Methods, Compositions, and Kits for Preparing Nucleic Acid Libraries

Abstract

The present invention relates to methods, compositions, and kits for extending polynucleotides and to methods, compositions, and kits for preparing sequencing libraries of polynucleotides, which methods, compositions, and kits involve generating a modified target polynucleotide on an adaptor template oligonucleotide and labeling one or both strands of the target sequence. The sequencing libraries are suitable for massively parallel sequencing and comprise a plurality of double-stranded nucleic acid molecules.

Description

BACKGROUND OF THE INVENTION

[0002] The present invention relates to methods and compositions for extending target polynucleotides. An adaptor sequence is added to the 3' end of a single-stranded nucleic acid.

[0003] Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, although the technology can generate the nucleotides of hundreds of billions of DNA sequences in a single experiment, an error rate of ~1% results in hundreds of millions of sequencing errors. These scattered errors become a serious problem when performing "deep sequencing" on mixtures with genetic heterogeneity (such as tumors) or mixed microbial populations.

[0004] To overcome the limitations of sequencing accuracy, several methods have been reported. Duplex sequencing (Schmitt, et al PNAS 109:14508-14513) is one of them. This method greatly reduces errors by independently labeling and sequencing each of the two strands of a DNA duplex. Since the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors only result in mutations in one strand and can therefore be ignored as technical errors. Kinde et al reported another method called the Safe-SeqS (PNAS 2011 Jun 7; 108(23):9530-5). The key to this method lies in (i) assigning a unique identifier (UID) to each template molecule, (ii) amplifying each uniquely labeled template molecule to create a UID family, and (iii) performing redundant sequencing on the amplification products. A mutation is only considered to be present (a "supermutant") when ≥95% of the PCR fragments with the same UID contain the same mutation. U.S. Patents US8,722,368, US8,685,678, and US8,742,606 describe methods for sequencing polynucleotides attached to degenerate base regions to determine / estimate the number of different starting polynucleotides. However, these methods cannot be easily used for targeted amplicon sequencing and often involve ligation to attach the degenerate base region. For targeted amplicon-based enrichment and sequencing, it is not easy to sequence fusion or translocation events. In addition, since the primer sites cannot be sequenced, it is sometimes difficult to design suitable primer pairs to cover hotspots without losing some unsequenced regions. For multiplex amplification in a single tube, overlapping regions cannot be amplified, resulting in the loss of sequencing information in the primer binding regions of the target polynucleotides. For small-sized target fragments (such as plasma DNA, small RNA, or miRNA), it is also difficult to design a pair of primers because there is not much space for primer sequence design.

[0005] Targeted next-generation sequencing often involves the analysis of large, complex fragments and is achieved by multiplex PCR (simultaneously amplifying different target DNA sequences in a single PCR reaction). However, the results obtained with multiplex PCR are often complicated by artifacts of the amplification products. These include false-negative results attributable to reaction failure and false-positive results attributable to non-specific priming events (such as the amplification of spurious products). Since the likelihood of non-specific priming increases with each additional primer pair, conditions must be adjusted as needed when adding individual primer sets.

[0006] Detailed description

[0007] To facilitate understanding of the present invention, a number of terms are defined below.

[0008] As used herein, "sample" refers to any substance that contains or is suspected of containing nucleic acids and includes samples of tissue or fluid isolated from one or more individuals. In particular, nucleic acid samples can be obtained from a single cell, an organism, or a combination of organisms selected from viruses, bacteria, fungi, plants, and animals. Preferably, nucleic acid samples are obtained from mammals. In a preferred embodiment, the mammal is a human. Nucleic acid samples can be obtained from samples of a subject's body fluid or tissue biopsy, or from cultured cells. Body fluids can be selected from whole blood, serum, plasma, urine, sputum, bile, feces, bone marrow, lymph fluid, semen, breast exudate, bile, saliva, tears, bronchial washing, gastric washing, spinal fluid, synovial fluid, peritoneal fluid, pleural effusion, and amniotic fluid. A "single sample" can be a single cell (which can be a T cell or a B cell), while multiple samples can be many blood cells in a blood sample.

[0009] As used herein, the term "nucleotide sequence" refers to a homopolymer or heteropolymer of deoxyribonucleotides, ribonucleotides, or other nucleic acids.

[0010] As used herein, the term "nucleotide" generally refers to the monomeric components of a nucleotide sequence, although in addition to nucleotides, the monomers can also be nucleosides and / or nucleotide analogs, and / or modified nucleosides (such as amino-modified nucleosides). In addition, "nucleotide" includes non-naturally occurring analog structures. Nucleotides can be deoxyribonucleotides, ribonucleotides, or other nucleic acids.

[0011] As used herein, the term "nucleic acid" refers to at least two nucleotides covalently linked together. Although in some cases nucleic acid analogs may be included that may have alternative backbones, nucleic acids will generally contain phosphodiester bonds. By definition, nucleic acids can be single-stranded or double-stranded, or contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, genomic and cDNA, RNA, a mixture of both DNA and RNA, or a DNA-RNA hybrid, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases (including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine, hypoxathanine, etc.). References to "DNA sequence" or "RNA sequence" can include single-stranded and double-stranded DNA or RNA. Unless the context indicates otherwise, a specific sequence refers to the single-stranded DNA or RNA of such a sequence, the duplex (double-stranded DNA or RNA) of such a sequence with its complementary sequence, and / or the complementary sequence of such a sequence.

[0012] As used herein, "polynucleotide" and "oligonucleotide" are types of "nucleic acid" and generally refer to primer or oligomer fragments to be detected. There is no intended distinction in length between the terms "nucleic acid", "polynucleotide" and "oligonucleotide", and these terms will be used interchangeably. The terms "nucleic acid", "DNA" and "RNA" and similar terms also include nucleic acid analogs. Oligonucleotides are not necessarily derived from any existing or natural sequence per se, but can be generated in any manner (including chemical synthesis, DNA replication, reverse transcription, or combinations thereof).

[0013] As used herein, the terms "target sequence", "target nucleic acid", "target polynucleotide" and "nucleic acid of interest" are used interchangeably and refer to the desired region to be amplified, to be detected, or to be amplified and detected, or which is the object of hybridization with a complementary oligonucleotide, polynucleotide (e.g., a blocking oligomer), or primer extension process. The target sequence can consist of DNA, RNA, their analogs, or combinations thereof. The target sequence can be single-stranded or double-stranded. During an extension process, a target polynucleotide that forms a hybrid duplex with an oligonucleotide (template) can be referred to as a "primer", or a target nucleic acid that forms a hybrid duplex with a primer can also be referred to as a "template". The template serves as a pattern for the synthesis of a complementary polynucleotide. The target sequence can be derived from any living or once-living organism (including but not limited to prokaryotes, eukaryotes, plants, animals, and viruses), as well as synthetic target sequences and / or recombinant target sequences, or combinations thereof.

[0014] As used herein, "primer" refers to an oligonucleotide or polynucleotide (whether naturally occurring or synthetically produced) that, when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (i.e., in the presence of nucleotides and an agent for polymerization, and at a suitable temperature and in a suitable buffer), is capable of serving as a starting point for synthesis. Such conditions include the presence of four or more different deoxyribonucleoside triphosphates and a polymerization-inducing agent (such as DNA polymerase, and / or RNA polymerase, and / or reverse transcriptase), in a suitable buffer (a "buffer" includes substituents that are cofactors, or that affect pH, ionic strength, etc.), and at a suitable temperature. The primers herein are selected to be substantially complementary to the strand of each specific sequence to be extended. This means that the primers must have sufficient complementarity to hybridize to their respective strands. Non-complementary nucleotides may be present.

[0015] As used herein, the term "complementary" refers to the ability of two nucleotide sequences to specifically bind to each other, either randomly or by design, through hydrogen bonding of their purine and / or pyrimidine bases to form a duplex nucleic acid complex according to the customary Watson-Crick rules. It can also refer to the ability of nucleotide sequences (which may include modified nucleotides, or analogs of deoxyribonucleotides and ribonucleotides, or combinations thereof) to specifically bind to each other according to rules other than the customary Watson-Crick rules to form alternative nucleic acid duplex structures.

[0016] As used herein, the terms "hybridization" and "annealing" are interchangeable and refer to the process by which two nucleotide sequences that are complementary to each other bind together to form a duplex sequence or segment.

[0017] The terms "duplex" and "double-stranded" are interchangeable and refer to a structure formed as a result of hybridization between two complementary nucleic acid sequences. Such a duplex can be formed by the complementary binding of two DNA segments to each other, two RNA segments to each other, or a DNA segment to an RNA segment, or by the complementary binding of two segments consisting of a mixture of RNA and DNA, the latter structure also being referred to as a hybrid duplex. Either or both members of such a duplex can contain modified nucleotides and / or nucleotide analogs as well as nucleoside analogs. As disclosed herein, such a duplex is formed as a result of the binding of one or more blocking oligonucleotides to a sample sequence.

[0018] As used herein, the terms "wild-type nucleic acid", "normal nucleic acid", "nucleic acid having normal nucleotides", "wild-type DNA", and "wild-type template" are used interchangeably and refer to a polynucleotide having a nucleotide sequence that is considered normal or unaltered.

[0019] As used herein, the terms "mutant polynucleotide", "mutant nucleic acid", "variant nucleic acid", and "nucleic acid having variant nucleotides" refer to a polynucleotide having a nucleotide sequence that is different from the nucleotide sequence of the corresponding wild-type polynucleotide. The nucleotide sequence difference of the mutant polynucleotide compared to the wild-type polynucleotide is referred to as a nucleotide "mutation", "variant nucleotide", or "variation". The term "one or more variant nucleotides" also refers to one or more nucleotide substitutions, one or more nucleotide deletions, one or more nucleotide insertions, one or more nucleotide methylations, and / or one or more nucleotide modifications.

[0020] As used herein, "amplification" refers to using any amplification procedure to increase the concentration or copy number of a specific nucleic acid sequence within a nucleic acid sequence mixture. Amplification can be linear amplification or exponential amplification.

[0021] The term "amplification product" or "amplicon" refers to a fragment of DNA or RNA amplified by a polymerase using primers in an amplification method.

[0022] The term "primer extension product" refers to a fragment of DNA or RNA extended by a polymerase using one or a pair of primers in a reaction, which can involve one pass extension (e.g., first-strand cDNA synthesis), or multiple extension cycles (which can be linear amplification, or cDNA synthesis), or many extension cycles (which can be exponential amplification (such as PCR)).

[0023] The term "compatible" refers to a primer sequence or a portion of a primer sequence that is the same as, or substantially the same as, complementary, substantially complementary, or similar to the PCR primer sequence / sequencing primer sequence used in a massively parallel sequencing platform.

[0024] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology, which are within the skill of those in the art. In one aspect, the present invention provides a removable template oligonucleotide (RTO) for generating a polynucleotide library, the removable template oligonucleotide (RTO) comprising:

[0025] (a) a 3' random sequence;

[0026] (b) a blocker moiety attached to the 3' end, the blocker moiety rendering the RTO non-extendable;

[0027] (c) Universal sequence from 5’ to the random sequence; and

[0028] (b) Nucleotide sequence / modification (NSM) recognizable by an agent,

[0029] wherein the RTO is used as a template, not incorporated into the reaction product, and is destroyed / removed after the reaction,

[0030] wherein the nucleotide sequence / modification facilitates the removal of the RTO.

[0031] In one embodiment, the 3’ blocker moiety is the same as the NSM. The 3’ blocker moiety and the NSM can be biotin, and the agent is avidin or streptavidin. For example, through digestion to generate strand cleavage or through affinity purification, the NSM allows the removal of the RTO. The RTO molecule includes one or more NSM moieties, and one or more NSM moieties render the RTO degradable or non-interfering and non-competitive in one or more reactions after the extension reaction, wherein the moieties are recognizable by an agent that facilitates the digestion / removal of the RTO.

[0032] The removable template oligonucleotide (RTO) can alternatively be referred to as an adaptor template oligonucleotide (ATO). The terms RTO and ATO can be used interchangeably and refer to an oligonucleotide that serves as a template for extending the end of a target in order to modify the target by adding a known sequence through polymerase extension. The term adaptor template oligonucleotide (ATO) or removable template oligonucleotide (RTO) refers to a population of sequences that have a common (universal) region between each member of the population and a randomly variable sequence (referred to as N, where N is each of the four bases). Due to the random nature of the 3’ end, the population of adaptor template oligonucleotides (ATO) refers to a plurality of different sequences.

[0033] The present disclosure provides an adaptor template oligonucleotide (ATO) for extending a polynucleotide, the adaptor template oligonucleotide (ATO) comprising:

[0034] (a) A 3’ random sequence;

[0035] (b) A blocker attached to the 3’ end, the blocker rendering the ATO non-extendable; and

[0036] (c) A universal sequence from 5’ to the random sequence;

[0037] wherein the ATO is used as a template to direct an extension reaction by a polymerase.

[0038] The term universal sequence refers to the entirety of the ATO from its 5’ end to the first nucleotide of the random sequence or the target-specific sequence, and is so named because it is the “universal sequence” present on all ATOs.

[0039] The ATO molecule may also include one or more moieties that render the ATO degradable or non-interfering and non-competitive in one or more reactions after the extension reaction, wherein the moieties are recognizable by an agent that facilitates digestion / removal of the ATO.

[0040] The present disclosure provides an adaptor template oligonucleotide (ATO) for extending a polynucleotide, the adaptor template oligonucleotide (ATO) comprising:

[0041] (a) a 3' random sequence of 3 to 36 'N' bases;

[0042] (b) a 3' end with a blocker that renders the ATO non-extendable;

[0043] (c) a universal sequence from 5' to the random sequence;

[0044] (d) an optional modified nucleotide or linkage that renders the ATO resistant to 3'-exonuclease cleavage; and

[0045] (e) a moiety that renders the ATO degradable.

[0046] In one embodiment, the moiety is a uracil nucleotide, wherein the agent is dU-glycosylase, or dU-glycosylase and apurinic / apyrimidinic endonuclease, which are capable of digesting / removing the ATO after the first extension reaction.

[0047] In another embodiment, the moiety is a ribonucleotide, wherein the ribonucleotide is incorporated into the ATO during oligonucleotide synthesis in place of any nucleotide or all nucleotides; wherein the agent is ribonuclease, which is capable of digesting / removing the ATO after the first extension reaction.

[0048] The ATO can be an RNA oligonucleotide, or a DNA oligonucleotide, or a combination of a DNA oligonucleotide and an RNA oligonucleotide.

[0049] The ATO can be a combination of one or more different ATos. The combination of ATos can differ in sequence. The combined ATos can differ in design. The combined ATos can differ in function. As used herein, the term "ATO" can refer to a combination of one or more ATos, any sequence of an ATO, any design of any combination of ATos having the design features of an ATO, any combination of any combination of ATos having a function. When using a combination of one or more ATos, there may be variations within the universal sequence of the ATos used, and in these cases the term universal sequence is also used.

[0050] In another embodiment, the degradable portion is a sequence recognizable by a restriction enzyme, and the agent is a restriction enzyme.

[0051] The universal sequence may include an RNA polymerase promoter sequence. Any RNA polymerase (such as T7 RNA polymerase, or T3 RNA polymerase, or SP6 RNA polymerase, or a combination thereof) can be used. The universal sequence may include an RNA polymerase promoter sequence and / or a priming site located 3' of the promoter sequence. The priming site provides a primer binding sequence for subsequent amplification.

[0052] The universal sequence can be double-stranded or partially double-stranded. Protecting the universal sequence as a double-stranded region prevents hybridization with the target polynucleotide and the random 3' end of the ATO. In one embodiment, the ATO includes a 5' stem portion sequence that is complementary or partially complementary to all or a portion of the universal sequence, which can form a stem-loop structure or a broken stem-loop structure. The ATO molecule can include, in 5' to 3' order: a 5' stem portion, an RNA polymerase sequence, a priming site sequence, and a 3' random / degenerate sequence, a mixture of random / degenerate sequences and a sequence-specific sequence, or a sequence-specific sequence. The RNA polymerase sequence can be located in the loop portion, or in a portion of the stem and a portion of the loop, or in the stem. The loop portion can include a non-replicable linkage. Alternatively, the loop portion may not include a non-replicable linkage. Alternatively, the stem portion can include a non-replicable linkage. If the 5' of the stem portion includes an additional sequence, there may be a non-replicable linkage between the stem portion and the additional sequence. Alternatively, the stem portion may not include a non-replicable linkage. In another embodiment, the 5' stem portion includes a non-replicable linkage. The non-replicable linkage can be selected from (but not limited to) the following groups: C3 interarm phosphoramidite, or triethylene glycol interarm, or 18-atom hexaethylene glycol interarm, or 1',2'-dideoxyribose (d-spacer (dSpacer)).

[0053] The double-stranded stem portion can include one or more non-complementary regions, where one or more non-complementary regions in the universal sequence strand include one or more random degenerate sequences, or specifically designed mismatches. The stem portion can form two or more broken segments separated by one or more non-replicable linkages. The stem portion can form two or more broken segments separated by one or more mismatched base pairs.

[0054] In another embodiment, the ATO includes an upper single strand that is complementary or partially complementary to all or a portion of the lower strand containing the universal sequence. The 5' end of the upper single strand can include a phosphate group.

[0055] The ATO may include one or more affinity binding moieties attached at any position of the ATO. The affinity binding moiety may be biotin.

[0056] The general sequence of the ATO may include one or more random sequences or one or more sequence-specific sequences as additional unique identifier (UID) sequences. One or more UID sequences may be located within the stem of the ATO. One or more UID sequences may be located within the loop of the ATO. One or more UID sequences may be located within both the stem and the loop of the ATO, and two or more UIDs may be present within both the stem and / or the loop of the ATO.

[0057] The ATO sequence may include at any position one or more non-canonical nucleotides (non-dA, non-dG, non-dT, non-dC) that are either naturally occurring nucleotides or artificial nucleotides. One or more non-canonical nucleotides may be universal nucleotides. The non-canonical nucleotides may include inosine bases. The 3' random sequence may partially or completely include non-canonical nucleotides. The general sequence may partially or completely include non-canonical nucleotides.

[0058] The 3' end of the ATO may include one or more modified nucleotides or linkages that render the ATO resistant to the 3'-exonuclease activity of DNA polymerase. The modified linkage contains a phosphorothioate bond.

[0059] The ATO may also include a specific sequence 3' of the random sequence, wherein the specific sequence is capable of hybridizing to a specific position of a target polynucleotide or to a specific sequence that is not designed for a specific target, and a portion of the 3' random / degenerate sequence serves as a template on which the polynucleotide is extended by a polymerase. The 3' random sequence of the ATO may be divided into two or more portions by a specifically designed specific sequence, and a portion of the 3' random / degenerate / target-specific sequence serves as a template on which the target polynucleotide is extended by a polymerase.

[0060] The present disclosure also provides a composition, which comprises at least one nucleic acid polymerase and one or more adapter-template oligonucleotides (ATO) from any combination or mixture of the described adapter-template oligonucleotides (ATO). The nucleic acid polymerase can be a DNA polymerase, or an RNA polymerase, or a reverse transcriptase, or a mixture of any combination of DNA polymerase, RNA polymerase and reverse transcriptase. Preferably, the polymerase has strand displacement activity. The polymerase can have 3'-to-5' exonuclease activity. The polymerase can be a mixture of one or more different DNA polymerases, or a mixture of one or more RNA polymerases, or a combination of one or more DNA polymerases or RNA polymerases. The polymerase is a template-dependent polymerase, rather than a non-template-dependent polymerase.

[0061] The present disclosure provides a method for extending a target polynucleotide, the method comprising incubating the target polynucleotide with the composition described herein under conditions sufficient to allow the 3' end of the target polynucleotide to be extended using one or more ATO as a template (referred to as the "ATO reaction"), wherein one or more ATO can hybridize to any position or a specific position of the target polynucleotide. On the other hand, the method further comprises degrading one or more ATO after the extension of the target polynucleotide.

[0062] The first ATO reaction can be an extension reaction, wherein the target polynucleotide is extended as a primer and one or more ATO are used as a template. Alternatively, the first ATO reaction can be an extension-ligation reaction (nick filling reaction), wherein the target polynucleotide is extended and ligated to the 5' stem portion or upper strand of one or more ATO. Alternatively, the first reaction can be only a ligation reaction, wherein the target polynucleotide hybridizes to the 3' random sequence portion of one or more ATO and is directly ligated to the 5' portion or upper strand of one or more ATO.

[0063] The ATO comprises one or more moieties that render the ATO degradable, wherein the one or more moieties can be recognized by an agent that facilitates the digestion / removal of the ATO. Alternatively, the ATO comprises one or more moieties that render the ATO non-interfering and / or non-competitive in the reaction after the extension reaction.

[0064] In some embodiments, the ATO molecule includes a dU base and can be degraded by incubating with dU-glycosylase (which creates an abasic site) and then incubating at a temperature above 80 °C (introducing a break within the abasic site), or by incubating with a mixture of dU-glycosylase and apurinic / apyrimidinic endonuclease. The provided methods and compositions include ATO having a dU base and incubating with dU-glycosylase to degrade the ATO molecule, or incubating with dU-glycosylase and then incubating at a temperature above 80 °C to degrade the ATO molecule, or incubating ATO with a mixture of dU-glycosylase and apurinic / apyrimidinic endonuclease. In a further aspect, the ATO includes ribonucleotides and can be degraded by ribonuclease under conditions that satisfy ribonuclease activity. In a related aspect, the ribonuclease is selected from the group consisting of ribonuclease H (RNase H), ribonuclease HII (RNase HII), ribonuclease A (RNase A), and ribonuclease T1 (RNase T1).

