a linker and terminating at the 3'-oligonucleotide end, comprising a modified nucleotide comprising an oligo-modified nucleic acid analog composition.

22. A method for modifying a nucleic acid, comprising contacting a single-stranded nucleic acid with a modified nucleotide, wherein the modified nucleotide is deoxyribose, a 5'-phosphate group bound to the deoxyribose, and a single-stranded oligonucleotide adapter bound to the deoxyribose and terminating at the 5'-oligonucleotide end, contacting the single-stranded nucleic acid with the modified nucleotide, using a polymerase to incorporate the modified nucleotide into the 3'-end of the single-stranded nucleic acid via the 5'-phosphate group to generate an extended single-stranded nucleic acid, annealing a primer comprising a recognition site for the 5'-region of the single-stranded oligonucleotide adapter, and extending the primer to synthesize a complementary strand of the single-stranded nucleic acid.

23. A nucleic acid fragment, comprising a single-stranded nucleic acid fragment or a double-stranded nucleic acid fragment, and a modified nucleotide bound to the 3'-end of the nucleic acid fragment, the modified nucleotide comprising an oligonucleotide adapter that is bound to a ribose by a linker at a first end and terminates at a 5'- or 3'-oligonucleotide end at a second end.

24. A method for modifying a nucleic acid, Contacting a double-stranded nucleic acid with a modified nucleotide, wherein the modified nucleotide comprises: deoxyribose; a 5'-phosphate group attached to the deoxyribose; a single-stranded oligonucleotide adapter attached to the deoxyribose via a linker at a first end and terminating at a second end with a 3'-oligonucleotide terminus; and contacting the double-stranded nucleic acid with the modified nucleotide; incorporating the modified nucleotide into the 3'-end of the first strand of the double-stranded nucleic acid via the 5'-phosphate group to generate an extended first strand; annealing a primer comprising a recognition site for a 3'-region of the single-stranded oligonucleotide adapter; extending the primer using a polymerase having 5'-to-3' exonuclease activity to synthesize a complementary strand of the single-stranded oligonucleotide adapter while degrading a 5'-portion of the second strand of the double-stranded nucleic acid. A method comprising:

25. A method for modifying a nucleic acid, comprising: contacting a single-stranded RNA with a plurality of single-stranded oligonucleotides comprising a 3'-random portion and a 5'-fixed sequence portion such that a 3'-random portion of one of the plurality of single-stranded oligonucleotides anneals to the 3'-end of the single-stranded RNA and the 5'-fixed sequence portion does not anneal to the single-stranded RNA; incorporating a modified nucleotide into the 3'-end of the single-stranded RNA using the fixed sequence portion as a template, wherein the modified nucleotide comprises: deoxyribose; a 5'-phosphate group attached to the deoxyribose; a single-stranded oligonucleotide adapter attached to the deoxyribose and terminating with a free 3'-end; and incorporating the modified nucleotide into the 3'-end of the single-stranded RNA. A method comprising:

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