[0065] In other embodiments, the moiety can be a modified nucleotide or nucleotide analogue. The modified nucleotide can be an atypical nucleotide (non-dA, non-dG, non-dT, non-dC), and the atypical nucleotide is a naturally occurring nucleotide or an artificial nucleotide. The atypical nucleotide can be a universal nucleotide. The atypical nucleotide can include an inosine base, where inosine is used in place of the guanine position in the ATO sequence, where deoxyinosine preferentially directs the incorporation of dC by DNA polymerase into the growing nascent strand, and where it is expected and understood that other residues may not be incorporated frequently.

[0066] The modified nucleotide / analogue can be present in the 5' universal sequence (5' universal portion) or in the 3' random / degenerate portion, or the modified nucleotide / analogue can be present in both the 5' universal sequence and the 3' random sequence (3' random portion).

[0067] The moiety can make the base-pairing bonds weaker or can be a modification that is degradable / removable after the first extension ATO reaction. Due to the weak binding, ATO cannot interfere with the normal primer binding to the same template in subsequent reactions or cannot compete with the normal primer for binding to the same template. In addition, due to the digestion of the agent, ATO cannot interfere with subsequent reactions.

[0068] Modifications that provide intermolecular hydrogen bonds with base-pairing weaker than standard canonical base-pairing can be any naturally occurring nucleotide or artificial nucleotide analogue. A preferred naturally occurring atypical nucleotide used in ATO is inosine, which can be used in place of dG to pair with dC, but is weaker than dG:dC.

[0069] Nucleic acid analogs are compounds that are similar (structurally) to naturally occurring RNA and DNA. Nucleic acids are chains of nucleotides, which consist of three parts: a phosphate backbone, a pentose sugar, ribose or deoxyribose, and one of four nucleobases. Analogs can have any of these alterations. Generally, among other things, analog nucleobases confer different base pairing properties and base stacking properties. Examples include universal bases that can pair with all four canonical bases and phospho-sugar backbone analogs that affect strand properties. The 3'-random portion of an ATO that acts as a template and hybridizes to any position of a polynucleotide can include one or more universal bases.

[0070] A universal base is an analog compound that can interact in a weak base pair in place of any of the four DNA bases. Commonly used universal bases can be 3-nitropyrrole, 5-nitroindole, or 2'-deoxyinosine. Inosine shows a slight bias in nucleotide hybridization, where dI:dC is preferred over other pairings.

[0071] Canonical bases can have a carbonyl or amine group on the carbon around the nitrogen atom farthest from the glycosidic bond, which allows them to perform base pairing (Watson-Crick base pairing) via hydrogen bonds (amine with keto, purine with pyrimidine).

[0072] Universal bases can pair with any other base indifferently, but generally greatly reduce the melting temperature of the sequence; examples include 2'-deoxyinosine (inosine monophosphate) and its derivatives, nitroazole analogs, and hydrophobic aromatic non-hydrogen bonding bases (strong stacking effect). The present disclosure has explored this property to make ATOs containing inosine have a lower Tm than normal oligonucleotides, such that the ATO does not interfere with subsequence reactions where the annealing temperature is higher than the Tm of the universal portion of the ATO.

[0073] Deoxyinosine (a naturally occurring base) is considered the first "universal" base, meaning that it can perform base pairing with the other natural bases A, C, G, and T. In fact, studies on deoxyinosine have shown that although it does not self-aggregate like deoxyguanosine, it acts as a specific analog of deoxyguanosine.

[0074] The weakly paired nucleotides in the common part of ATO include modified nucleotides, nucleotide analogs, or / and modified linkages, which can be (but are not limited to) inosine, dinosine, or phosphorothioate bonds, methylphosphonate linkages. Any modification of nucleotides or / and linkages can be used as long as it provides weak pairing compared to natural nucleotides or linkages. The weak pairing in the common part of ATO can also be related to the primers used in subsequent reactions. If the weakly paired nucleotides are normal natural nucleotides, the primers used in subsequent reactions can include strongly paired nucleotides, which are modified nucleotides or / and modified linkages. Modified nucleotides providing strong pairing ability or weak pairing ability can include (but are not limited to) LNA, O-me RNA, 2-amino-dA, 2-thiol-dT, 2-aminopurine, 2’FluoRNA base, AP-dC, C5-propargyl analogs and methyl analogs of dC and dT, deoxyinosine, deoxyuridine, or superbase nucleotides (Epoch Bioscience).

[0075] ATO can include a 5’ stem portion sequence that is complementary to a part of the common sequence and is capable of forming a stem-loop structure,

[0076] wherein the loop portion can include a non-replicable linkage.

[0077] The non-replicable linkage can be selected from (but is not limited to) the following groups: C3 spacer phosphoramidite, or triethylene glycol spacer, or 18-atom hexaethylene glycol spacer, or 1’,2’-dideoxyribose (d spacer).

[0078] The 5' stem portion sequence can include a sequence that is complementary or substantially complementary to a portion of the 5' universal sequence (preferably the portion adjacent to the random sequence). In one embodiment, the loop portion can include a non-replicable linkage, wherein the polymerase in the ATO reaction can possess strand displacement activity or 5' to 3' exonuclease activity. Desirably, the target polynucleotide hybridizes only to the 3' random / degenerate / target-specific sequence of the ATO and not to the 5' universal sequence. This is achieved by a stem-loop structure that prevents the target polynucleotide from hybridizing to the universal sequence of the ATO in the ATO reaction. In the ATO reaction, the target polynucleotide hybridizes to the 3' random sequence and is extended by a mixture of DNA polymerase, RNA polymerase, reverse transcriptase, or any combination thereof. The extended strand displaces the stem portion and terminates at the non-replicable linkage. Additionally, in the reaction following the first ATO reaction, the stem-loop structure prevents ATO competition for binding to the modified target polynucleotide, such that any added primer can effectively bind to the modified target polynucleotide for amplification. In this embodiment, the stem portion and the non-replicable linkage serve as the portions that render the ATO non-interfering and non-competitive in the reaction following the ATO reaction. The ATO can include or can not include other modifications (such as one or more uracil nucleotides, or ribonucleotides) that can be digested after the ATO reaction.

[0079] In another embodiment, the loop portion of the ATO can include nucleotides (such as one or more uracil nucleotides, or ribonucleotides) that can be digested. In the ATO reaction, the target polynucleotide hybridizes to the 3' random sequence and is extended by DNA polymerase. The extended strand contacts the 5' end of the stem strand containing a 5' phosphate group and is ligated by DNA ligase. The reaction includes a DNA polymerase with nick filling activity (such as polymerase 1 or the Klenow fragment) and DNA ligase. The ATO molecule can include multiple uracil nucleotides or ribonucleotides. After the ATO reaction, the ATO molecule can be digested.

[0080] In yet another embodiment, the loop portion of the ATO can include nucleotides (such as uracil nucleotides or ribonucleotides) that can be digested. In the first reaction, the target polynucleotide hybridizes to the 3' random sequence adjacent to the 5' end of the stem strand and is ligated to the 5' end of the stem strand by DNA ligase. The 5' universal sequence portion can include multiple uracil nucleotides or ribonucleotides. After the first reaction, the 5' universal sequence portion can be digested.

[0081] In yet another embodiment, the ATO comprises an upper single strand that is complementary or substantially complementary to a part or all of the universal sequence (which is capable of forming a partial double-stranded structure). Desirably, the target polynucleotide hybridizes only to the 3' random sequence of the ATO and not to the 5' universal sequence. This is achieved by the double-stranded structure provided by the upper strand, which prevents the target polynucleotide from hybridizing to the universal sequence of the ATO in the first ATO reaction. The ATO may include other modifications (such as uracil nucleotides or ribonucleotides) that can be digested after the first reaction. In the first ATO reaction, the target polynucleotide hybridizes to the 3' random sequence and is extended by DNA polymerase. The extended strand may displace the upper strand. Alternatively, the extended strand contacts the 5' end of the upper strand having a 5' phosphate group at the 5' end and is then ligated by DNA ligase. The reaction includes a DNA polymerase with nick filling activity (such as polymerase 1 or Klenow fragment) and DNA ligase. The 5' universal sequence portion may include a plurality of uracil nucleotides or ribonucleotides. After the first reaction, the 5' universal sequence portion may be digested. On the other hand, in the first reaction, the target polynucleotide hybridizes to the 3' random sequence adjacent to the 5' end of the upper strand and is ligated to the 5' end of the upper strand by DNA ligase. The 5' universal sequence portion and the 3' random portion may include a plurality of uracil nucleotides. After the first reaction, the ATO molecule may be digested.

[0082] In an embodiment where ligation is used, the 5' end of the upper single strand may include a phosphate group and the 3' end of the upper single strand may include biotin. In an embodiment where extension is used without ligation, the 5' end of the upper single strand does not include a phosphate group, the 3' end of the upper single strand does not include biotin, but the upper single strand may include nucleotides (such as uracil nucleotides) that can be digested.

[0083] The random sequence portion can have any length, with the length ranging from 3 to 48 nucleotides (preferably ranging from 3 to 36 nucleotides, or most preferably ranging from 12 to 30 nucleotides). Specifically, the random sequence portion has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more nucleotides. The random sequence can include completely random nucleotides, where any one of the four nucleotides can be present at any position. Alternatively, degenerate nucleotides can be present at some positions. The random sequence portion can include some specific (non-random) nucleotides at some specific positions. For example, the 3'-terminal nucleotide can be a specific nucleotide (such as a T residue, or an A residue, or a G residue, or a C residue). The specific 3'-terminal nucleotide is selected for convenient and low-cost attachment of modifications (such as biotin, a spacer arm, or a phosphate blocker). The random sequence can include degenerate nucleotides or semi-degenerate nucleotides or completely random nucleotides as well as specific nucleotides, with the presence of random nucleotides being dominant. The random sequence can also include naturally occurring or artificially modified nucleotides. These modified nucleotides can be universal bases as described above. The random sequence can have sequence bias, such as making the entire sequence rich in C and G residues, meaning that more than 50% of the nucleotides are composed of C and G, or making the entire sequence rich in A and T residues, meaning that more than 50% of the nucleotides are composed of A and T. The random sequence can be partially or completely replaced by a target-specific sequence.

[0084] ATO hybridizes to the target polynucleotide through the random sequence under non-stringent conditions, where the 3'-end of the polynucleotide hybridizes to the random sequence, and in one embodiment, extension is carried out using ATO as a template.

[0085] As described, the random sequence provides the function of serving as a template for hybridization to the target polynucleotide, and ATO is used as a template to direct the extension of the target polynucleotide. In addition, the random sequence also provides a second function as a UID (unique identifier, molecular barcode) in next-generation sequencing.

[0086] In another embodiment, the ATO molecule can include a 3' appended specific sequence located 3' of the random sequence portion. The 3' appended specific sequence can include a sequence capable of hybridizing to a specific location of the target polynucleotide. The 3' appended specific sequence can include a restriction enzyme recognition sequence, which, when hybridized to the target sequence, can cause a nick in the target polynucleotide by an enzyme.

[0087] The 3' end of the ATO can be attached with a blocking group that renders the ATO non-extendable. Any blocking group can be used. Generally, the 3' end of the ATO will be "blocked" to prohibit the ATO from acting as a primer, unless extension of the ATO is desired in the design (in which case, the 3' will not be blocked and extension will be allowed). "Blocking" can be achieved by attaching a chemical moiety (such as biotin or a phosphate group) to the 3'-hydroxyl of the last nucleotide, which, depending on the group selected, can serve a dual purpose by also acting as an affinity capture moiety for subsequent removal or capture of the ATO after the ATO reaction. Blocking can also be achieved by removing the 3'OH or by using a nucleotide lacking a 3'OH (such as a dideoxynucleotide). The blocking group can be selected from the group consisting of: at least one ribonucleotide, at least one deoxynucleotide, a C3 spacer arm, a phosphate, a dideoxynucleotide, an amino group, and a reverse deoxythymidine.

[0088] The ATO reaction preferably contains a polymerase having 3' to 5' exonuclease activity, and thus the ATO molecule preferably includes a modified nucleotide or linkage at the 3' end that prevents digestion by the polymerase. Any resistant modification can be used. One example is that the 3' end includes a modified linkage between the final nucleotides (preferably the last two nucleotides) at this position, such that it includes a resistant moiety (such as a phosphorothioate), rather than a conventional phosphodiester.

[0089] In one embodiment, the 5' universal sequence portion is part of a sequence that provides the function of serving as a template for the extension of the target polynucleotide, the product of which is equivalent to the operation of adding an adaptor for sequencing library preparation via ligation. However, the extended sequence (similar to an adaptor) is synthesized in a new way and is not attached with any added oligonucleotide. The 5' universal sequence portion includes a primer sequence or a primer-binding sequence that is compatible with next-generation (or third-generation) sequencing (NGS) or other massively parallel sequencing. For example, the 5' universal sequence portion can include a sequencing primer sequence for the Illumina platform and / or an anchored primer sequence for the Illumina platform.

[0090] The 5'-universal sequence portion can form a stem-loop structure or a hairpin structure, where the stem portion end is close to the 5'-end of the random sequence portion. The 5'-end of the stem can form a 5'-overhang. The stem portion can have any length. The stem portion can have any length in the range from 3 to 30 nucleotides (preferably 4 to 24 nucleotides). The stem portion can be fully double-stranded or preferably non-fully double-stranded. The stem portion can include one or more unpaired regions. One or more unpaired regions can include a random sequence that functions as a UID (molecular barcode) in NGS. The stem portion can include a sequence that can be recognized and cleaved by an enzyme (e.g., a restriction enzyme site). Stem formation can prevent hybridization of the universal sequence with the target polynucleotide. The loop portion can have any length. The loop portion can have any length in the range from 0 to 36 nucleotides (preferably 1 to 30 nucleotides). The loop can partially or fully include nucleotide analogs or other chemical linkages that are non-replicable by polymerase, such as abasic sites, hexanediol (HEG) monomers, 18-atom hexaethylene glycol spacers, or 1',2'-dideoxyribose (d-spacers). The non-replicable linker prevents polymerase-mediated extension on the 5'-portion of the ATO.

[0091] The ATO molecule includes nucleotides selected from the group consisting of: 2'-deoxythymidine 5'-monophosphate (dTMP), 2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-monophosphate (dAMP), 2'-deoxycytidine 5'-monophosphate (dCMP), 2'-deoxyuridine 5'-monophosphate (dUMP), thymidine monophosphate (TMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), base analogs, and combinations thereof. It is also contemplated that the ATO includes modified nucleotides or linkage modifications as defined herein.

[0092] A modified oligonucleotide or a modified polynucleotide may include one or more sugars and / or one or more internucleotide linkages of the nucleotide units in the oligonucleotide or polynucleotide and is replaced by a "non-naturally occurring" group. In one aspect, this embodiment contemplates peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of the polynucleotide is replaced by an amide-containing backbone. The modified oligonucleotide / polynucleotide backbone may contain phosphorus atoms and may include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates. The modified oligonucleotide or modified polynucleotide may also contain one or more substituted sugar moieties. Further modifications include those that extend the genetic code (such as, but not limited to, Iso-dC and Iso-dG). Iso-dC and Iso-dG are chemical variants of cytosine and guanine, respectively. Iso-dC will hydrogen bond with Iso-dG but not with dG. Similarly, Iso-dG will base pair with Iso-dC but not with dC. In one aspect, the modification of the sugar includes locked nucleic acid (LNA).

[0093] The target polynucleotide may be fragmented naturally or artificially, randomly or specifically, or in a combination of random and specific manners, and after hybridization with a random sequence or a target-specific sequence of ATO, the 3' end of the fragmented target polynucleotide is extended. The combination of the random 3' end sequence of the target polynucleotide and the extended portion on the random template provides a unique identifier (UID, molecular barcode) sequence, which can be used to assemble sequencing reads into families. In addition, ATO may include one or more additional UIDs located in the universal sequence. The additional UID may be located in a loop, near the stem portion and 5' of the random sequence portion. The additional UID may be located in the stem, at any position between the 3' random sequence and the loop. The additional UID may have any length. The additional UID may have any length, which may range from 2 to 48 nucleotides (preferably ranging from 3 to 36 nucleotides). The additional UID may include completely random nucleotides, where any one of the four nucleotides may be present at any position. Alternatively, degenerate nucleotides may be present at some positions.

[0094] The ATO molecule may include atypical nucleotides, analogs or modifications that can be recognized and cleaved by an agent. The atypical nucleotides are selected from the group consisting of dUMP, dIMP, and 5-OH-Me-dCMP. The agent capable of cleaving the base portion of the atypical nucleotide is an N-glycosylase. The N-glycosylases are selected from the group consisting of uracil N-glycosylase (UNG), hypoxanthine-N-glycosylase, and hydroxymethylcytosine-N-glycosylase. When the atypical nucleotide is dUMP, the enzyme capable of cleaving the base portion of the atypical nucleotide is UNG. When the atypical nucleotide is dUMP, the enzyme capable of cleaving the base portion of the atypical nucleotide is UNG, and the phosphodiester backbone is cleaved with DMED. In one embodiment, uracil nucleotides are incorporated into the ATO in place of thymine nucleotides during oligonucleotide synthesis. The ATO comprising atypical nucleotides can be synthesized in the presence of two or more different atypical nucleotides, thereby synthesizing an ATO comprising two or more different atypical nucleotides. When the ATO comprising atypical nucleotides is synthesized in the presence of three canonical nucleotides and one atypical nucleotide, or all four canonical nucleotides and one atypical nucleotide, the atypical nucleotide is provided in a proportion suitable for degrading the ATO after the ATO reaction. Generally, base excision repair enzymes can be selected from the group consisting of DNA glycosylases, AP endonucleases, and deoxyphosphodiesterases. Preferably, the DNA glycosylases can be selected from the group consisting of uracil-DNA glycosylase, 3-methyladenine DNA glycosylase, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, and thymine mismatch-DNA glycosylase. More preferably, the DNA glycosylase is uracil-DNA glycosylase. Uracil-DNA glycosylase (UDG) or uracil-N-glycosylase (UNG) is an enzyme that catalyzes the release of free uracil from single-stranded DNA and double-stranded DNA of more than 6 base pairs.

[0095] In one embodiment, the ATO molecule may include ribonucleotides, wherein ribonucleotides are incorporated into the ATO in place of any nucleotide or all nucleotides during oligonucleotide synthesis; wherein the agent is a ribonuclease that is capable of degrading / removing the ATO after the ATO reaction. The ATO can be RNA throughout its length or can be partially composed of RNA. Any portion of the ATO can be RNA, preferably the universal sequence portion is RNA.

[0096] In another embodiment, the ATO sequence may include a restriction enzyme recognition sequence located in the universal sequence or in the 3' appended specific sequence; wherein the agent is a restriction enzyme that is capable of degrading / removing the ATO after the ATO reaction. The restriction enzyme recognition sequence can be located in the stem portion of the ATO and can be cleaved by the restriction enzyme.

[0097] In another embodiment, the ATO molecule can include an affinity binding moiety attached at any position of the ATO; wherein the agent is a protein or an antibody that is capable of removing the ATO after the ATO reaction. For example, the affinity binding moiety is biotin; wherein the agent is avidin or streptavidin. In certain embodiments, the ATO can include one or more moieties incorporated into the 5' end or 3' end or any internal position of the ATO, and incorporating one or more moieties into the 5' end or 3' end or any internal position of the ATO allows for affinity removal of the ATO from the reaction mixture after the ATO reaction. Preferred affinity moieties are those that can specifically interact with a cognate ligand. For example, the affinity moiety can include biotin, digoxigenin, etc. Other examples of capture groups include ligands, receptors, antibodies, haptens, enzymes, chemical groups recognizable by an antibody, or aptamers. The affinity moiety can be immobilized on any desired substrate / solid-phase support. Examples of desired substrates include, for example, particles, beads, magnetic beads, optically-trapping beads, microtiter plates, slides, paper, test strips, gels, other matrices, nitrocellulose, or nylon. For example, when the capture moiety is biotin, the substrate can include streptavidin. In some cases, the solid-phase support is a bead. Examples of beads include (but are not limited to) streptavidin beads, agarose beads, magnetic beads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo-dT-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, and anti-fluorochrome microbeads.

[0098] While a portion of the random sequence of the RTO serves as a unique identifier (UID) sequence, the RTO can include additional UIDs located in the common sequence portion.

[0099] The present disclosure provides methods for generating a polynucleotide library, the methods comprising:

[0100] (i) using a target polynucleotide from a sample as a primer and using a removable template oligonucleotide (RTO) of any one of the above RTOs as a template to generate a modified target polynucleotide;

[0101] (ii) removing the RTO; and

[0102] (iii) using a first primer to generate a first complementary sequence (CS) of the modified target polynucleotide, the first primer including a common sequence,

[0103] wherein the generating operation includes extending a primer hybridized to a template by polymerase.

[0104] Accordingly, the present disclosure provides methods for extending a population of target polynucleotides having single-stranded 3' ends, the methods comprising:

[0105] (i) incubating a target polynucleotide with an adaptor template oligonucleotide (ATO), the adaptor template oligonucleotide (ATO) having:

[0106] (a) a 3’ random sequence;

[0107] (b) a 3’ end with a blocker that renders the ATO non-extendable; and

[0108] (c) a universal sequence from 5’ to the random sequence,

[0109] wherein the target polynucleotide hybridizes to the 3’ random sequence of the ATO;

[0110] (ii) performing polymerase extension on the target polynucleotide using the ATO as a template to generate an extended target polynucleotide having a 3’ universal sequence.

[0111] The method may further comprise (iii) generating a first complementary sequence (CS) of the modified target polynucleotide, wherein generating the first CS comprises performing polymerase extension from the 3’ universal sequence using the modified target polynucleotide as a template.

[0112] The present disclosure provides a method for extending a polynucleotide, the method comprising:

[0113] (i) generating a modified target polynucleotide by incubating the target polynucleotide with a composition as described above, wherein in an enzymatic first ATO reaction, the 3’ end of the target polynucleotide hybridizes to the 3’ random sequence of an adaptor template oligonucleotide (first ATO), wherein the 3’ end of the target polynucleotide is extended using the ATO as a template, and wherein if a 3’ overhang is present, the 3’ end of the target polynucleotide is trimmed before extension occurs.

[0114] In one embodiment, after extension of the 3’ end on the ATO, the modified polynucleotide is optionally incubated with a single-stranded DNA ligase to effect cyclization of the single-stranded modified polynucleotide.

[0115] The method further comprises (ii) generating a first complementary sequence (CS) of the modified target polynucleotide.

[0116] Step (ii) may comprise using the modified target polynucleotide as a template for one round of amplification, as a template for successive multiple rounds of amplification, as a template for successive multiple rounds of amplification separated by a purification step.

[0117] In one embodiment, the operation of generating the first CS includes using a first primer and performing extension using a modified target polynucleotide as a template, wherein the first primer hybridizes to the modified target polynucleotide and is extended by a polymerase. The first primer anneals to the 3'-extended universal region of the modified target polynucleotide. The first primer may include additional sequences compatible with the NGS platform, such as a 5'-tail containing the necessary sequences.

[0118] In another embodiment, the operation of generating the first CS includes performing in vitro transcription using an RNA polymerase with a double-stranded RNA polymerase promoter region in the modified target polynucleotide, and the double-stranded RNA polymerase promoter region in the modified target polynucleotide is generated by extension on an ATO containing an RNA polymerase promoter. In another embodiment, a double-stranded RNA polymerase of the modified target polynucleotide is generated by hybridizing nucleotides to the modified target polynucleotide after digesting and / or removing the ATO.

[0119] In yet another embodiment, the operation of generating the first CS includes heat-denaturing the modified target polynucleotide, self-annealing the 3'-stem-loop structure of the modified target polynucleotide, and self-priming to extend to form the first CS.

[0120] In yet another embodiment, the operation of generating the first CS includes annealing a target-specific primer to the modified target polynucleotide and performing extension by a polymerase. The target-specific primer anneals to the target sequence of interest. The double-stranded ends of the first CS can be ligated to an adaptor.

[0121] The method may further include digesting the ATO before or after the operation of generating the first complementary sequence (CS) of the modified target polynucleotide.

[0122] The method may further include performing affinity capture before or after the operation of generating the first complementary sequence (CS) of the modified target polynucleotide.

[0123] In the first ATO reaction, the method may include extension and ligation, wherein a DNA polymerase extends the 3'-end of the target, and a DNA ligase ligates the extended target sequence to the 5'-stem portion of the ATO or the upper single strand of the ATO.

[0124] The method may further include generating a modified first CS by incubating the first CS with the composition as described above, wherein in the enzymatic second ATO reaction, the 3' end of the first CS hybridizes to the 3' random sequence of an adaptor template oligonucleotide (second ATO), wherein the 3' end of the first CS is extended using the ATO as a template, wherein if a 3' overhang is present, the 3' end of the first CS is trimmed before the extension occurs, and wherein the second ATO includes a different 5' universal sequence from the first ATO.

[0125] The method may further include generating a modified first CS by ligating an adaptor to the product of step (ii).

[0126] The method may further include extending a second primer hybridized to the first CS or to the modified first CS to generate a second CS, wherein the second primer includes a target-specific portion or a universal sequence, or both a 3' target-specific sequence and a 5' universal sequence.

[0127] The method may further include splitting the reaction of the modified target polynucleotide into two separate reactions, wherein each reaction contains a primer complementary to the universal region of the modified target polynucleotide, wherein one reaction includes a target-specific second primer or a library of target-specific primers complementary to the forward strand of the target sequence, and the other reaction includes a target-specific second primer or a library of target-specific primers complementary to the reverse strand of the target sequence, and wherein the forward and reverse strands of the target sequence are complementary.

[0128] In another embodiment, the library of target-specific primers may contain a mixture of primers, wherein each individual primer may target the forward or reverse strand for different targets such that the final library may target different regions of both the forward and reverse strands. The forward and reverse strands of the target sequence are complementary, and the forward target-specific primers and the reverse target-specific primers are divided into two different primer libraries, provided that if two primers targeting the forward and reverse strands can function together as primers for PCR and generate unwanted PCR products, they are not added to the same library. Additionally, as described above, all references to the "forward" library and / or the "reverse" library allow each library to contain primers targeting both the forward and reverse strands.

[0129] The method may further include hybridizing a primer complementary to the universal region of the modified target polynucleotide and then performing one or more rounds of linear amplification to generate a first complementary sequence (CS). The linear amplification reaction product is split into two separate reactions, wherein each reaction contains a primer complementary to the universal region of the modified target polynucleotide, wherein one reaction includes a target-specific second primer or a library of target-specific primers complementary to the forward strand of the target sequence, and the other reaction includes a target-specific second primer or a library of target-specific primers complementary to the reverse strand of the target sequence, and wherein the forward and reverse strands of the target sequence are complementary.

[0130] The method may further include an end repair step of the double-stranded target polynucleotide, followed by ligation of a double-stranded adaptor. Then, the ligation product is hybridized with a primer complementary to the universal region of the target polynucleotide ligation product, followed by one or more rounds of linear amplification. The linear amplification reaction product is divided into two separate reactions, each reaction containing a primer complementary to the universal region of the modified target polynucleotide, one reaction including a target-specific second primer or a library of target-specific primers complementary to the forward strand of the target sequence, and the other reaction including a target-specific second primer or a library of target-specific primers complementary to the reverse strand of the target sequence, wherein the forward and reverse strands of the target sequence are complementary.

[0131] When the target polynucleotide in the sample is double-stranded and the second primer is a target-specific primer, the reaction for target-specific amplification after the ATO reaction may include dividing the ATO reaction product or the first CS product of linear amplification into two separate reactions, the forward reaction including one or more target-specific second primers complementary to the forward strand of the target sequence, and the reverse reaction including one or more target-specific second primers complementary to the reverse strand of the target sequence, wherein the forward and reverse strands of the target sequence are complementary.

[0132] In one aspect, the operation of generating the first CS includes performing one extension or linear amplification using a first primer, which is a universal primer targeting the 3'-extended universal portion of the polynucleotide. The linear amplification may have 1-30 cycles, or 2-25 cycles, or 3-24 cycles, or 4-23 cycles, or 5-22 cycles, 6-21 cycles, or 7-20 cycles, or 8-19 cycles, or 9-18 cycles, or 10-17 cycles.

[0133] In another aspect, if the target is RNA, the operation of generating the first CS includes performing a reverse transcription reaction using a reverse transcriptase.

[0134] The method may further include performing exponential amplification using a first primer and a second primer. The first primer may be a universal primer targeting the 3'-extended universal portion of the modified target polynucleotide; the second primer may be a universal primer targeting the 3'-extended universal portion of the first CS. Alternatively, the second primer is a target-specific primer annealing to the region of interest of the first CS. The second primer may be a set of multiple primers targeting multiple regions of interest. When the second primer is a target-specific primer, after linear amplification or exponential amplification using the second primer, a nested target-specific third primer is used for further amplification.

[0135] The first primer, the second primer, or the third primer may include a sample barcode (SBC) sequence and one or more additional universal sequences necessary for compatibility with the NGS platform.

[0136] The method may include fragmenting or fragmenting / labeling the target polynucleotide prior to the first ATO reaction.

[0137] In one embodiment, the operation of fragmenting and / or labeling the target polynucleotide includes contacting a double-stranded polynucleotide with a transposase bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and a universal sequence, and wherein the transposase / transposon DNA complex binds to a target position on the double-stranded polynucleotide and cleaves the double-stranded polynucleotide into multiple double-stranded fragments, each of which has transposon DNA bound to each 5' end of the double-stranded fragment. The method further includes the operation of heat-denaturing the fragmented target polynucleotide prior to the first ATO reaction. The transposase may be Tn5 transposase. The transposon DNA may include a barcode sequence and may include a priming site. The transposon DNA includes a double-stranded 19bp Tnp binding site and an overhang, wherein the overhang may include a UID and a priming site. The transposon DNA includes a double-stranded 19bp Tnp binding site and a nucleic acid stem-loop structure. The bound transposase may be removed from the double-stranded fragment prior to the first ATO reaction. The Tn5 transposase complexes with the transposon DNA, and the Tn5 transposase / transposon DNA complex binds to a target position along the double-stranded genomic DNA, cleaving the double-stranded genomic DNA into multiple double-stranded fragments. The transposon DNA includes a double-stranded 19bp Tn5 transposase (Tnp) binding site at one end and a long single-stranded overhang that contains a barcode region, a priming site, and other sequences. Upon transposition, the Tnp and the transposon DNA bind to each other and dimerize to form a transpososome. Then, the transpososome randomly captures the target polynucleotide or otherwise binds to the target polynucleotide. Then, the transposase in the transpososome cleaves the genomic DNA, with one transposase cleaving the upper strand and one transposase cleaving the lower strand to create DNA fragments. Thus, the transposon DNA is randomly inserted into the polynucleotide, leaving 9bp gaps at both ends of the transposition / insertion site. The resulting DNA fragments have a transposon DNA Tnp binding site attached to the 5' position of the upper strand and a transposon DNA Tnp binding site attached to the 5' position of the lower strand.

[0138] In another embodiment, the operation of fragmenting the target polynucleotide includes contacting double-stranded DNA with a CRISPR / Cas9 enzyme bound to a guide RNA, wherein the CRISPR / Cas9 / guide RNA complex binds to a region of the target polynucleotide determined by the sequence of the guide RNA. The CRISPR / Cas9 / guide RNA / DNA complex results in a double-strand break as determined by the sequence targeted by the guide RNA. In another embodiment, only single-strand breaks are induced.

[0139] The fragmentation operation can include the use of targeted fragmentation with a genome editing tool. The genome editing tool can include clustered regularly interspaced short palindromic repeats and CRISPR-associated protease 9 (CRISPR / Cas9). The enzyme can belong to Class I. Class I enzymes are type I enzymes, type III enzymes, or type IV enzymes. The enzyme can belong to Class II. Class II enzymes are type II enzymes, type V enzymes, or type VI enzymes. The enzyme can include any combination of Class I and Class II enzymes. The combination can form any combination of type I enzymes, type II enzymes, type III enzymes, type IV enzymes, type V enzymes, or type VI enzymes. A library of guide RNAs is used to target multiple regions of the genome to induce DNA breaks. The DNA break can be a single-strand break or a double-strand break. The DNA break can be a combination of double-strand breaks and single-strand breaks. The library consists of any number of guide RNAs.

[0140] In another embodiment, the operation of fragmenting and labeling a target polynucleotide includes contacting a single-stranded target polynucleotide with a random primer comprising a 5' universal sequence and a 3' random sequence, and extending the random primer on the target polynucleotide to generate a 5'-labeled fragmented polynucleotide.

[0141] The target polynucleotide or the fragmented target polynucleotide includes a free 3'-hydroxyl group. The target polynucleotide can be single-stranded DNA, or single-stranded RNA, or a combination of single-stranded RNA and single-stranded DNA.

[0142] One embodiment provides a method for extending a polynucleotide, the method comprising:

[0143] Mixing a target polynucleotide with a DNA polymerase and an adaptor template oligonucleotide (ATO) comprising a 3' random sequence as described above, the adaptor template oligonucleotide (ATO) comprising a blocked and modified 3' end to be tolerant to 3'-exonuclease activity;

[0144] Incubating the mixture under conditions that promote annealing, trimming the 3' overhang (if present), and extending to generate a modified target polynucleotide; and

[0145] Optionally degrading the ATO.

[0146] One embodiment provides a method for generating a sequencing library, the method comprising:

[0147] Mixing a target polynucleotide with one or more DNA polymerases and an adaptor template oligonucleotide (ATO) comprising a 3' random sequence as described above, the adaptor template oligonucleotide (ATO) comprising a blocked and modified 3' end to be tolerant to 3'-exonuclease activity;

[0148] Incubate the mixture under annealing-promoting conditions, trim the 3' overhangs (if any), and extend to generate a modified target polynucleotide;

[0149] Optionally degrade the ATO; and

[0150] Amplify the modified target polynucleotide using one, or two, or more rounds of linear amplification and / or exponential amplification with primers compatible with the NGS platform.

[0151] The method further includes the operation of fragmenting the target polynucleotide before mixing. The target polynucleotide can be a naturally occurring fragmented polynucleotide. The naturally occurring fragmented polynucleotide can be cell-free nucleic acid in plasma. The fragmented target polynucleotide can include contacting a double-stranded polynucleotide with a transposase bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and a universal sequence, wherein the transposase / transposon DNA complex binds to a target position on the double-stranded polynucleotide and cleaves the double-stranded polynucleotide into a plurality of double-stranded fragments, wherein each double-stranded fragment has transposon DNA bound to each 5' end of the double-stranded fragment.

[0152] Also provided is a method for generating a sequencing library, the method comprising:

[0153] Adding an adaptor sequence to a single-stranded target polynucleotide according to the ATO and composition as described above by extending the single-stranded target polynucleotide on an ATO template; and amplifying the adaptor-labeled target polynucleotide using primers compatible with the NGS platform, wherein the ATO includes an adaptor sequence in the universal portion. The adaptor sequence provides a primer sequence for both amplification and sequencing of nucleic acid fragments and, in some aspects, for next-generation sequencing applications. In a further aspect, the "adaptor sequence" is used as a promoter sequence for generating an RNA molecule, wherein the promoter sequence is (e.g., but not limited to) a T7 promoter sequence or an SP6 promoter sequence.

[0154] The present disclosure provides a method for generating a polynucleotide library, the method comprising:

[0155] (i) generating a modified target polynucleotide by using a target polynucleotide from a sample, the target polynucleotide from the sample hybridizing to the 3' random sequence of an adaptor template oligonucleotide (ATO) of any one of the above-mentioned ATOs (first ATO) in an enzymatic first reaction (which adds an adaptor sequence to the 3' end of the target polynucleotide); and

[0156] (ii) generating a first complementary sequence (CS) of the modified target polynucleotide using a first primer comprising a universal sequence and using the modified target polynucleotide as a template, wherein the first primer hybridizes to the template and is extended by a polymerase.

[0157] The target polynucleotide is preferably fragmented either naturally or artificially. The target polynucleotide can be any nucleic acid, such as DNA, cDNA, RNA, mRNA, small RNA, or microRNA, or any combination thereof. The target polynucleotide can include multiple target polynucleotides. Each of the multiple target polynucleotides can include a different sequence or the same sequence. One or more of the target polynucleotide or multiple target polynucleotides can include variant sequences.

[0158] Depending on the type of the target polynucleotide and ATO (which is DNA and / or RNA), the method can utilize reverse transcription or primer extension. The primer extension reaction can be a single primer extension step. The primer extension reaction can include extending one or more individual primers once. The primer extension reaction can include extending one or more individual primers in one step. In step (i), the 3'-end or the trimmed 3'-end of the target polynucleotide serves as a primer, and using ATO as a template, the primer is extended. In step (ii), the extended primer or the amplification primer is the first primer, which anneals to the 3'-extended portion of the target polynucleotide.

[0159] In one embodiment, in step (i), the 3'-end of the target polynucleotide serving as a primer hybridizes to ATO through the 3'-end of a sequence that can be randomly fragmented or specifically fragmented, and is trimmed by the 3'-to-5' exonuclease activity of a polymerase to remove the 3'-overhang (if present), and then extended using ATO as a template to generate a modified target polynucleotide. Due to the random nature of the 3'-random sequence portion of ATO, it may be difficult to obtain perfect hybridization between the 3'-end of the target polynucleotide and ATO by applying less stringent conditions. For example, a high concentration of ATO can be used, a low hybridization temperature (such as 4 °C) can be used, and / or multiple extension cycles and / or a longer hybridization time can be used. The extension can be carried out by any polymerase and / or any reverse transcriptase or a mixture of different polymerases. Preferably, the DNA polymerase can have 3'-to-5' exonuclease activity so that any 3'-overhang (if present) is digested (trimmed) and extension can be carried out. The DNA polymerase can contain strand displacement activity so that the stem-loop structure and the double-stranded universal portion of the ATO molecule can be opened and copied. Alternatively, the DNA polymerase can contain 5'-to-3' exonuclease activity so that the 5'-end of the stem-loop structure and the double-stranded universal portion of the ATO molecule can be digested, and the lower strand of ATO is copied. The DNA polymerase is preferably active at low temperatures. The polymerase can contain a mixture of different polymerases, which can have 3'-to-5' exonuclease activity, 5'-to-3' exonuclease activity, and / or strand displacement activity. The polymerases that can be used to implement the methods disclosed herein include, but are not limited to, DeepVentR TM DNA polymerase, LongAmpTM Taq DNA polymerase, Phusion TM High-fidelity DNA polymerase, Phusion TM Hot-start high-fidelity DNA polymerase, DNA polymerase, DyNAzyme TM II Hot-start DNA polymerase, Phire TM Hot-start DNA polymerase, Crimson LongAmp TM Taq DNA polymerase, DyNAzyme TM EXT DNA polymerase, LongAmp TM TaqDNA polymerase, Taq DNA polymerase with standard Taq (without Mg) buffer, Taq DNA polymerase with standard Taq buffer, Taq DNA polymerase with ThermoPol II (without Mg) buffer, Taq DNA polymerase with ThermoPol buffer, Crimson Taq TM DNA polymerase, Crimson Taq with (without Mg) buffer TM DNA polymerase, (exo-)DNA polymerase, Hemo KlenTaq TM , Deep VentR TM (exo-)DNA polymerase, AMV First Strand cDNA Synthesis Kit, M-MuLV First Strand cDNA Synthesis Kit, Bst DNA polymerase, full-length Bst DNA polymerase, large fragment Taq DNA polymerase with ThermoPol buffer, 9°Nm DNA polymerase, Crimson Taq TM DNA polymerase, Crimson Taq with (without Mg) buffer TM DNA polymerase, Deep VentR TM (exo-)DNA polymerase, Deep VentR TM DNA polymerase, DyNAzyme TM EXT DNA polymerase, DyNAzyme TM II Hot-start DNA polymerase, Hemo KlenTaq TM , Phusion TM High-fidelity DNA polymerase, Phusion TM Hot-start high-fidelity DNA polymerase, Sulfolobus DNA polymerase IV, TherminatorTM γ DNA polymerase, Therminator TM DNA polymerase, Therminator TM DNA polymerase II, Therminator TM DNA polymerase III, DNA polymerase, (exo-)DNA polymerases, Bsu DNA polymerase, large fragment Bst DNA polymerase, large fragment DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment, Klenow fragment (3’→5’exo-), phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase (unmodified), reverse transcriptases and RNA polymerases, AMV reverse transcriptase, M-MuLV reverse transcriptase, phi6 RNA polymerase (RdRP), SP6 RNA polymerase and T7 RNA polymerase.

[0160] Ligases that can be used to implement the methods of the present disclosure include, but are not limited to, T4 DNA ligase, T4 RNA ligase, E. coli DNA ligase, and E. coli RNA ligase.

[0161] Multiple cycles of extension can be carried out by thermal cycling of temperature (annealing, extension, and denaturation). The modified target polynucleotide has an extended 3’ portion, and the extended 3’ portion can include some random sequences and a universal sequence that provides a primer binding site. The extended 3’ portion can also include an additional UID.

[0162] In one embodiment, in step (i), if the universal portion of the ATO includes weakly paired nucleotides (such as inosine), it may not be necessary to remove the ATO after the first extension reaction. Otherwise, the ATO can be removed or digested from the reaction mixture by any means. For example, if the ATO includes uracil residues, the ATO is digested / removed by UNG digestion; if the ATO includes RNA, the ATO is digested / removed by ribonuclease digestion; if the ATO includes a restriction enzyme site, the ATO is digested / removed by restriction enzyme digestion; or if the ATO includes biotin, the ATO is removed by capture on streptavidin beads. In another embodiment, if the ATO includes a hairpin structure / stem-loop structure ( Figure 3 B and Figure 3 C), it may not be necessary to digest or remove the ATO from the reaction mixture because the hairpin structure of the ATO makes it impossible for the ATO to hybridize to the extended 3’ portion of the modified target polynucleotide.

[0163] In one embodiment, the first ATO reaction is a primer extension reaction, wherein a target polynucleotide serving as a primer is extended on an ATO template by a DNA polymerase. The DNA polymerase may include strand displacement activity or 5' to 3' exonuclease activity, wherein during the extension, the stem-loop structure is opened or the upper ATO strand is displaced or digested. Any polymerase may be used, such as Klenow exo- polymerase, Bst polymerase, or T4 DNA polymerase.

[0164] In another embodiment, the first reaction is an extension-ligation reaction, wherein a DNA polymerase extends the target, and a DNA ligase ligates the extended target sequence to the 5' stem portion of the ATO or the upper strand of the ATO. Any DNA polymerase and DNA ligase (e.g., Klenow fragment, T4 DNA ligase) may be used.

[0165] In another embodiment, the first reaction is a ligation reaction, wherein a DNA ligase ligates the target polynucleotide to the 5' stem portion of the ATO or the upper strand of the ATO. Any DNA ligase (e.g., T4 DNA ligase) may be used.

[0166] After the first reaction, the method may include an operation of digesting or removing a portion of the ATO by affinity capture.

[0167] The method may include an operation of generating a modified first CS by using a first CS, wherein the first CS hybridizes with the 3' random sequence of a second ATO of any of the above ATOs in an enzymatic reaction (which adds an adaptor sequence to the 3' end of the first CS), and the second ATO includes a different 5' universal sequence of the first ATO.

[0168] The method may further include an operation of generating a modified first CS by ligating a double-stranded adaptor to the product of step (ii).

[0169] In the step after digestion / removal of ATO or in the step without digestion / removal of ATO, an operation of generating a first complementary sequence (CS) of a modified target polynucleotide is performed. The first complementary sequence (CS) can be generated by primer extension. The primer can be a universal primer that can hybridize to and extend the 3'-extended portion of the modified target polynucleotide. Depending on the type of the target polynucleotide (which is DNA, RNA, or a combination of RNA and DNA), the method can utilize reverse transcription and / or primer extension by DNA polymerase and / or reverse transcriptase. The primer extension reaction can be a single primer extension step. Alternatively, the primer extension reaction can be a cycle of multiple linear amplifications using a first primer. The generated first CS includes a 5'-universal sequence and a 3'-complementary sequence of the target polynucleotide. The first primer includes a 3'-universal sequence that is the same as or substantially the same as the universal sequence portion of ATO. The first primer can also include a 5'-additional universal sequence portion that is compatible with the sequencing platform. The first primer can also include a sample barcode sequence (SBC) between the 3'-universal sequence and the 5'-additional universal portion.

[0170] In one embodiment, the method further includes: an operation of using the first CS as a primer and using a second adaptor template oligonucleotide (ATO) of any of the above oligonucleotides as a template to generate a modified first CS. The 3'-end of the first CS is extended on the ATO template to generate an extended first CS, and the extended first CS includes a second universal sequence in the 3'-end. After removing the second ATO, the first CS can be PCR amplified using two universal primers.

[0171] In another embodiment, the method further comprises: extending a second primer hybridized to the first CS or the modified first CS to form a second CS, wherein the second primer comprises a target-specific portion or a universal sequence, or both a 3' target-specific sequence and a 5' universal sequence. In one aspect, when the second primer is a target-specific primer, it may comprise a 3' target-specific portion with or without a 5' universal portion. The extension operation using the second primer may be a single extension or multiple cycles of linear amplification. Alternatively, step (ii) and this step group are combined into a single PCR reaction that uses the first primer to generate the first CS and uses the second primer to form the second CS, wherein the first CS and the second CS are generated simultaneously after the first PCR cycle. After the PCR reaction or linear amplification, the product can be purified, or the primers can be removed by digestion with a single-strand-specific nuclease. If the first primer contains a sample barcode, the purified PCR products from multiple samples can be pooled together. The purified (pooled) PCR products or linear amplification products are further PCR amplified using a nested target-specific third primer for the first CS and a universal primer for the second CS. The nested target-specific third primer for the first CS comprises a 3' target-specific portion and a 5' universal sequence portion, wherein the 5' universal sequence portion is compatible with the NGS platform. The PCR products are then purified for sequencing. In another aspect, when the second primer is a target-specific primer, it may comprise a 3' target-specific portion and a 5' universal portion, wherein the 5' universal portion is compatible with the NGS platform. Step (ii) and this step group are combined into a single PCR reaction that uses the first primer to generate the first CS and uses the second primer to form the second CS, wherein the first CS and the second CS are generated simultaneously after the first PCR cycle. The PCR products are then purified for sequencing.

[0172] When the original target polynucleotide in the sample is double-stranded, the reaction of extending the second primer hybridized to the first CS can be divided into two separate reactions. The forward reaction comprises a target-specific second primer complementary to the forward strand of the target sequence, and the reverse reaction comprises a target-specific second primer complementary to the reverse strand of the target sequence, wherein the forward strand and the reverse strand of the target sequence are complementary.

[0173] In step (i) or step (ii), the extension operation may comprise a single extension or linear amplification using the first primer or the second primer. In step (ii), the extension operation may comprise exponential amplification using the first primer and the second primer.

[0174] The first primer may comprise a sample barcode (SBC) sequence and an additional 5' universal sequence compatible with the NGS platform.

[0175] In one aspect, the present disclosure provides a method for extending a target polynucleotide, the method comprising: mixing the target polynucleotide with a polymerase and an ATO molecule comprising an NGS adapter sequence and a cleavable sequence; performing a trimming and extension reaction, heat inactivating the polymerase, and then incubating with a single-strand specific circular ligase; optionally including a cleavage reaction or an amplification reaction having a reverse primer and a forward primer, and performing one of them to dissociate the circular molecule into a complete linear NGS library molecule.

[0176] In another aspect, the method provides a method for extending a target polynucleotide, the method comprising: mixing the target polynucleotide with a polymerase and an ATO comprising an NGS adapter sequence; performing a trimming and extension reaction, and then incubating with a first primer complementary to the universal NGS adapter sequence, a DNA polymerase, and dNTPs to perform an extension reaction to generate a CS and a double-stranded substrate molecule; performing ligation with T4 DNA ligase and a blunt adapter or a T-tailed adapter, the blunt adapter or the T-tailed adapter being formed by annealing two oligonucleotides comprising an NGS adapter sequence and a truncated complement and a 3' phosphate, wherein one oligonucleotide is ligated to the 5' phosphate of the modified target polynucleotide molecule to complete the linear NGS library molecule.

[0177] The present disclosure also provides a method for accurately determining the sequence of a target polynucleotide, the method comprising:

[0178] (i) sequencing at least one of the amplified second CSs in any of the above methods;

[0179] (ii) aligning at least two sequences containing the same UID from (i) and / or aligning the same target sequences of two reactions, each reaction generating sequence information of one strand or a complementary strand of a duplex target sequence; and

[0180] (iii) determining the consensus sequence and / or identical variant sequences of the two reactions based on (ii), wherein the consensus sequence and / or variant sequences accurately represent the target polynucleotide sequence.

[0181] The present disclosure also provides a kit for generating a polynucleotide library, the kit comprising an adapter template oligonucleotide (ATO) of any of the above ATos and primers compatible with an NGS platform.

[0182] The kit comprises the composition as described above.

[0183] A kit for generating a polynucleotide library comprises the adapter template oligonucleotide (ATO) as described above, a polymerase, and primers compatible with an NGS platform.

[0184] The target polynucleotide is a polynucleotide, a modified polynucleotide, or a combination thereof as described below. In various embodiments, the target polynucleotide is DNA, RNA, or a combination thereof. In another embodiment, the target polynucleotide is a chemically treated nucleic acid, including but not limited to embodiments in which the substrate polynucleotide is bisulfite-treated DNA for detecting methylation status by NGS.

[0185] The target polynucleotides are obtained from naturally occurring sources, or they can be synthetic. Naturally occurring sources are RNA and / or genomic DNA from prokaryotes or eukaryotes. For example but not limited to, the source can be human, mouse, virus, plant, or bacterium. In various aspects, the target polynucleotide is extended at the 3'-end with an adapter sequence for use in assays involving microarrays and creating libraries for next-generation nucleic acid sequencing.

[0186] If the source of the target polynucleotide is genomic DNA or RNA or both, then in some embodiments, the genomic DNA or RNA or both are fragmented before they are extended. The fragmentation operation of genomic DNA / RNA is a general procedure known to those skilled in the art and is carried out in vitro by, for example but not limited to: shearing (nebulizing) DNA / RNA, cutting DNA / RNA with an endonuclease, sonication of DNA / RNA, heating DNA / RNA, irradiating DNA / RNA with an α-radiation source, β-radiation source, γ-radiation source, or other radiation source, by light, by chemical cleavage of DNA / RNA in the presence of metal ions, by free radical cleavage, and combinations thereof. The fragmentation operation of genomic DNA / RNA can also occur in vivo, for example but not limited to due to apoptosis, radiation, and / or exposure to asbestos. According to the methods provided herein, the population of target polynucleotides does not need to have a uniform size. Thus, the methods of the present disclosure are effective for populations of target polynucleotide fragments of different sizes.

[0187] As used herein, "target polynucleotide complementary sequence (CS)" is a polynucleotide that includes a sequence complementary to the target sequence or its complementary sequence (the complementary sequence of the sequence complementary to the target sequence). In some embodiments, the target polynucleotide complementary sequence includes a first complementary sequence. The "first complementary sequence" is a polynucleotide reverse transcribed from the target polynucleotide or a polynucleotide formed by primer extension reaction on the target polynucleotide or RNA polynucleotide, which is transcribed by RNA polymerase from a double-stranded RNA polymerase promoter of a modified target polynucleotide. The modified target polynucleotide is a target polynucleotide extended on an ATO including a random sequence and a universal sequence.

[0188] The target polynucleotide complementary sequence includes a second complementary sequence. The "second complementary sequence" is a polynucleotide including a sequence complementary to the first complementary sequence. The target polynucleotide complementary sequence may include a UID. For example, the first complementary sequence may include a random sequence of ATO and a UID provided by the 3'-terminal portion of the randomly fragmented target polynucleotide.

[0189] The second primer or the third primer may be a group of multiple second primers or third primers. Each second primer or third primer among the multiple second primers or third primers is simultaneously extended and extended in the same reaction chamber.

[0190] The amplification step using the target-specific second primer can be divided into two reactions: a forward reaction and a reverse reaction. The forward reaction includes annealing a forward group of multiple target-specific second primers to the first strand of multiple target CSs from one sample, while the reverse reaction includes annealing a reverse group of multiple target-specific second primers to the second strand of multiple target CSs from the same sample. The primers for generating the PCR product in nested PCR may include a universal primer targeting the 5'-universal sequence portion of the first primer and a third group of multiple target-specific primers annealing to the second strand of multiple target sequences, where the third group of target-specific primers (internal primers) is nested within the group of target-specific second primers (external primers). The universal primers in the forward reaction and the reverse reaction may be the same.

[0191] The reaction mixture may include multiple reactions for more than one sample (which may be two samples, three samples, or more than three samples, or more than ten samples). Different samples can be processed in parallel together. Each sample may include two reactions: a forward reaction and a reverse reaction. After step (ii), it is preferably to mix different sample reactions (all forward reactions or all reverse reactions), where the consistency of each sample is assigned by the first primer having SBC in the amplification. All forward reactions or reverse reactions after step (ii) can be carried out in one mixture.

[0192] The method further includes an operation of analyzing the NGS reads of the forward reaction and the reverse reaction derived from two different strands representing the target sequence. The analyzing operation includes generating an error-corrected consensus sequence by the following operations: (i) grouping into families containing the same random sequence identifier (UID) sequence; (ii) removing target sequences of the same family having one or more nucleotide positions where the target sequence is inconsistent with most members; and (iii) checking whether the same mutation appears in the two reactions representing different strands of the target sequence.

[0193] The method further includes an operation of analyzing NGS reads from forward and reverse reactions derived from two different strands representing a target sequence, and the analysis operation includes an operation of generating a consensus sequence by grouping into families containing the same random sequence identifier (UID) sequence and counting the number of families. The method provides an accurate count of the original target nucleic acid quantity present in the sample.

[0194] The method can be used to quantify starting molecules, and compared with other samples or between forward and reverse reactions, the operation of counting UID families of target sequences can provide accurate counting information.

[0195] The purpose of the UID is twofold. The first is to assign a unique UID to each original DNA template molecule or RNA template molecule. The second is the amplification of each uniquely labeled template so as to generate many progeny molecules having the same UID sequence (defined as a UID family). If a mutation pre-exists in the template molecule used for amplification, then that mutation should be present in each progeny molecule containing that UID.

[0196] The universal primer can contain one, or two, or more terminal thiophosphate esters to render it resistant to any exonuclease activity. The universal primer can also contain a 5' grafting sequence necessary for hybridization to an NGS flow cell (e.g., an Illumina GA IIx flow cell). Finally, the universal primer can contain an index sequence between the grafting sequence and the universal tag sequence. This index sequence enables PCR products from multiple different individuals to be analyzed simultaneously in the same flow cell chamber of a sequencer.

[0197] The target nucleic acid sequence of the sample can include a nucleic acid fragment or gene that contains one or more variant nucleotides and can be selected from the group consisting of one or more disease-associated SNPs / deletions / insertions, one or more chromosomal rearrangements, trisomies, or one or more cancer genes, one or more drug resistance genes, and one or more virulence genes. Disease-associated genes can include but are not limited to cancer-associated genes and genes related to genetic diseases. The sample can be genomic DNA, circulating nucleic acid, RNA, mRNA, small RNA, microRNA, or FFPE DNA or RNA.

[0198] One or more variant nucleotides in the diagnostic region of the target polynucleotide sequence can include one or more nucleotide substitutions, chromosomal rearrangements, deletions, insertions, and / or abnormal methylation.

[0199] DNA methylation is an important epigenetic modification of the genome. Aberrant DNA methylation can cause the silencing of tumor suppressor genes and is common in many human cancer cells. To detect the presence of any aberrant methylation in a target polynucleotide, a pretreatment should be performed before implementing this method. Preferably, the nucleic acid sample should be chemically modified by bisulfite treatment, which converts cytosine to uracil, but not methylated cytosine (i.e., 5-methylcytosine, which is resistant to this treatment and remains as cytosine). Due to these modifications, the method can be applied to the detection of one or more aberrant methylations in the target nucleic acid. In another embodiment, the modification by bisulfite treatment occurs after the ATO reaction. In another embodiment, the modification by bisulfite treatment occurs after the generation of the first CS.

[0200] The present disclosure provides methods for analyzing the presence and / or amount and / or frequency of mutations or polymorphisms at multiple loci of different target nucleic acid sequences in a biological sample. In another aspect, the present disclosure provides methods for analyzing chromosomal abnormalities (e.g., trisomy) in a biological sample. Next-generation sequencing, digital PCR, microarray, or other high-throughput analyses can be performed after the ATO reaction. The number of multiplexing of target loci can be more than 5, or more than 10, or more than 30, or more than 50, or more than 100, or more than 500, more than 1000, or even more than 2000.

[0201] When the concentration of mutants in the sample is very low (e.g., there is one or two mutants in the sample), after dividing the sample nucleic acid into two reactions, only one reaction may contain the mutant. Comparison of the two-strand sequences from the two reactions can reveal that only one reaction may contain the mutation. If more than one read family contains the same mutation, it is classified as a true mutation even if the mutation appears in only one reaction.

[0202] In another embodiment, for one or more subsequent rounds of amplification, the modified target polynucleotide can be amplified by linear amplification or exponential amplification before dividing the sample nucleic acid into two reactions. Due to the increased copy number of all original molecules, comparison of the two-strand sequences from the two reactions is more likely to reveal the presence of mutations.

[0203] In another embodiment, the modified target polynucleotide is not divided into two reactions, and the presence of mutations is detected only in one of the two strands.

[0204] In another embodiment, the target polynucleotide is not divided into two reactions, and the two strands are amplified separately and sequentially to allow analysis of both strands.

[0205] It has been well documented that free DNA is released from dying tumor cells into the blood of patients with various types of cancer. Studies have shown that circulating tumor DNA can be used as a non-invasive biomarker to detect the presence of malignancies, track treatment response, or monitor recurrence. However, current detection methods have significant limitations. Next-generation sequencing (NGS) methods have revolutionized genomic exploration by allowing simultaneous sequencing of hundreds of billions of base pairs at a fraction of the time and cost of traditional methods. However, when aiming to identify rare mutations in genetically heterogeneous mixtures such as tumors and plasma, an error rate of ~1% results in hundreds of millions of sequencing errors, which is unacceptable. The method of the present invention overcomes these limitations of sequencing accuracy. Relatively excessive background wild-type DNA can mask cfDNA with mutations; detection has proven challenging. By independently labeling and sequencing each original DNA duplex, the method greatly reduces errors.

[0206] The method of the present invention can significantly improve the accuracy of massively parallel sequencing. The method can be easily used to identify rare mutations in a population of DNA templates. Each of the two strands of a target template in a sample is uniquely labeled and independently sequenced. The operation of comparing the sequences of the two strands results in either agreement or disagreement with each other. Agreement provides confidence in scoring the mutation as a true positive.

[0207] After sequencing, members of each read family are identified and grouped based on having the same UID tag sequence. Then, the sequences of the uniquely UID-labeled families are compared with one or both strands of the target sequence to create a consensus sequence. This step filters out random errors introduced during sequencing or PCR to produce a set of sequences, each of which is derived from a separate single-stranded DNA molecule.

[0208] In addition to its application for highly sensitive detection of rare DNA variants, the barcoded random sequence identifiers in the target-specific primers can also be used for single molecule counting to accurately determine relative or absolute DNA copy numbers and / or RNA copy numbers. Since the labeling occurs prior to primary amplification, the relative abundance of variants in the population can be accurately assessed given that the proportional representation is not affected by amplification bias.

[0209] By labeling each target sequence with a random sequence identifier and sequencing both strands, the method of the present invention greatly reduces errors. By grouping the uniquely labeled sequences that have been sequenced; removing target sequences of the same family that have one or more nucleotide positions where the target sequence disagrees with most members of the family; and the same mutation that appears in both populations will be a true mutation, the analysis provides an error-corrected consensus sequence.

[0210] The method can be used to detect mutations in any sample (such as FFPE or blood). Accurate counting of sequencing reads reflecting the original molecules present in the sample provides information for prenatal testing of copy number variations or chromosomal abnormalities.

[0211] The reagents used in the method of the present invention can be packaged into a kit. The kit contains one or more ATOs, one or more polymerases, one or more primers in separate containers or in a single master mixture container. The kit may also contain other appropriately packaged reagents, as well as materials required for extension, amplification, enrichment (e.g., buffers, dNTPs, and / or polymerization means), materials required for detection and analysis (e.g., enzymes), and instructions for performing the assay.

[0212] BRIEF DESCRIPTION OF THE DRAWINGS

[0213] Figure 1A A schematic diagram depicting an illustrative embodiment is shown. The target polynucleotide (one or more PCR products, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), ATO molecules hybridize to the single-stranded target polynucleotide sequence at one or more positions. The 3'-end of the target polynucleotide is hybridized to the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the priming site. After step (i), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (ii), a first universal primer is added to hybridize to the modified target polynucleotide and extended to generate a first complementary sequence (CS).

[0214] Figure 1BDepicts a schematic diagram of an illustrative embodiment. In step (i), the ATO hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The 3' end of the target polynucleotide hybridizes to the 3' random sequence portion of the ATO, is trimmed (if any 3' overhangs are present), and is extended using the ATO as a template. The ATO includes a stem-loop structure, and the loop portion includes a non-replicable linkage. The extension displaces the 5' stem portion sequence and terminates at the non-replicable linkage. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3' universal sequence as the priming site. After step (i), the ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. In step (ii), a first universal primer is added to hybridize to the modified target polynucleotide and is extended to generate a first complementary sequence (CS).

[0215] Figure 1C Depicts a schematic diagram of an illustrative embodiment. In step (i), the ATO hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The 3' end of the target polynucleotide hybridizes to the 3' random sequence portion of the ATO, is trimmed (if any 3' overhangs are present), and is extended using the ATO as a template. The ATO includes a stem-loop structure, and the loop portion includes a modification (such as uracil nucleotides) that can be digested or cleaved, allowing disruption of the hairpin. The extended strand contacts the 5' stem portion sequence and ligates to the 5' stem portion containing a 5' phosphate group. Extension-ligation generates a modified target polynucleotide that includes a random sequence as the UID and a 3' universal sequence as the priming site. In some embodiments, the 3' end of one strand of the target polynucleotide hybridizes to the 3' random sequence portion of the ATO immediately adjacent to the 5' stem portion of the ATO and ligates to the 5' stem portion of the ATO by DNA ligation. This ligation can occur without the use of DNA polymerase. After step (i), the ATO can be cleaved at the modification, and the lower portion of the ATO can be digested or removed by affinity capture. In step (ii), a first universal primer is added to hybridize to the modified target polynucleotide and is extended to generate a first complementary sequence (CS).

[0216] Figure 1DDepicts a schematic diagram of an illustrative embodiment. In step (i) of the reaction, ATO hybridizes to a single-stranded target polynucleotide sequence at one or more positions. The 3' end of one strand of the target polynucleotide hybridizes to the 3' random sequence portion of ATO, is trimmed (if any 3' overhangs are present), and is extended using ATO as a template. ATO includes a double-stranded structure, a lower portion of ATO containing a random sequence, and a separate upper portion. The universal portion of ATO is formed by two sequences designed to anneal to form a double-stranded region. By a DNA polymerase having strand displacement activity or 5' to 3' exonuclease activity, the extension of the target nucleotide displaces or digests the upper portion sequence. The extension generates a modified target polynucleotide, which includes a random sequence as the UID and a 3' universal sequence as the priming site. ATO can be digested after the first reaction. In some embodiments, the 3' end of one strand of the target polynucleotide hybridizes to the 3' random sequence portion of ATO, is trimmed (if any 3' overhangs are present), and is extended using ATO as a template. The extended strand contacts the 5' upper portion sequence and ligates to the 5' upper portion containing a 5' phosphate group. The extension-ligation generates a modified target polynucleotide, which includes a random sequence as the UID and a 3' universal sequence as the priming site. In some embodiments, the 3' end of one strand of the target polynucleotide hybridizes to the 3' random sequence portion of ATO immediately adjacent to the 5' end of the upper portion of ATO and ligates to the 5' upper portion of ATO. This ligation can occur without using a DNA polymerase. After step (i), ATO or the lower portion of ATO can be removed by digestion or affinity capture. In step (ii), a first universal primer is added to hybridize to the modified target polynucleotide and is extended to generate a first complementary sequence (CS).

[0217] Figure 2 Depicts a schematic diagram of an illustrative embodiment.

[0218] (A) A target-specific second primer is provided to hybridize to the first CS and is extended to generate a second CS. The target-specific second primer includes a 3' target-specific portion and a 5' universal portion. The extension operation can be a single extension, or multiple cycles of linear amplification, or PCR amplification using both the first primer and the second primer. Optionally, a universal primer compatible with the NGS platform can be used to further PCR amplify the second CS.

[0219] (B) A target-specific second primer is provided to hybridize to the first CS and is extended to generate a second CS. The target-specific second primer includes a 3' target-specific portion and may or may not have a 5' universal portion. The extension operation can be a single extension, or multiple cycles of linear amplification, or PCR amplification using both the first primer and the second primer. The second CS is further PCR amplified using nested target-specific third primers and universal primers compatible with the NGS platform. Optionally, the second CS can be further PCR amplified using universal primers compatible with the NGS platform.

[0220] (C) The first primer is annealed to the added adapter sequence of the target polynucleotide and extended to generate double-stranded DNA of the first CS. The double-stranded DNA of the first CS is ligated to the adapter via double-stranded ligation by a DNA ligase. Only the CS strand needs to be ligated. Optionally, the ligation product can be affinity captured on a solid support. After ligation, the product can be amplified by two universal primers.

[0221] (D) The target polynucleotide is bisulfite treated, which converts unmethylated cytosine (C) to uracil and leaves methylated cytosine intact. The ATO hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The 3' end of one strand of the target polynucleotide is hybridized to the 3' random sequence portion of the ATO, trimmed (if any 3' overhangs are present), and extended using the ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3' universal sequence as the priming site. The ATO can be digested after the first reaction. The modified target polynucleotide hybridizes to a universal primer whose 3' end is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence) and to a library or one or more target-specific primers that include a 3' target-specific portion and may or may not have an additional 5' universal portion. Both mixtures are amplified by one or more cycles of PCR amplification using both the universal primer and one or more target-specific primers. The amplified product can be directly used for next-generation sequencing or can be further processed to produce a product suitable for next-generation sequencing.

[0222] (E) ATO hybridizes with the single-stranded target polynucleotide sequence at one or more positions. The 3'-end of one strand of the target polynucleotide is hybridized with the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs exist), and extended using ATO as a template. The extension generates a modified target polynucleotide, which includes a random sequence as the UID and a 3'-universal sequence as the priming site. ATO can be digested after the first reaction. The modified target polynucleotide then undergoes bisulfite treatment. The modified target polynucleotide hybridizes with a universal primer, the 3'-end of which is designed to hybridize with the universal sequence of the modified target polynucleotide (with or without an additional 5'-universal sequence), and a library or one or more target-specific primers, which include a 3'-target-specific portion and have an additional 5'-universal portion or no additional 5'-universal portion. Both the universal primer and one or more target-specific primers are used to amplify the two mixtures by one or more cycles of PCR amplification. The amplified product can be directly used for next-generation sequencing or can be further processed to produce a product suitable for next-generation sequencing.

[0223] (F) Hybridize the first CS with the second ATO and generate a modified first CS with an adaptor sequence added to the 3'-end of the first CS. The first CS can be generated by linear amplification using a first primer that includes biotin. Optionally, after linear amplification, the first CS is captured by avidin affinity and non-first CSs are removed by washing. The second ATO is added and the extension reaction is repeated as the first reaction. After the extension reaction, the unreacted products are washed away and the products are amplified by two universal primers targeting both ends of the universal sequence. The universal primer targeting the 5'-end of the first CS can be a nested primer.

[0224] (G) Divide the modified target polynucleotide into one or more aliquots, combine each aliquot with a universal primer, the 3'-portion of which is designed to hybridize with the universal sequence of the modified target polynucleotide and has a 5'-tail containing the sequences necessary for next-generation sequencing, add different target-specific primers or a library of target-specific primers, and design the primers to amplify the target region of the target polynucleotide. The target-specific primers include a 3'-target-specific portion and a 5'-universal portion containing the sequences necessary for next-generation sequencing. Both the universal primer and one or more target-specific primers are used to amplify the mixture by one or more cycles of PCR amplification. The amplified products will be PCR products (each of which has the amplified target region of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0225] (H) Combine the modified target polynucleotide with a universal primer, which is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence), and add a target-specific primer or a library of target-specific primers. The target-specific primers include a 3' target-specific sequence, with or without a 5' universal sequence. Using both the universal primer and one or more target-specific primers, amplify the mixture by one or more cycles of PCR amplification. The amplified product will be a PCR fragment that has the amplified target region of the original polynucleotide, with or without a 3' universal sequence and a 5' universal sequence. The first amplified product can be purified to remove reagents that are no longer needed from the first PCR reaction. Combine the PCR product with a second nested universal primer, the 3' portion of which is designed to hybridize to the universal sequence of the first PCR product and has a 5' tail containing sequences necessary for next-generation sequencing, and add a different set of nested target-specific primers or a library of nested target-specific primers. The nested target-specific primers include a 3' target-specific portion and a 5' universal portion containing sequences necessary for next-generation sequencing. Using both the universal primer and one or more target-specific primers, amplify the mixture by one or more cycles of PCR amplification. The amplified product is a PCR product that has the amplified target region of the original polynucleotide and will contain all the necessary sequences compatible with next-generation sequencing.

[0226] Figure 3 A schematic diagram illustrating an exemplary embodiment is depicted. The adapter template oligonucleotide comprises:

[0227] (A) ATO other than any combination of the design components mentioned in (A-T), which includes a 3' random sequence, a degenerate sequence, a random sequence with nucleotide bias, or a target-specific sequence; a blocker moiety is attached to the extreme 3' end, which renders the ATO non-extendable; a universal sequence 5' to the random sequence; and one or more portions of nucleotide sequence / modification (NSM) that can be recognized by an agent.

[0228] (B) ATO other than any combination of the design components mentioned in (A-T), which includes a hairpin structure / stem-loop structure, where the stem portion sequence is indicated. The loop portion may include a non-replicable linkage, or one or more cleavable nucleotides.

[0229] (C) ATO other than any combination of the design components mentioned in (A-T), which includes a hairpin structure / stem-loop structure, where the stem portion sequence is indicated. The stem portion may include one or more non-replicable linkages, or one or more cleavable nucleotides.

[0230] (D) An ATO other than any combination of the design components mentioned in (A - U), which includes a hairpin structure / stem - loop structure, where the stem - part sequence is indicated. The stem part may include one or more non - replicable linkages, or one or more cleavable nucleotides. The loop region may contain a random sequence, a degenerate sequence, a random sequence with nucleotide bias, or a target - specific sequence capable of serving as a unique identifier.

[0231] (E) An ATO with two separate strands, other than any combination of the design components mentioned in (A - J), which includes an upper separate strand that is complementary or substantially complementary to all or part of the lower strand of the ATO, and the lower part of the ATO is equivalent to the ATO in (A).

[0232] (F) An ATO other than any combination of the design components mentioned in (A - U), where the universal site consists entirely or in part of a sequence designed to act as an RNA polymerase promoter.

[0233] (G) An ATO other than any combination of the design components mentioned in (A - U), where the universal site consists in part of a sequence designed to act as an RNA polymerase promoter and a separate sequence designed to act as a priming site.

[0234] (H) An ATO other than any combination of the design components mentioned in (A - U), which includes a hairpin structure / stem - loop structure, where the stem - part sequence is indicated, the loop part may contain non - replicable linkages, one or more cleavable nucleotides, and has a sequence designed to act as an RNA polymerase promoter.

[0235] (I) An ATO other than any combination of the design components mentioned in (A - U), which includes a hairpin structure / stem - loop structure, where the stem part is divided into two or more regions separated by a random sequence, a degenerate sequence, or a random sequence with nucleotide bias, and the two or more regions are spanned by non - replicable linkages or random sequences, degenerate sequences, or random sequences with nucleotide bias of equivalent length.

[0236] (J) An ATO other than any combination of the design components mentioned in (A - U), which includes a 3’ sequence designed to be target - specific, a random sequence, a degenerate sequence, or a random sequence with nucleotide bias from 5’ to the target - specific sequence, and a universal sequence from 5’ to the random sequence.

[0237] (K) ATO other than any combination of the design components mentioned in (A-U), wherein the lower strand contains a universal site, the universal site being partially composed of a sequence designed to act as an RNA polymerase promoter and a separate sequence designed to act as a priming site, and the upper strand is a second oligonucleotide that is partially or fully complementary to the lower strand portion capable of forming a double-stranded RNA polymerase promoter.

[0238] (L) ATO other than any combination of the design components mentioned in (A-U), which includes a hairpin structure / stem-loop structure, wherein the stem portion sequence is indicated. The loop region may contain a random sequence, a degenerate sequence, a random sequence with nucleotide bias, or a target-specific sequence capable of serving as a unique identifier, and includes a non-replicable linkage, or one or more cleavable nucleotides.

[0239] (M) ATO other than any combination of the design components mentioned in (A-U), which includes a hairpin structure / stem-loop structure, wherein the stem portion sequence is indicated. The stem portion may include one or more non-replicable linkages, or one or more cleavable nucleotides. The loop region may contain a random sequence, a degenerate sequence, a random sequence with nucleotide bias, or a target-specific sequence capable of serving as a unique identifier.

[0240] (N) ATO other than any combination of the design components mentioned in (A-U), which includes a hairpin structure / stem-loop structure, wherein the stem portion is divided into two or more regions separated by a random sequence, a degenerate sequence, or a random sequence with nucleotide bias, and the two or more regions are spanned by a non-replicable linkage or a random sequence, a degenerate sequence, or a random sequence with nucleotide bias of equivalent length. The loop portion may include a non-replicable linkage, or one or more cleavable nucleotides.

[0241] (O) ATO other than any combination of the design components mentioned in (A-U), which has a 3' random sequence, a degenerate sequence, or a random sequence with nucleotide bias, and a primer suitable for allowing extension of the outermost 3' end of the ATO.

[0242] (P) ATO other than any combination of the design components mentioned in (A-U), which has a 3' target-specific sequence and a primer suitable for allowing extension of the outermost 3' end of the ATO.

[0243] (Q) ATO other than any combination of the design components mentioned in (A-U), which includes a hairpin structure / stem-loop structure, wherein the stem portion sequence is indicated. The loop portion may include a non-replicable linkage, or one or more cleavable nucleotides. The 5' of the ATO includes a ligation acceptor (such as phosphate).

[0244] (R) ATO other than any combination of the design components mentioned in (A-U), which includes a hairpin structure / stem-loop structure, where the stem portion sequence is indicated. The loop portion may include non-replicable linkages, or one or more cleavable nucleotides. The 5’ of the ATO includes an affinity purification moiety (such as biotin).

[0245] (S) Linear ATO other than any combination of the design components mentioned in (A-U), which has a 5’ including a linking receptor (such as phosphate).

[0246] (T) Linear ATO other than any combination of the design components mentioned in (A-U), which has a 5’ including an affinity purification moiety (such as biotin).

[0247] (U) Linear ATO other than any combination of the design components mentioned in (A-U), which consists only of a random sequence having blocked 3’ and 5’ phosphates to prevent extension.

[0248] Figure 4 A schematic diagram depicting illustrative embodiments is shown. The process of enzymatic fragmentation using a nuclease can be replaced by fragmentation by means of a transposase or the operation of physically shearing DNA by sonication. In step (i), the target double-stranded polynucleotide (DNA) is incubated with transposon DNA and a transposase. Once the random transposition reaction has been completed, the target polynucleotide will contain multiple copies of the transposon DNA, which generates free 3’ ends on the target polynucleotide and free 5’ ends of the transposon DNA. In step (ii), the ATO molecule hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The free 3’ end generated due to the random transposition of one strand of the target polynucleotide is hybridized to the 3’ random sequence portion of the ATO, trimmed (if any 3’ overhangs are present), and extended using the ATO as a template. The extension generates a modified target polynucleotide, which includes the 5’ universal sequence from the transposon DNA, the region of the target polynucleotide 3’ to the transposon DNA, the random sequence 3’ to the target polynucleotide as the UID, and the 3’ second universal sequence as the priming site. After step (ii), the ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. In step (iii), the modified target polynucleotide is used as starting material for complementary strand (CS) generation, PCR amplification, or other downstream processes.

[0249] Figure 5Depicts a schematic diagram of an illustrative embodiment. The target polynucleotide (a PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), ATO molecules hybridize randomly with the single-stranded target polynucleotide sequence at one or more positions. In step (ii), the 3'-end of the ATO molecule is extended using the target polynucleotide as a template. The extension generates a modified copy of the target polynucleotide, which includes a 5'-universal sequence, a random sequence as the UID, and a copy of the target polynucleotide at the 3'-end. After step (ii), the modified copy of the target polynucleotide is purified to remove unused ATO. In some embodiments, the ATO is not digested or removed. In step (iii), a second ATO hybridizes with the modified target polynucleotide at one or more positions. The 3'-end of the modified copy of the target polynucleotide is hybridized with the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and extended using the ATO as a template. The extension generates a doubly modified target polynucleotide, which includes a 3'-universal site and a 5'-universal site that can serve as primer sites, within which are two different random sequences as the UID, and in the center is a copy of the target polynucleotide.

[0250] Figure 6 Depicts a schematic diagram of an illustrative embodiment. The target polynucleotide (a PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), ATO molecules designed with target-specific sequences hybridize with the single-stranded target polynucleotide sequence at one or more positions. In step (ii), the 3'-end of the ATO molecule is extended using the target polynucleotide as a template. The extension generates a modified copy of the target polynucleotide, which includes a 5'-universal sequence, a random sequence as the UID, and a copy of the target polynucleotide at the 3'-end. After step (ii), the modified copy of the target polynucleotide is purified to remove unused ATO. In some embodiments, the ATO is not digested or removed. In step (iii), a second ATO hybridizes with the modified target polynucleotide at one or more positions. The 3'-end of the modified copy of the target polynucleotide is hybridized with the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and extended using the ATO as a template. The extension generates a doubly modified target polynucleotide, which includes a 3'-universal site and a 5'-universal site that can serve as primer sites, within which are two different random sequences as the UID, and in the center is a copy of the target polynucleotide.

[0251] Figure 7A schematic diagram depicting an illustrative embodiment is shown. The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before the single-stranded target polynucleotide randomly hybridizes with ATO molecules at one or more positions. The 3'-end of the target polynucleotide is hybridized with the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the priming site.

[0252] Figure 8 A schematic diagram depicting an illustrative embodiment is shown. (a) The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before the single-stranded target polynucleotide randomly hybridizes with ATO molecules at one or more positions, where ATO contains an RNA polymerase promoter sequence. The 3'-end of the target polynucleotide is hybridized with the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a double-stranded sequence region between the target polynucleotide and ATO, and the modified target polynucleotide includes a random sequence as the UID and a 3'-universal sequence containing an RNA polymerase promoter. After step (i), optionally, the modified target polynucleotide is purified to remove unused ATO. In some embodiments, ATO is not digested or removed. In step (ii), the double-stranded RNA polymerase promoter of the modified target polynucleotide is used to generate an RNA copy (the first complementary sequence) of the modified target polynucleotide.

[0253] (b) The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before the single-stranded target polynucleotide randomly hybridizes with the ATO molecule at one or more positions. The ATO contains an RNA polymerase promoter sequence. The 3'-end of the target polynucleotide is hybridized with the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and extended using the ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the priming site, which contains complementary regions capable of forming a hairpin at the 3'-end of the modified target polynucleotide. After step (i), the ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. In step (ii), the 3'-end of the modified target polynucleotide is allowed to form a small hairpin by annealing to itself, and then the 3'-end is used as a primer that, once extended, will generate a first complementary sequence that includes a double-stranded RNA polymerase promoter. In step (iii), the double-stranded RNA polymerase promoter of the modified target polynucleotide is used to generate an RNA copy (the first complementary sequence) of the modified target polynucleotide.

[0254] Figure 9 A schematic diagram illustrating an illustrative embodiment is depicted. The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before the single-stranded target polynucleotide randomly hybridizes with the ATO molecule at one or more positions. The 3'-end of the target polynucleotide is hybridized with the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and extended using the ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the priming site, which contains complementary regions capable of forming a hairpin at the 3'-end of the modified target polynucleotide. After step (i), the ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. In step (ii), the 3'-end of the modified target polynucleotide is allowed to form a small hairpin by annealing to itself, and then the 3'-end is used as a primer that, once extended, will generate a first complementary sequence.

[0255] Figure 10A schematic diagram depicting an illustrative embodiment is shown. The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before random hybridization of the single-stranded target polynucleotide with ATO molecules at one or more positions. The 3'-end of the target polynucleotide is hybridized to the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the priming site. After step (i), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (ii), a target-specific primer is added to the modified target polynucleotide, and the modified target polynucleotide is extended by polymerase until the 5'-end of the modified target polynucleotide is reached. In step (iii), a double-stranded adaptor is mixed with the modified target polynucleotide and a suitable enzyme, and then the adaptor is ligated to the 5'-double-stranded DNA of the modified target polynucleotide.

[0256] Figure 11 A schematic diagram depicting an illustrative embodiment is shown. To measure the efficiency of 3'-extension of the target polynucleotide to generate the modified target, polynucleotide quantitative PCR (qPCR) is used. The target polynucleotide used is a single-stranded DNA oligonucleotide of known length and sequence (internal control (IC)). The target polynucleotide used is also genomic DNA mixed with 10% by amount of a single-stranded DNA oligonucleotide of known length and sequence (internal control (IC)). Once the modified target polynucleotide has been generated, the two modified target polynucleotides are independently used as templates for 3 different qPCR reactions, one reaction with two primers (forward primer 1 and reverse primer 1) located within the target polynucleotide sequence, one reaction with a primer located within the target polynucleotide sequence and a primer present in the universal sequence added to the modified target polynucleotide (forward primer 2 and reverse primer 2), and another reaction with a primer located within the target polynucleotide sequence and a primer at the end of the universal sequence added to the modified target polynucleotide that has a tail (which is non-homologous to the modified target polynucleotide) (forward primer 3 and reverse primer 3), all of these reactions containing a dual-labeled qPCR probe located within the target polynucleotide (probe). When comparing the 'CT' value when detecting the fluorescence amplification signal of the primer pairs located within the IC and the universal sequence, the 'CT' value when detecting the fluorescence amplification signal of the IC reference-specific primer shows the efficiency of 3'-extension of the target polynucleotide in proportion. Since the 'CT' values are very similar, the efficiency can be interpreted as being between 50 - 100%.

[0257] Figure 12 Depicts a schematic diagram of an illustrative embodiment. The target polynucleotide (PCR product from any source, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is fragmented before random hybridization with ATO molecules at one or more positions. The 3'-end of the target polynucleotide is hybridized to the 3'-random sequence portion of ATO, trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID and a 3'-universal sequence as the primer site, which contains complementary regions capable of forming a hairpin at the 3'-end of the modified target polynucleotide. In step (ii), the 3'-end of the modified target polynucleotide is allowed to form a small hairpin by annealing to itself. In step (iii), the 3'-end is then used as a primer, which, once extended, will generate the first complementary strand. After step (iii), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (iv), the double-stranded DNA of the first CS is ligated to an adaptor via double-stranded ligation by DNA ligase. In step (v), the remaining ATO is digested, and the hairpin is disrupted by incubating the reaction mixture with a mixture of dU-glycosylase, apurinic / apyrimidinic endonuclease, and S1 nuclease. Then, the final double-stranded product is directly used for sequencing on a compatible next-generation sequencer (e.g., MiSeq).

[0258] Figure 13Depicts a schematic diagram of an illustrative embodiment. All steps used in the generation of a targeted amplicon next-generation sequencing set from starting materials to sequencing data analysis are depicted. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is enzymatically fragmented. In step (II), the size distribution of the fragmented target polynucleotide is determined using a high-sensitivity bioanalyzer chip. In step (iii), the fragmented target polynucleotide randomly hybridizes with ATO molecules at one or more positions. The 3' end of the target polynucleotide is hybridized to the 3' random sequence portion of ATO, trimmed (if any 3' overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a 3' random sequence as a UID and a 3' universal sequence as a primer site. In step (iii), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (iv), the modified target polynucleotide, a universal primer designed to bind to a universal site on ATO, a gene-specific primer pool, a suitable buffer, a suitable enzyme, dNTPs, and other additives are combined and used for exponential amplification of the modified target polynucleotide. In step (v), the PCR product is purified. In step (vi), the first PCR product, a second universal primer designed to bind to a universal site in the PCR product, a second nested gene-specific primer pool, a suitable buffer, a suitable enzyme, dNTPs, and other additives are combined and used for exponential amplification of the modified target polynucleotide. In step (vii), the second PCR product is purified. In step (viii), the size distribution of the final sequencing library is determined using a high-sensitivity bioanalyzer chip. Then, the library is sequenced using a MiSeq sequencer with 150bp paired-end sequencing. Subsequently, the sequencing data is analyzed using a combination of the bwa aligner and a custom data filtering python script. Data graph (H) represents the size distribution of the insert fragments determined from the sequencing data generated by MiSeq, (I) represents the distribution of the read sequences across all primers present in the library used in the exponential amplification, and (J) shows the number of "barcode families" identified in the sequencing data with a family size of at least 3 read sequences.

[0259] Figure 14FIG. depicts a schematic diagram of an illustrative embodiment. All steps used in the generation of a whole-genome DNA library from starting materials to a final library are depicted. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the target polynucleotide is enzymatically fragmented. In step (II), the size distribution of the fragmented target polynucleotide is determined using a high-sensitivity bioanalyzer chip. In step (iii), the fragmented target polynucleotide randomly hybridizes with ATO molecules at one or more positions. The 3' end of the target polynucleotide is hybridized with the 3' random sequence portion of ATO, trimmed (if any 3' overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a 3' random sequence as a UID and a 3' universal sequence as a priming site. In step (iii), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (iv), the modified target polynucleotide, a universal primer designed to bind to the universal site on ATO, a suitable buffer, a suitable enzyme, dNTPs, and other additives are combined and used to linearly amplify the modified target polynucleotide to generate a first CS (complementary sequence). Then, the product is optionally purified. In step (v), the linear CS randomly hybridizes with ATO molecules at one or more positions. The 3' end of the linear CS is hybridized with the 3' random sequence portion of ATO, trimmed (if any 3' overhangs are present), and extended using ATO as a template. The extension generates a modified CS that includes a random sequence as a UID at each end, different 3' universal sequences and 5' universal sequences as priming sites. In step (vi), ATO is digested or removed by affinity capture. In some embodiments, ATO is not digested or removed. In step (vii), the modified CS product, two different universal primers designed to bind to the universal sites at the 5' and 3' ends of the modified CS product, the necessary buffer, enzyme, dNTPs, and other additives are combined and used to exponentially amplify the modified CS product. In step (viii), the second PCR product is purified. In step (ix), the size distribution of the final sequencing library is determined using a high-sensitivity bioanalyzer chip.

[0260] Figure 15A schematic diagram depicting an illustrative embodiment is shown. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the single-stranded target polynucleotide randomly hybridizes with ATO molecules at one or more positions. The 3'-end of the target polynucleotide hybridizes with the 3'-random sequence portion of ATO, is trimmed (if any 3'-overhangs are present), and extended using ATO as a template. The extension generates a modified target polynucleotide that includes a variable-length random sequence as the UID and a 3'-universal sequence as the priming site. In step (ii), the modified target polynucleotide is purified to remove unutilized ATO. In some embodiments, ATO is not digested or removed. In step (iii), the modified target polynucleotide, a universal primer designed to bind to a universal site in the PCR product, a gene-specific primer, a suitable buffer, a suitable enzyme, dNTPs, and other additives are combined and used for exponential amplification of the modified target polynucleotide.

[0261] Figure 16 A schematic diagram depicting an illustrative embodiment is shown. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown.

[0262] (A) The modified target polynucleotide is divided into two approximately equal aliquots. A universal primer is added to each of these aliquots, the 3'-end of which is designed to hybridize with the universal sequence of the modified target polynucleotide and has a 5'-tail containing the sequences necessary for next-generation sequencing. Different target-specific primers or a library of target-specific primers are added to each aliquot, and the primers in each library are designed to amplify either the forward or reverse strand of the target region of the target polynucleotide. The target-specific primers include a 3'-target-specific portion and a 5'-universal portion containing the sequences necessary for next-generation sequencing. Using both the universal primer and one or more target-specific primers, the two separate mixtures are amplified with multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (each of which has amplified one or the other of the strands of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0263] (B) Divide the modified target polynucleotide into two approximately equal aliquots. Add a universal primer to each of these aliquots, which is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence). Add different target-specific primers or a library of target-specific primers to each aliquot, and the primers in each library are designed to amplify the forward or reverse strand of the target region of the target polynucleotide. The target-specific primers include a 3' target-specific sequence, with or without a 5' universal sequence. Using both the universal primer and the library of target-specific primers, amplify the two separate mixtures by multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (with or without 3' and 5' universal sequences), each of which has amplified one or the other of the strands of the original polynucleotide. The first amplified product can be purified to remove reagents no longer needed from the first PCR reaction. Combine each of the two separate libraries of PCR products with a second universal primer, the universal primer used in the first PCR, or a nested universal primer, the 3' of which is designed to hybridize to the universal sequence of the first PCR product and has a 5' tail containing the sequences necessary for next-generation sequencing, and add different nested target-specific primers or a library of nested target-specific primers, where the nested target-specific primers include a 3' target-specific portion and a 5' universal portion containing the sequences necessary for next-generation sequencing. Using both the universal primer and the library of target-specific primers, amplify the two separate mixtures by multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (each of which has amplified one or the other of the strands of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0264] (C) Combine the modified target polynucleotide with a universal primer that is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence). The modified target polynucleotide is then linearly amplified by one or more rounds of amplification. The linear amplification product can be purified to remove reagents that are no longer needed from the linear amplification reaction. The linear amplification product is divided into two approximately equal aliquots. A universal primer is added to each of these aliquots, the 3' of which is designed to hybridize to the universal sequence of the modified target polynucleotide and has a 5' tail containing sequences necessary for next-generation sequencing. Different target-specific primers or target-specific primer pools are added to each aliquot, each pool containing primers that are designed to amplify the target regions of the forward or reverse strand of the target polynucleotide. The target-specific primers include a 3' target-specific portion and a 5' universal portion containing sequences necessary for next-generation sequencing. Using both the universal primer and the primer pools, the two separate mixtures are amplified with multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (each of which has amplified one or the other of the strands of the original polynucleotide) and will contain all of the necessary sequences compatible with next-generation sequencing.

[0265] (D) Combine the modified target polynucleotide with a universal primer that is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence). Then, linearly amplify the modified target polynucleotide by one or more rounds of amplification. The linear amplification product can be purified to remove reagents that are no longer needed from the first PCR reaction. Divide the linear amplification product into two approximately equal aliquots. Add a universal primer to each of these aliquots that is designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence). Add a different target-specific primer or a library of target-specific primers to each aliquot, where each library contains primers that are designed to amplify the target region of the forward or reverse strand of the target polynucleotide. The target-specific primers include a 3' target-specific sequence and have a 5' universal sequence or do not have a 5' universal sequence. Using both the universal primer and the primer library, amplify the two separate mixtures by multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (with or without 3' and 5' universal sequences) where each has amplified one or the other of the strands of the original polynucleotide. The first amplification product can be purified to remove reagents that are no longer needed from the first PCR reaction. Combine each of the two separate libraries of PCR products with a second nested universal primer, or the universal primer used in the first amplification, where the 3' of the primer is designed to hybridize to the universal sequence of the first PCR product and has a 5' tail containing the sequences necessary for next-generation sequencing, and add a different nested target-specific primer or a library of nested target-specific primers, where the nested target-specific primers include a 3' target-specific portion and a 5' universal portion containing the sequences necessary for next-generation sequencing. Using both the universal primer and the primer library, amplify the two separate mixtures by multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (where each has amplified one or the other of the strands of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0266] (E) Combine the modified target polynucleotide with a universal primer designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence), add a target-specific primer or a library of target-specific primers designed to amplify either the forward or reverse strand of the target polynucleotide. The target-specific primers include a 3' target-specific sequence and have a 5' universal sequence or no 5' universal sequence. Using both the universal primer and one or more target-specific primers, amplify the mixture by multiple cycles of PCR amplification. The amplified product will be a library of PCR products (with or without 3' and 5' universal sequences) that has amplified either one or the other strand of the original polynucleotide. Purify the first amplification product to remove all unused single-stranded primers. Combine the purified first amplification product with a universal primer designed to hybridize to the universal sequence of the modified target polynucleotide (with or without an additional 5' universal sequence), add a target-specific primer or a library of target-specific primers, and one or more target-specific primers are designed to amplify either the forward or reverse strand (not targeted in the first amplification reaction) of the target polynucleotide. If the forward strand was targeted in the first reaction, the reverse strand is targeted in the second reaction. The target-specific primers include a 3' target-specific sequence and have a 5' universal sequence or no 5' universal sequence. Using both the universal primer and one or more target-specific primers, amplify the mixture by multiple cycles of PCR amplification. Purify the second amplification product to remove all unused primers. Divide the second amplification product into two approximately equal aliquots. Add to each of these aliquots a second universal primer whose 3' end is designed to hybridize to the universal sequence of the modified target polynucleotide and has a 5' tail containing the sequences necessary for next-generation sequencing. Add to each aliquot a different set of target-specific nested primers or a library of target-specific nested primers designed to amplify either the forward or reverse strand of the target polynucleotide. The target-specific primers include a 3' target-specific portion and a 5' universal portion containing the sequences necessary for next-generation sequencing. Using both the universal primer and one or more target-specific primers, amplify the two separate mixtures by multiple cycles of PCR amplification. The amplified products will be two separate libraries of PCR products (each of which has amplified either one or the other strand of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0267] (F) Combine the modified target polynucleotide with universal primers designed to hybridize to the universal sequence of the modified target polynucleotide having an additional 5' universal sequence. Add a target-specific primer or a library of target-specific primers, which are designed to amplify either the forward or reverse strand of the target polynucleotide. The target-specific primers include a 3' target-specific sequence and a 5' universal sequence. Amplify the mixture by multiple cycles of PCR using both the universal primers and one or more target-specific primers. The amplified products will be a library of PCR products (having 5' and 3' universal sequences), each of which has amplified either one or the other of the strands of the original polynucleotide. Purify the first amplification product to remove all unused primers. Combine the purified first amplification product with universal primers designed to hybridize to the universal sequence of the modified target polynucleotide having an additional 5' universal sequence. Add a target-specific primer or a library of target-specific primers, where one or more target-specific primers are designed to amplify the forward or reverse strand of the target polynucleotide (the one not targeted in the first amplification reaction). If the forward strand was targeted in the first reaction, then the reverse strand is targeted in the second reaction. The target-specific primers include a 3' target-specific sequence and a 5' universal sequence. Amplify the mixture by multiple cycles of PCR using both the universal primers and one or more target-specific primers. Purify the second amplification product to remove all unused primers. Mix the second amplification product with two universal primers, the 3' ends of which are designed to hybridize to the universal sequences at the 3' and 5' ends of the amplification product and which have 5' tails containing sequences necessary for next-generation sequencing. Amplify the two mixtures by multiple cycles of PCR using the two universal primers. The amplified products will be a library of PCR products (each of which has independently amplified both strands of the original polynucleotide) and will contain all the necessary sequences compatible with next-generation sequencing.

[0268] Figure 17 A schematic diagram illustrating an illustrative embodiment is depicted. A DNA target polynucleotide is shown. The process of enzymatic fragmentation using a nuclease, or the operation of physical shearing of DNA by sonication, or the use of a transposase can be replaced by the individual or combined targeting of genomic editing tools such as the Cas genes of the enzymes and types I, II, III of the CRISPR subtypes, or a combination thereof. For example, incubate the CRISPR / Cas9 enzyme with a mixture of DNA and one or more guide RNAs. This results in the association of the DNA, the CRISPR / Cas9 enzyme, and the guide RNA, whereby the guide RNA targets double-stranded or single-stranded cleavage of the DNA. Then, the targeted fragmented DNA can be purified and used in any subsequent downstream process (such as the first ATO reaction).

[0269] Figure 18Depicts a schematic diagram of an illustrative embodiment. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. In step (i), the ATO molecule hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The 3'-end of the target polynucleotide is hybridized to the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and extended using the ATO as a template. The extension generates a modified target polynucleotide that includes a random sequence as the UID. After step (i), the ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. In step (ii), the target polynucleotide undergoes an end-repair process followed by ligation to a double-stranded adaptor. The product of this ligation reaction will be a modified target polynucleotide that contains a short 5'-ligation sequence and a longer universal 3'-ligation sequence. It can then be used in downstream processes.

[0270] Figure 19 Depicts a schematic diagram of an illustrative embodiment. The target polynucleotide (PCR product, or DNA, or RNA, or any mixture thereof) can be double-stranded, and only one of the two strands (the forward strand) is shown. The depiction exploits the ability of polymerase to use only DNA (not RNA) as a primer for the extension of polynucleotides. In the first round, the ATO molecule hybridizes to the single-stranded target polynucleotide sequence at one or more positions. The 3'-end of the target polynucleotide is hybridized to the 3'-random sequence portion of the ATO, trimmed (if any 3'-overhangs are present), and only the DNA is extended using the ATO as a template and a polymerase that cannot use an RNA primer. The ATO is digested or removed by affinity capture. In some embodiments, the ATO is not digested or removed. The 3'-end of the modified target DNA polynucleotide is allowed to form a small hairpin by annealing to itself, and then the 3'-end is used as a primer that, once extended, will generate the first complementary strand. The double-stranded DNA with the hairpin will function to preferentially allow the duplex to reform during the second round of ATO hybridization. The 3'-end of the target RNA polynucleotide is hybridized to the 3'-random sequence portion of the ATO and extended using the ATO as a template and a polymerase. The 3'-end of the modified target RNA polynucleotide is allowed to form a small hairpin by annealing to itself, and then the 3'-end is used as a primer that, once extended, will generate the first complementary strand. Then, the hairpin can be digested. The resulting DNA:DNA hybrid and RNA:DNA hybrid can then form templates for further downstream processing. Such as generating a DNA sequencing library, or an RNA sequencing library, or a DNA+RNA sequencing library by selectively amplifying DNA and / or RNA.

[0271] The following paragraphs are provided as clauses and should not be regarded as claims.

[0272] According to a first aspect, the present invention provides:

[0273] 1. A removable template oligonucleotide (RTO) for generating a polynucleotide library, the removable template oligonucleotide (RTO) comprising:

[0274] (a) A 3' random sequence;

[0275] (b) A blocking moiety attached to the 3' end, the blocking moiety rendering the RTO non-extendable;

[0276] (c) A universal sequence from 5' to the random sequence; and

[0277] (b) A nucleotide sequence / modification (NSM) recognizable by an agent,

[0278] wherein the RTO serves as a template, is not incorporated into the reaction product, and is destroyed / removed after the reaction,

[0279] wherein the NSM facilitates the removal of the RTO.

[0280] 2. The removable template oligonucleotide according to clause 1, wherein the NSM is a uracil nucleotide, wherein the uracil nucleotide is incorporated into the RTO during oligonucleotide synthesis in place of a thymine nucleotide; wherein the agent is uracil-DNA glycosylase (UNG), which is capable of destroying / removing the RTO after the reaction ends.

[0281] 3. The removable template oligonucleotide according to clause 1, wherein the NSM is a ribonucleotide, wherein the ribonucleotide is incorporated into the RTO during oligonucleotide synthesis in place of any nucleotide or all nucleotides; wherein the agent is ribonuclease, which is capable of destroying / removing the ATO after the reaction ends.

[0282] 4. The removable template oligonucleotide according to clause 1, wherein the NSM is a restriction enzyme recognition sequence located in the universal sequence; wherein the agent is a restriction enzyme, which is capable of destroying / removing the RTO after the reaction ends.

[0283] 5. The removable template oligonucleotide according to clause 1, wherein the NSM is an affinity binding moiety attached at any point of the RTO; wherein the agent is a protein or an antibody, which is capable of removing the RTO after the reaction ends.

[0284] 6. The removable template oligonucleotide according to clause 5, wherein the affinity binding moiety is biotin; wherein the agent is avidin.

[0285] 7. The removable template oligonucleotide according to any one of clauses 1-6, wherein the RTO comprises an additional unique identifier (UID) sequence located in the universal sequence.

[0286] 8. A removable template oligonucleotide as described in any one of clauses 1-7, wherein the RTO comprises a portion forming a stem-loop structure.

[0287] 9. A method for generating a polynucleotide library, the method comprising:

[0288] (i) Using a target polynucleotide from a sample as a primer and using a removable template oligonucleotide (RTO) as described in any one of clauses 1-8 as a template to generate a modified target polynucleotide;

[0289] (ii) Removing the RTO; and

[0290] (iii) Using a first primer to generate a first complementary sequence (CS) of the modified target polynucleotide, the first primer comprising a universal sequence,

[0291] wherein the generating operation comprises extending a primer hybridized to a template by a polymerase.

[0292] 10. The method as described in clause 9, the method further comprising: using the first complementary sequence as a primer and generating an operation of a modified first CS using a second removable template oligonucleotide (RTO) as described in any one of clauses 1-8 as a template.

[0293] 11. The method as described in clause 9 or 10, the method further comprising: extending a second primer hybridized to the first CS or the modified first CS to form an operation of a second CS, wherein the second primer comprises a target-specific portion or a universal sequence, or both a 3'-target-specific sequence and a 5'-universal sequence.

[0294] 12. The method as described in clause 11, wherein when the target polynucleotide in the sample is double-stranded, the operation of extending the second primer hybridized to the first CS comprises dividing the first CS into two separate reactions, wherein the first reaction comprises a target-specific second primer complementary to the first strand of the target sequence, and the second reaction comprises a target-specific second primer complementary to the second strand of the target sequence, wherein the first strand and the second strand of the target sequence are complementary.

[0295] 13. The method as described in clause 11 or 12, wherein the extending operation comprises linear amplification using the first primer or the second primer.

[0296] 14. The method as described in clause 11 or 12, wherein the extending operation comprises exponential amplification using the first primer and the second primer.

[0297] 15. The method as described in any one of clauses 9-14, wherein the first primer comprises a sample barcode (SBC) sequence and an additional universal sequence compatible with an NGS platform.

[0298] 16. The method according to any one of clauses 9 - 15, wherein when the second primer is a target - specific primer, after linear amplification or exponential amplification using the second primer, an overlapping target - specific third primer is used for further amplification.

[0299] 17. A method for accurately determining the sequence of a target polynucleotide, the method comprising:

[0300] (i) sequencing at least one of the amplified second CSs according to any one of clauses 9 - 16;

[0301] (ii) aligning at least two sequences containing the same UID from (i) and / or aligning the same target sequences of two reactions, wherein each reaction generates sequence information of one strand or the complementary strand of a double - stranded target sequence; and

[0302] (iii) determining the consensus sequence and / or identical variant sequences of the two reactions based on (ii), wherein the consensus sequence and / or variant sequences accurately represent the target polynucleotide sequence.

[0303] 18. A kit for generating a polynucleotide library, the kit comprising a removable template oligonucleotide (RTO) according to any one of clauses 1 - 8 and primers compatible with an NGS platform.

[0304] According to the second aspect, the present invention provides:

[0305] 1. An adaptor template oligonucleotide (ATO) for generating a polynucleotide library, comprising:

[0306] (a) a 3’ random sequence;

[0307] (b) a blocker moiety attached to the 3’ end, the blocker moiety causing the ATO to be non - extendable;

[0308] (c) a universal sequence from 5’ to the random sequence; and

[0309] (b) a nucleotide sequence / modification (NSM) that renders the ATO non - interfering and non - competitive;

[0310] wherein the ATO is used as a template to direct a first extension reaction and is not incorporated into the reaction product.

[0311] 2. The adaptor template oligonucleotide according to clause 1, wherein the NSM is an atypical nucleotide (non - dA, non - dG, non - dT, non - dC), and the atypical nucleotide is a naturally occurring nucleotide or an artificial nucleotide.

[0312] 3. The adaptor template oligonucleotide according to clause 2, wherein the atypical nucleotide is a universal nucleotide.

[0313] 4. The adaptor template oligonucleotide as described in clause 2, wherein the atypical nucleotide includes inosine base, wherein inosine is used to replace the normal guanine position in the ATO sequence, and deoxyinosine preferentially directs the incorporation of dC into the growing nascent strand by DNA polymerase.

[0314] 5. The adaptor template oligonucleotide as described in clause 1, wherein the NSM can be recognized by an agent that facilitates the digestion / removal of ATO.

[0315] 6. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is uracil nucleotide, and uracil nucleotide is incorporated into ATO during oligonucleotide synthesis to replace thymine nucleotide; the agent is uracil-DNA glycosylase (UNG), which can destroy / remove ATO after the end of the first extension reaction.

[0316] 7. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is ribonucleotide, and ribonucleotide is incorporated into ATO during oligonucleotide synthesis to replace any nucleotide or all nucleotides; the agent is ribonuclease, which can destroy / remove ATO after the end of the first extension reaction.

[0317] 8. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is a restriction enzyme recognition sequence located in the universal sequence; the agent is a restriction enzyme, which can destroy / remove ATO after the end of the first extension reaction.

[0318] 9. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is an affinity binding moiety attached at any point of ATO; the agent is a protein or antibody, which can remove ATO after the end of the first extension reaction.

[0319] 10. The adaptor template oligonucleotide as described in clause 9, wherein the affinity binding moiety is biotin; the agent is avidin.

[0320] 11. The adaptor template oligonucleotide as described in any one of clauses 1-10, wherein ATO includes an additional unique identifier (UID) sequence located in the universal sequence.

[0321] 12. The adaptor template oligonucleotide as described in any one of clauses 1-10, wherein ATO includes a part that forms a stem-loop structure.

[0322] 13. A method for generating a polynucleotide library, the method comprising:

[0323] (i) Generating a modified target polynucleotide by using a target polynucleotide from a sample as a primer and using an adaptor template oligonucleotide (ATO) as described in any one of clauses 1-12 as a template; and

[0324] (ii) Generating a first complementary sequence (CS) of the modified target polynucleotide by using a first primer comprising a universal sequence and using the modified target polynucleotide as a template,

[0325] wherein the generating operation comprises extending a primer hybridized to the template by polymerase.

[0326] 14. The method according to clause 13, the method further comprising: an operation of generating a modified first CS by using the first complementary sequence as a primer and using a second adaptor template oligonucleotide (ATO) as described in any one of clauses 1-12 as a template and extending the first CS on the ATO template.

[0327] 15. The method according to clause 13, wherein step (i) further comprises an operation of removing the ATO.

[0328] 16. The method according to clause 13, 14 or 15, the method further comprising: an operation of extending a second primer hybridized to the first CS or the modified first CS to form a second CS, wherein the second primer comprises a target-specific part or a universal sequence, or both a 3'-target-specific sequence and a 5'-universal sequence.

[0329] 17. The method according to clause 16, wherein when the target polynucleotide in the sample is double-stranded, the operation of extending the second primer hybridized to the first CS comprises dividing the first CS into two separate reactions, wherein the first reaction comprises a target-specific second primer complementary to the first strand of the target sequence, and the second reaction comprises a target-specific second primer complementary to the second strand of the target sequence, wherein the first strand and the second strand of the target sequence are complementary.

[0330] 18. The method according to clause 13, 14, 16 or 17, wherein the extending operation comprises linear amplification.

[0331] 19. The method according to clause 16, wherein the extending operation comprises exponential amplification using the first primer and the second primer.

[0332] 20. The method according to any one of clauses 13-19, wherein the first primer comprises a sample barcode (SBC) sequence and an additional universal sequence compatible with the NGS platform.

[0333] 21. The method according to any one of clauses 13-19, wherein when the second primer is a target-specific primer, after linear amplification or exponential amplification using the second primer, a nested target-specific third primer is used for further amplification.

[0334] 22. A method for accurately determining the sequence of a target polynucleotide, the method comprising:

[0335] (i) sequencing at least one of the amplified second CSs described in any one of clauses 13 - 21;

[0336] (ii) aligning at least two sequences with the same UID from (i) and / or aligning the same target sequences of two reactions, wherein each reaction generates sequence information of one strand or the complementary strand of the duplex target sequence; and

[0337] (iii) determining the consensus sequence and / or identical variant sequences of the two reactions based on (ii), wherein the consensus sequence and / or variant sequences accurately represent the target polynucleotide sequence.

[0338] 23. A kit for generating a polynucleotide library, the kit comprising the adaptor template oligonucleotide (ATO) described in any one of clauses 1 - 12 and primers compatible with an NGS platform.

[0339] According to the third aspect, the present invention provides:

[0340] 1. An adaptor template oligonucleotide (ATO) for generating a polynucleotide library, comprising:

[0341] (a) a 3’ random sequence;

[0342] (b) a blocker moiety attached to the 3’ end, the blocker moiety rendering the ATO non - extendable;

[0343] (c) a universal sequence from 5’ to the random sequence; and

[0344] (b) a nucleotide sequence / modification (NSM);

[0345] wherein the ATO is used as a template to direct the first reaction, and all or part of the ATO is not incorporated into the first reaction product, and the NSM renders the ATO non - interfering and non - competitive in the reactions after the first reaction.

[0346] 2. The adaptor template oligonucleotide according to clause 1, wherein the NSM is an atypical nucleotide (non - dA, non - dG, non - dT, non - dC), and the atypical nucleotide is a naturally occurring nucleotide or an artificial nucleotide.

[0347] 3. The adaptor template oligonucleotide according to clause 2, wherein the atypical nucleotide is a universal nucleotide.

[0348] 4. The adaptor template oligonucleotide as described in clause 2, wherein the atypical nucleotide includes inosine base, wherein inosine is used to replace the guanine position in the ATO sequence, and deoxyinosine preferentially guides the incorporation of dC into the growing nascent strand by DNA polymerase.

[0349] 5. The adaptor template oligonucleotide as described in clause 1, wherein the NSM can be recognized by an agent that facilitates the digestion / removal of ATO.

[0350] 6. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is uracil nucleotide, and the uracil nucleotide is incorporated into ATO during oligonucleotide synthesis to replace thymine nucleotide; the agent is uracil-DNA glycosylase (UNG), which can destroy / remove ATO after the end of the first extension reaction.

[0351] 7. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is ribonucleotide, and the ribonucleotide is incorporated into ATO during oligonucleotide synthesis to replace any nucleotide or all nucleotides; the agent is ribonuclease, which can destroy / remove ATO after the end of the first extension reaction.

[0352] 8. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is a restriction enzyme recognition sequence located in the universal sequence; the agent is a restriction enzyme, which can destroy / remove ATO after the end of the first extension reaction.

[0353] 9. The adaptor template oligonucleotide as described in clause 5, wherein the NSM is an affinity binding moiety attached at any point of ATO; the agent is a protein or antibody, which can remove ATO after the end of the first extension reaction.

[0354] 10. The adaptor template oligonucleotide as described in clause 9, wherein the affinity binding moiety is biotin; the agent is avidin.

[0355] 11. The adaptor template oligonucleotide as described in any one of clauses 1-10, wherein the ATO includes an additional unique identifier (UID) sequence located in the universal sequence.

[0356] 12. The adaptor template oligonucleotide as described in clause 1, wherein the ATO includes a 5' stem partial sequence complementary to a part of the universal sequence, which can form a stem-loop structure.

[0357] 13. The adaptor template oligonucleotide as described in clause 12, wherein the loop part includes a non-replicable linkage.

[0358] 14. The adaptor template oligonucleotide as described in clause 13, wherein the non-replicable linker is a C3 spacer phosphoramidite, or a triethylene glycol spacer, or an 18-atom hexaethylene glycol spacer, or a 1’,2’-dideoxyribose (d-spacer).

[0359] 15. The adaptor template oligonucleotide as described in clause 12, wherein the loop portion comprises nucleotides that can be digested.

[0360] 16. The adaptor template oligonucleotide as described in clause 12, wherein the 5’ end of the stem portion sequence comprises a phosphate group.

[0361] 17. The adaptor template oligonucleotide as described in clause 1, wherein the ATO comprises an upper single strand complementary to a part of the universal sequence, which is capable of forming a partially double-stranded structure.

[0362] 18. The adaptor template oligonucleotide as described in clause 17, wherein the upper single strand comprises nucleotides that can be digested.

[0363] 19. The adaptor template oligonucleotide as described in clause 17, wherein the 5’ end of the upper single strand comprises a phosphate group.

[0364] 20. The adaptor template oligonucleotide as described in clause 17, wherein the 3’ end of the upper single strand comprises biotin.

[0365] 21. A method for generating a polynucleotide library, the method comprising:

[0366] (i) generating a modified target polynucleotide by using a target polynucleotide from a sample, the target polynucleotide from the sample hybridizing with the 3’ random sequence of an adaptor template oligonucleotide (ATO) (first ATO) as described in any one of clauses 1-20 in an enzymatic first reaction, and the enzymatic first reaction adding an adaptor sequence to the 3’ end of the target polynucleotide; and

[0367] (ii) using a first primer comprising a universal sequence and using the modified target polynucleotide as a template to generate a first complementary sequence (CS) of the modified target polynucleotide, wherein the first primer hybridizes with the template and is extended by a polymerase.

[0368] 22. The method as described in clause 21, wherein the first reaction is a primer extension reaction, wherein the target polynucleotide serves as a primer and is extended on the ATO template by a DNA polymerase.

[0369] 23. The method as described in clause 22, wherein the DNA polymerase has strand displacement activity, and wherein during the extension, the stem-loop structure is opened or the upper ATO strand is displaced.

[0370] 24. The method as described in clause 22, wherein the first reaction is an extension-ligation reaction, wherein DNA polymerase extends the target, and DNA ligase ligates the extended target sequence to the 5'-stem portion of the ATO or the upper strand of the ATO.

[0371] 25. The method as described in clause 22, wherein the first reaction is a ligation reaction, wherein DNA ligase ligates the target sequence to the 5'-stem portion of the ATO or the upper strand of the ATO.

[0372] 26. The method as described in clause 21, the method further comprising, after the first reaction, an operation of digesting a part of the ATO or removing a part of the ATO by affinity capture.

[0373] 27. The method as described in clause 21, the method further comprising an operation of generating a modified first CS by using a first CS, the first CS hybridizing with the 3'-random sequence of a second ATO as described in any one of clauses 1-20 in an enzymatic reaction, the enzymatic reaction adding an adaptor sequence to the 3'-end of the first CS, wherein the second ATO includes a different 5'-universal sequence compared with the first ATO.

[0374] 28. The method as described in clause 21, the method further comprising an operation of generating a modified first CS by ligating a double-stranded adaptor to the product of step (ii).

[0375] 29. The method as described in clause 21, 27 or 28, the method further comprising an operation of extending a second primer hybridizing with the first CS or the modified first CS, thereby forming a second CS, wherein the second primer includes a target-specific portion or a universal sequence, or both a 3'-target-specific sequence and a 5'-universal sequence.

[0376] 30. The method as described in clause 29, wherein when the target polynucleotide in the sample is double-stranded and the second primer is a target-specific primer, the operation of extending the second primer hybridizing with the first CS includes dividing the first CS into two separate reactions, wherein the forward reaction includes a target-specific second primer complementary to the forward strand of the target sequence, and the reverse reaction includes a target-specific second primer complementary to the reverse strand of the target sequence, wherein the forward strand and the reverse strand of the target sequence are complementary.

[0377] 31. The method as described in any one of clauses 21-30, wherein the operation of generating the first CS includes linear amplification with 1-30 cycles.

[0378] 32. The method as described in any one of clauses 21-31, wherein if the target is RNA, the operation of generating the first CS includes a reverse transcription reaction using reverse transcriptase.

[0379] 33. The method according to any one of clauses 21-32, wherein the extension operation includes exponential amplification using a first primer and a second primer.

[0380] 34. The method according to any one of clauses 21-33, wherein when the second primer is a target-specific primer, and after linear or exponential amplification using the second primer, a nested target-specific third primer is used for further amplification.

[0381] 35. The method according to any one of clauses 21-34, wherein the first primer or the fourth primer targeting a universal adaptor sequence includes a sample barcode (SBC) sequence and an additional universal sequence compatible with the NGS platform.

[0382] 36. A method for accurately determining the sequence of a target polynucleotide, the method comprising:

[0383] (i) sequencing at least one of the amplified first CS or the amplified second CS according to any one of clauses 21-35;

[0384] (ii) aligning at least two sequences having the same UID from (i) and / or aligning the same target sequences of two reactions, wherein each reaction generates sequence information of one strand or the complementary strand of a double-stranded target sequence; and

[0385] (iii) determining the consensus sequence and / or the same variant sequences of the two reactions based on (ii), wherein the consensus sequence and / or the variant sequences accurately represent the target polynucleotide sequence.

[0386] 37. A kit for generating a polynucleotide library, the kit comprising the adaptor template oligonucleotide (ATO) according to any one of clauses 1-36 and primers compatible with the NGS platform.

[0387] According to the fourth aspect, the present invention provides:

[0388] 1. An adaptor template oligonucleotide (ATO) for extending a polynucleotide, comprising:

[0389] (a) a 3' random sequence;

[0390] (b) a blocker attached to the 3' end, the blocker rendering the ATO non-extendable; and

[0391] (c) a universal sequence from the 5' to the random sequence;

[0392] wherein the ATO is used as a template to direct an extension reaction by a polymerase.

[0393] 2. The adaptor template oligonucleotide as described in clause 1, the adaptor template oligonucleotide (ATO) further comprises one or more moieties, and the one or more moieties render the ATO degradable or non-interfering and non-competitive in the reaction after the extension reaction, wherein the moieties can be recognized by an agent that facilitates the digestion / removal of the RTO.

[0394] 3. The adaptor template oligonucleotide as described in clause 2, wherein the moiety is a uracil nucleotide, and the agent comprises dU-glycosylase, which is capable of digesting / removing the ATO after the first extension reaction.

[0395] 4. The adaptor template oligonucleotide as described in clause 2, wherein the moiety is a ribonucleotide, and the ribonucleotide is incorporated into the ATO during oligonucleotide synthesis to replace any nucleotide or all nucleotides; the agent is ribonuclease, which is capable of digesting / removing the ATO after the first extension reaction.

[0396] 5. The adaptor template oligonucleotide as described in clause 1, wherein the ATO is an RNA oligonucleotide.

[0397] 6. The adaptor template oligonucleotide as described in clause 1, wherein the ATO is a DNA oligonucleotide.

[0398] 7. The adaptor template oligonucleotide as described in clause 1, wherein the ATO is a combination of a DNA oligonucleotide and an RNA oligonucleotide.

[0399] 8. The adaptor template oligonucleotide as described in clause 2, wherein the moiety is a restriction enzyme recognition sequence, and the agent is a restriction enzyme.

[0400] 9. The adaptor template oligonucleotide as described in clause 1, wherein the universal sequence comprises a sequence capable of serving as an RNA polymerase promoter.

[0401] 10. The adaptor template oligonucleotide as described in clause 9, wherein the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.

[0402] 11. The adaptor template oligonucleotide as described in clause 9, wherein the universal sequence comprises a 5' RNA polymerase promoter sequence and a priming site, and the priming site is located 3' of the RNA polymerase promoter sequence.

[0403] 12. The adaptor template oligonucleotide as described in clause 1, wherein the universal sequence is double-stranded or partially double-stranded.

[0404] 13. The adaptor template oligonucleotide as described in clause 12, wherein the ATO comprises a 5' stem partial sequence complementary or partially complementary to a part or all of the universal sequence, which is capable of forming a stem-loop structure.

[0405] 14. The adaptor template oligonucleotide as described in clause 13, wherein the ATO comprises, in the 5' to 3' order: a 5' stem portion, an RNA polymerase sequence, a priming site sequence, and a 3' random / degenerate sequence.

[0406] 15. The adaptor template oligonucleotide as described in clause 13, wherein the RNA polymerase sequence is located in the loop portion.

[0407] 16. The adaptor template oligonucleotide as described in clause 13, wherein the loop portion comprises a non-replicable linkage.

[0408] 17. The adaptor template oligonucleotide as described in clause 13, wherein the loop portion does not comprise a non-replicable linkage.

[0409] 18. The adaptor template oligonucleotide as described in clause 13, wherein if the 5' of the stem portion comprises an additional sequence, there is a non-replicable linkage between the stem portion and the additional sequence.

[0410] 19. The adaptor template oligonucleotide as described in clause 13, wherein the 5' stem portion comprises a non-replicable linkage.

[0411] 20. The adaptor template oligonucleotide as described in clauses 13 - 19, wherein the non-replicable linkage is a C3 spacer phosphoramidite, or a triethylene glycol spacer, or a 18-atom hexaethylene glycol spacer, or a 1',2'-dideoxyribose (d spacer).

[0412] 21. The adaptor template oligonucleotide as described in clause 13, wherein the double-stranded stem portion comprises a non-complementary region, and the non-complementary region in the universal sequence strand comprises a random sequence.

[0413] 22. The adaptor template oligonucleotide as described in clause 13, wherein the stem portion forms two or more broken segments separated by one or more non-replicable linkages.

[0414] 23. The adaptor template oligonucleotide as described in clause 13, wherein the stem portion forms two or more broken segments separated by one or more mismatched base pairs.

[0415] 24. The adaptor template oligonucleotide as described in clause 12, wherein the ATO comprises an upper single strand that is complementary or partially complementary to the universal sequence.

[0416] 25. The adaptor template oligonucleotide as described in clause 24, wherein the 5' end of the upper single strand comprises a phosphate group.

[0417] 26. The adaptor template oligonucleotide as described in clause 1, wherein the ATO further comprises an affinity binding moiety attached at any position of the ATO.

[0418] 27. An adaptor template oligonucleotide as described in clause 26, wherein the affinity binding moiety is biotin.

[0419] 28. An adaptor template oligonucleotide as described in any of the preceding clauses, wherein the 5' end comprises a phosphate group.

[0420] 29. An adaptor template oligonucleotide as described in any of the preceding clauses, wherein the universal sequence comprises a random sequence as an additional unique identifier (UID) sequence.

[0421] 30. An adaptor template oligonucleotide as described in clause 29, wherein the ATO comprises an additional UID within the loop segment.

[0422] 31. An adaptor template oligonucleotide as described in clause 29, wherein the ATO comprises an additional UID within the stem segment.

[0423] 32. An adaptor template oligonucleotide as described in clause 1, wherein the ATO sequence comprises non - canonical nucleotides (non - dA, non - dG, non - dT, non - dC), and the non - canonical nucleotides are naturally occurring nucleotides or artificial nucleotides.

[0424] 33. An adaptor template oligonucleotide as described in clause 32, wherein the non - canonical nucleotides are universal nucleotides.

[0425] 34. An adaptor template oligonucleotide as described in clause 32, wherein the non - canonical nucleotides comprise inosine bases.

[0426] 35. An adaptor template oligonucleotide as described in clause 32, wherein the 3' random sequence comprises non - canonical nucleotides.

[0427] 36. An adaptor template oligonucleotide as described in clause 32, wherein the universal sequence comprises non - canonical nucleotides.

[0428] 37. An adaptor template oligonucleotide as described in clause 1, wherein the 3' end comprises one or more modified nucleotides or linkages that render the ATO resistant to the 3' exonuclease activity of DNA polymerase.

[0429] 38. An adaptor template oligonucleotide as described in clause 37, wherein the modified linkage is a phosphorothioate bond.

[0430] 39. An adaptor template oligonucleotide as described in clause 1, the adaptor template oligonucleotide (ATO) further comprises a specific sequence 3' of the random sequence, wherein the specific sequence is capable of hybridizing with the specific sequence of a polynucleotide, and a portion of the 3' random / degenerate sequence serves as a template on which the polynucleotide is extended by a polymerase.

[0431] 40. A composition, the composition comprising at least one nucleic acid polymerase and the adaptor template oligonucleotide (ATO) as described in any one of the preceding clauses.

[0432] 41. The composition as described in clause 40, wherein the nucleic acid polymerase is a DNA polymerase.

[0433] 42. The composition as described in clause 41, wherein the DNA polymerase has strand displacement activity.

[0434] 43. The composition as described in clause 41, wherein the DNA polymerase has 3' to 5' exonuclease activity.

[0435] 44. The composition as described in clause 41, wherein the DNA polymerase is a template-dependent polymerase, rather than a non-template-dependent polymerase.

[0436] 45. A method for extending a target polynucleotide, the method comprising:

[0437] (i) generating a modified target polynucleotide by incubating the target polynucleotide with the composition as described in any one of the preceding clauses, wherein in the enzymatic first ATO reaction, the 3' end of the target polynucleotide hybridizes with the 3' random sequence of the adaptor template oligonucleotide (first ATO), wherein the 3' end of the target polynucleotide is extended using the ATO as a template, and wherein if a 3' overhang exists, the 3' end of the target polynucleotide is trimmed before the extension occurs.

[0438] 46. The method as described in clause 45, the method further comprising (ii) an operation of generating a first complementary sequence (CS) of the modified target polynucleotide.

[0439] 47. The method as described in clause 46, wherein the operation of generating the first CS comprises extension using a first primer and using the modified target polynucleotide as a template, wherein the first primer hybridizes with the template and is extended by a polymerase.

[0440] 48. The method as described in clause 46, wherein the operation of generating the first CS comprises in vitro transcription from the double-stranded promoter region in the modified target polynucleotide using an RNA polymerase, and the modified target polynucleotide is generated by extension on an ATO containing an RNA polymerase promoter.

[0441] 49. The method as described in clause 46, wherein the operation of generating the first CS comprises heat denaturing the modified target polynucleotide, annealing the 3' stem-loop structure of the modified target polynucleotide, and self-priming to extend to form the first CS.

[0442] 50. The method as described in clause 46, wherein the operation of generating the first CS includes annealing a target-specific primer to the modified target polynucleotide and extension by a polymerase.

[0443] 51. The method as described in any one of clauses 46-50, the method further includes an operation of digesting ATO before or after generating the first complementary sequence (CS) of the modified target polynucleotide.

[0444] 52. The method as described in any one of clauses 46-51, the method further includes an operation of affinity capture before or after generating the first complementary sequence (CS) of the modified target polynucleotide.

[0445] 53. The method as described in clause 45, wherein the first ATO reaction includes extension and ligation, wherein the DNA polymerase extends the 3' end of the target, and the DNA ligase ligates the extended target sequence to the 5' stem portion of ATO or the upper single strand of ATO.

[0446] 54. The method as described in clause 46, the method further includes an operation of generating a modified first CS by incubating the first CS with the composition as described in any one of clauses 35-39, wherein in the enzymatic second ATO reaction, the 3' end of the first CS hybridizes with the 3' random sequence of the adaptor template oligonucleotide (second ATO), wherein the 3' end of the first CS is extended using ATO as a template, wherein if there is a 3' overhang, the 3' end of the first CS is trimmed before the extension occurs, and the second ATO includes a different 5' universal sequence of the first ATO.

[0447] 55. The method as described in clause 46, the method further includes an operation of generating a modified first CS by ligating an adaptor to the product of step (ii).

[0448] 56. The method as described in any one of clauses 46-55, the method further includes an operation of extending a second primer hybridized to the first CS or the modified first CS to form a second CS, wherein the second primer includes a target-specific portion or a universal sequence, or both a 3' target-specific sequence and a 5' universal sequence.

[0449] 57. The method as described in clause 56, wherein when the target polynucleotide in the sample is double-stranded and the second primer is a target-specific primer, the operation of extending the second primer hybridized to the first CS includes dividing the first CS into two separate reactions, wherein the forward reaction includes a target-specific second primer complementary to the forward strand of the target sequence, and the reverse reaction includes a target-specific second primer complementary to the reverse strand of the target sequence, and the forward strand and the reverse strand of the target sequence are complementary.

[0450] 58. The method as described in clause 47, wherein the operation of generating the first CS includes linear amplification with 1 to 30 cycles or more cycles.

[0451] 59. The method as described in any one of clauses 47 or 49, wherein if the target is RNA, the operation of generating the first CS includes a reverse transcription reaction using a reverse transcriptase.

[0452] 60. The method as described in any one of clauses 56 - 59, the method further includes exponential amplification using a first primer and a second primer.

[0453] 61. The method as described in any one of clauses 56 - 60, wherein when the second primer is a target - specific primer, after linear amplification or exponential amplification using the second primer, a nested target - specific third primer is used for further amplification.

[0454] 62. The method as described in any one of clauses 56 - 61, wherein the first primer or the third primer includes a sample barcode (SBC) sequence and an additional universal sequence compatible with the NGS platform.

[0455] 63. The method as described in clause 45, the method includes an operation of fragmenting the target polynucleotide before the first ATO reaction.

[0456] 64. The method as described in clause 63, wherein the operation of fragmenting the target polynucleotide includes contacting a double - stranded polynucleotide with a transposase bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and a universal sequence, wherein the transposase / transposon DNA complex binds to a target position on the double - stranded polynucleotide and cuts the double - stranded polynucleotide into multiple double - stranded fragments, and wherein each double - stranded fragment has transposon DNA bound to each 5' end of the double - stranded fragment.

[0457] 65. The method as described in clause 63, wherein the fragmenting operation includes the use of targeted fragmentation using a genome - editing tool.

[0458] 66. The method as described in clause 65, wherein the genome - editing tool includes clustered regularly interspaced short palindromic repeats and CRISPR - associated protease 9 (CRISPR / Cas9).

[0459] 67. The method as described in clause 63, which includes an operation of heat - denaturing the fragmented target polynucleotide before the first ATO reaction.

[0460] 68. The method as described in clause 64, wherein the transposase is Tn5 transposase.

[0461] 69. The method as described in clause 63, wherein the operations of fragmenting and labeling the target polynucleotide include contacting a single-stranded polynucleotide with a random primer comprising a 5' universal sequence and a 3' random sequence, and extending the random primer on the target polynucleotide to generate a 5'-labeled fragmented polynucleotide.

[0462] 70. The method as described in clause 45, wherein the target polynucleotide comprises a free 3'-hydroxyl group.

[0463] 71. The method as described in clause 45, wherein the target polynucleotide is single-stranded DNA, or single-stranded RNA, or a combination of single-stranded RNA and single-stranded DNA.

[0464] 72. A method for extending a target polynucleotide, the method comprising:

[0465] Mixing the target polynucleotide with a DNA polymerase and an adaptor template oligonucleotide (ATO) comprising a 3' random sequence, the adaptor template oligonucleotide (ATO) having a blocked and modified 3' end to be resistant to 3'-exonuclease activity;

[0466] Incubating the mixture under conditions that promote annealing, trimming the 3' overhang (if present), and extending to generate a modified target polynucleotide; and

[0467] Optionally degrading the ATO.

[0468] 73. A method for generating a sequencing library, the method comprising:

[0469] Mixing the target polynucleotide with a DNA polymerase and an adaptor template oligonucleotide (ATO) comprising a 3' random sequence, the adaptor template oligonucleotide (ATO) being blocked at the 3' end and modified to be resistant to 3'-exonuclease activity;

[0470] Incubating the mixture under conditions that promote annealing, trimming the 3' overhang (if present), and extending to generate a modified target polynucleotide;

[0471] Optionally degrading the ATO; and

[0472] Amplifying the modified target polynucleotide using a primer compatible with an NGS platform.

[0473] 74. The method as described in clause 73, the method comprising an operation of fragmenting the target polynucleotide before mixing.

[0474] 75. The method as described in clause 73, wherein the target polynucleotide is a naturally occurring fragmented polynucleotide.

[0475] 76. The method as described in clause 75, wherein the naturally occurring fragmented polynucleotide is the circulating free nucleic acid in plasma.

[0476] 77. The method as described in clause 74, wherein the operation of fragmenting the target polynucleotide includes contacting the double-stranded polynucleotide with a transposase bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and a universal sequence, wherein the transposase / transposon DNA complex binds to the target position on the double-stranded polynucleotide and cleaves the double-stranded polynucleotide into multiple double-stranded fragments, and wherein each double-stranded fragment has transposon DNA bound to each 5' end of the double-stranded fragment.

[0477] 78. A method for generating a sequencing library, the method comprising:

[0478] adding an adaptor sequence to the single-stranded target polynucleotide defined in any one of clauses 45-69 by extending the single-stranded target polynucleotide on the ATO; and

[0479] amplifying the adaptor-labeled target polynucleotide using a primer compatible with the NGS platform.

[0480] 79. A kit comprising the composition described in any one of clauses 1 to 44.

[0481] 80. A kit for generating a polynucleotide library, the kit comprising an adaptor template oligonucleotide (ATO) defined in any one of clauses 1-41, a polymerase, and a primer compatible with the NGS platform. Examples

[0482] Example 1

[0483] Using deoxyribonucleic acid (DNA) as the target polynucleotide for generating a next-generation sequencing library (e.g., but not limited to, compatible with an Illumina next-generation sequencer) using one or more adaptor template oligonucleotides having a stem-loop structure, wherein the loop includes a chemical spacer (e.g., but not limited to, a C3 spacer, a C18 spacer).

[0484] Materials

[0485] Target polynucleotide, human gDNA (BIO-35025)

[0486] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0487] Adapter Template Oligonucleotide (ATO) 1-014, Adapter Template Oligonucleotide (ATO) 1-015, Adapter Template Oligonucleotide (ATO) 1-016, Adapter Template Oligonucleotide (ATO) 1-017, Adapter Template Oligonucleotide (ATO) 1-018 (Table 1)

[0488] TAQ DNA Polymerase (NEB, M0273L)

[0489] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0490] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L)

[0491] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0212L)

[0492] dNTP (NEB, N0447s)

[0493] Enzyme (NEB, M5505S)

[0494] High-Fidelity DNA Polymerase (NEB, M0530S)

[0495] Phusion Buffer (NEB, M0530S)

[0496] Primers 2-001, 2-002, 2-003 (Table 2)

[0497] Ammonium Sulfate (SIGMA, A4418)

[0498] Agencourt AMPure XP (Beckman Coulter, A63881)

[0499] Agilent Bioanalyzer High Sensitivity Kit (Product ID 5067-4626)

[0500] Method

[0501] One or more adapter template oligonucleotides are annealed to deoxyribonucleic acid

[0502] An appropriate amount of deoxyribonucleic acid (DNA) of any length (fragmented or unfragmented, single-stranded or double-stranded) is mixed with an appropriate amount of one or more adapter template oligonucleotides (one or more ATOs; e.g., but not limited to, hairpin-forming oligonucleotides) in a suitable buffer. As Figure 14As depicted in part (A) herein, in this example, human gDNA was fragmented using a fragmentation kit to generate fragmented DNA between 100 - 400 bp. A total of 25 ng was mixed with 50 picomoles of each of four ATOs (1 - 001, 1 - 002, 1 - 003, 1 - 004) (a total of 200 picomoles of ATO) in H2O to a final volume of 15 μl. The mixture was heated at a suitable temperature (in this example, 95 °C) for a suitable time (in this example, 2 minutes), or the mixture was heated at other temperatures or times that can cause double-stranded nucleic acids to denature into single strands. As Figure 14 As depicted in part (B) herein, subsequently, the nucleic acid and the mixture of one or more ATOs were cooled (in this example, cooled for 2 minutes to 4 °C), or the nucleic acid and the mixture of one or more ATOs were cooled at other suitable temperatures or times that can promote annealing between the nucleic acid and one or more ATOs.

[0503] Target polynucleotide extension by using the target polynucleotide as a primer and one or more adaptor template oligonucleotides as templates

[0504] A suitable amount of one or more DNA polymerases (with or without proofreading activity, with or without strand displacement activity) (such as but not limited to, Bst polymerase, DNA polymerase I, large (Klenow) fragment, Taq DNA polymerase, or Phusion) mixed with one or more suitable buffers and suitable dNTPs was combined with the DNA - ATO mixture. In this example, a total of 2.5 units of Klenow and 2.5 units of Taq DNA polymerase, 2 μl of 10× Taq DNA polymerase buffer, and 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) were combined with the DNA - ATO mixture to a final volume of 20 μl. In this example, to further promote annealing of the target nucleic acid and one or more ATOs in the presence of one or more DNA polymerases and one or more buffers, the mixture was incubated at a temperature below 25 °C for 30 minutes, or the mixture was incubated at other suitable temperatures or times that can promote annealing between the nucleic acid and one or more ATOs. In this example, to initiate the extension of the target polynucleotide using the ATO sequence as a template, the incubation temperature was increased to 37 °C for 30 minutes, or the incubation temperature was increased to other suitable temperatures that can promote DNA polymerase activity. The product is a modified target polynucleotide. As Figure 14 As depicted in part (C) herein.

[0505] Degradation of one or more adaptor template oligonucleotides

[0506] By adding a combination of (e.g., but not limited to) dU-glycosylase and apurinic / apyrimidinic endonuclease, one or more adaptor template oligonucleotides are selectively degraded (in this example, 2 units of USER enzyme are added to 20 μl of the modified target polynucleotide reaction mixture), followed by sequential incubation at 37 °C for 30 minutes and then at 25 °C for 15 minutes (in this example), or sequential incubation at other suitable temperatures and times that can promote enzyme activity. Any suitable purification method can be used to optionally remove one or more degraded ATOs. As Figure 14 depicted in part (D) of

[0507] Single extension or linear amplification of the modified target polynucleotide

[0508] The purified or unpurified target polynucleotide extension product (modified target polynucleotide) is combined with the following: a primer complementary to the conserved (universal) sequence now present at the 3' of the target DNA molecule, a DNA polymerase with or without proofreading activity (e.g., but not limited to, Phusion DNA polymerase), one or more suitable buffers, and suitable dNTPs. In this example, all USER-treated modified target polynucleotides (purified or unpurified) are combined with the following: 50 picomoles of a primer (2-001) complementary to the universal sequence now present at the 3' end of the target DNA molecule, 2 units of Phusion DNA polymerase, 10 μl of 5× Phusion DNA polymerase buffer, and 12 nanomoles of each dNT (dATP / dTTP / dCTP / dGTP). Then, the mixture is thermocycled to perform a single extension or linear amplification of the target nucleic acid extension product to generate a first complementary sequence (CS), in this example, 98 °C for 30 seconds, 5 cycles of 98 °C for 5 seconds, 60 °C for 1 minute, and 72 °C for 1 minute, and then 72 °C for 2 minutes. Then, the linear CS product can be optionally purified by any suitable method (e.g., using magnetic beads). As Figure 14 depicted in part (E) of

[0509] One or more second adaptor template oligonucleotides are annealed to the first complementary strand

[0510] Mix the first CS product with an appropriate amount of one or more second adaptor template oligonucleotides (such as, but not limited to, oligonucleotides forming a hairpin) in a suitable buffer. In this example, mix the first CS product with 200 picomoles of ATO (1 - 018) in H2O to a final volume of 15 μl. Heat the mixture at a suitable temperature (in this example, 95 °C) for a suitable time (in this example, 2 minutes), or heat the mixture at other temperatures or times that can cause double-stranded nucleic acids to denature into single strands. In this example, subsequently cool the mixture of nucleic acid and one or more ATOs for 2 minutes to 4 °C, or cool the mixture of nucleic acid and one or more ATOs at other suitable temperatures or times that can promote annealing between the nucleic acid and one or more ATOs. As Figure 14 depicted in part (F) of

[0511] Perform first complementary strand extension to form a modified first complementary strand by using the first complementary strand as a primer and one or more second adaptor template oligonucleotides as templates

[0512] Combine an appropriate amount of one or more DNA polymerases (with or without proofreading activity, with or without strand displacement activity) (such as, but not limited to, Taq DNA polymerase or Phusion) mixed with one or more suitable buffers and suitable dNTPs with the one or more DNA - ATO mixtures. In this example, combine a total of 2.5 units of Klenow and 2.5 units of TAQ DNA polymerase, 2 μl of 10×TAQ DNA polymerase buffer, and 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) with the DNA - ATO mixture to a final volume of 20 μl. To further promote annealing of the target nucleic acid and one or more ATOs in the presence of one or more DNA polymerases and one or more buffers, incubate the mixture in this example at a temperature below 25 °C for 30 minutes, or incubate at other temperatures or times that can promote annealing between the nucleic acid and one or more ATOs. In this example, to promote extension of the target nucleic acid using one or more ATO sequences as templates, increase the incubation temperature to 37 °C for 30 minutes, or increase the incubation temperature to other suitable temperatures that can promote DNA polymerase activity. As Figure 14 depicted in part (G) of

[0513] Degradation of one or more adaptor template oligonucleotides

[0514] By adding a combination of (e.g., but not limited to) dU-glycosylase and apurinic / apyrimidinic endonuclease, selectively degrading one or more adaptor template oligonucleotides (in this example, 2 units of USER enzyme were added to 20 μl of the modified target polynucleotide reaction mixture), followed by sequential incubation (in this example, 37 °C for 30 minutes and then 25 °C for 15 minutes), or sequential incubation at other suitable temperatures and times that can promote enzyme activity. Any suitable purification method can be used to optionally remove one or more degraded ATOs. As Figure 14 depicted in the (H) portion of

[0515] Exponential amplification of the modified first complementary strand product by PCR

[0516] Combine the modified first CS product (purified or unpurified) with the following: two primers (one primer having sequence similarity to an extension sequence formed by one or more first ATOs and designed to bind to the extension sequence formed by one or more first ATOs and will contain sequences compatible with and necessary for sequencing using next-generation sequencing techniques (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence), and the second primer having sequence similarity to an extension sequence formed by one or more second ATOs and designed to bind to the extension sequence formed by one or more second ATOs and also contain sequences compatible with and necessary for sequencing using next-generation sequencing techniques (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence)), a proofreading polymerase (e.g., but not limited to, Phusion DNA polymerase, Q5), one or more suitable buffers, and suitable dNTPs, as well as other suitable or necessary additives (e.g., but not limited to, DMSO, betaine, and ammonium sulfate). In this example, combine the purified modified first CS product with 50 picomoles of each of the two primers (2-002 and 2-003), 2 units of Phusion DNA polymerase, 10 μl of 5× Phusion DNA polymerase buffer, 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP), and 1 micromole of ammonium sulfate to a final volume of 50 μl. Then, thermocycler the mixture to exponentially amplify the modified first CS product. In this example, 98 °C for 30 seconds, 12 cycles of 98 °C for 5 seconds, 60 °C for 1 minute, and 72 °C for 1 minute, then 72 °C for 2 minutes. Then, the amplification product can be purified by any suitable method (e.g., using magnetic beads). The final amplification product will be compatible with next-generation sequencing on, for example, an Illumina platform. As Figure 14 depicted in part (I) of

[0517] Results

[0518] Using an Agilent Bioanalyzer High Sensitivity Kit to evaluate the final complete sequencing library, we were able to demonstrate that the final product of library preparation had a size distribution equivalent to that expected given the distribution of the input material. As Figure 14 depicted in part (J) of

[0519] Conclusion

[0520] By combining the first ATO reaction using fragmented gDNA, a linear amplification process to generate multiple first complementary strands, a second ATO reaction using the linear amplification product as the target polynucleotide, and subsequent global amplification, we have successfully verified the use of this technical method in the generation of sequencing libraries.

[0521] Example 2

[0522] Using deoxyribonucleic acid (DNA) as the target polynucleotide for generating a targeted amplicon next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more adaptor template oligonucleotides with hairpins (e.g., but not limited to, C3 spacer, C18 spacer), where the hairpins are generated using chemical spacers.

[0523] Materials

[0524] Target polynucleotide, human gDNA (BIO-35025)

[0525] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0526] Adaptor Template Oligonucleotide (ATO) 1-006 (Table 1)

[0527] TAQ DNA Polymerase (NEB, M0273L)

[0528] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0529] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0530] dNTP (NEB, N0447s)

[0531] High-Fidelity DNA Polymerase (NEB, M0530S)

[0532] Phusion Buffer (NEB, M0530S)

[0533] Enzyme (NEB, M5505S)

[0534] Primers 2-004, 2-005 (Table 2)

[0535] Ammonium Sulfate (SIGMA, A4418)

[0536] Agencourt AMPure XP (Beckman Coulter, A63881)

[0537] Agilent High Sensitivity DNA Kit (Product ID 5067 - 4626)

[0538] SYBR TM Green I Nucleic Acid Gel Stain (Invitrogen, S7563)

[0539] Method

[0540] Annealing of one or more adaptor template oligonucleotides to deoxyribonucleic acid

[0541] As in Example 1, in H2O with a total of 25 ng of fragmented target polynucleotide, 100 picomoles of ATO1 - 006 (100 picomoles of ATO in total) to a final volume of 7.5 μl. As Figure 13 depicted in parts (A) and (B) of

[0542] Extension of target deoxyribonucleic acid by using the target deoxyribonucleic acid as a primer and one or more adaptor template oligonucleotides as templates

[0543] As in Example 1, combine a total of 1.25 units of Klenow and 1.25 units of TAQ DNA polymerase, 1 μl of 10×TAQ DNA polymerase buffer, and 5 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) with the DNA - ATO mixture to a final volume of 10 μl. As Figure 13 depicted in part (C) of

[0544] Degradation of one or more adaptor template oligonucleotides

[0545] As in Example 1. As Figure 13 depicted in part (D) of

[0546] First exponential amplification of modified target polynucleotide by PCR

[0547] Combine the modified target polynucleotide (purified or unpurified) with the following: a universal primer complementary to a universal sequence initially on one or more ATOs and now present at the 3'-end of the target polynucleotide, a library of target-specific primers designed to target regions of DNA containing a mutation of interest (e.g., but not limited to, one or more single nucleotide polymorphisms, one or more insertions / deletions, one or more DNA fusions), an appropriate amount of one or more DNA polymerases (with or without proofreading activity) (e.g., but not limited to, Taq DNA polymerase, or Phusion), one or more suitable buffers, suitable dNTPs, and other suitable or necessary additives (e.g., but not limited to, DMSO, betaine, and ammonium sulfate). In this example, 5 μl of the modified target polynucleotide, 50 picomoles of 2-004, 100 picomoles of the library of target-specific primers, 12 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP), 2 units of Phusion DNA polymerase, 10 μl of 5× Phusion buffer, and 1 micromole of ammonium sulfate are combined in a final volume of 50 μl. The mixture is then thermocycled to amplify the selected region of the target nucleic acid extension product, in this example, 98 °C for 1 minute, 15 cycles of 98 °C for 5 seconds, 60 °C for 5 minutes, and 72 °C for 30 seconds, then 72 °C for 2 minutes. As Figure 13 depicted in part (E) of

[0548] Optional second exponential amplification of the first exponential amplification product by PCR

[0549] Combine the purified first exponential amplification product with the following: a second set of nested universal primers (which have sequence similarity to the universal sequences introduced by the first universal primers and are designed to bind to the universal sequences introduced by the first universal primers, which will contain sequences compatible with and necessary for sequencing using next-generation sequencing techniques (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence)), a second library of target primers designed to target regions of DNA (which contains or nests relative to the first primer library, which also targets regions of DNA containing mutations of interest and also contains sequences compatible with and necessary for compatibility with next-generation sequencing techniques (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequencing primer sites)), a proofreading polymerase (e.g., but not limited to, Phusion DNA polymerase, Q5), one or more suitable buffers, suitable dNTPs, and other suitable or necessary additives (e.g., but not limited to, DMSO, betaine, and ammonium sulfate). In this example, 10 μl of the purified first amplification product, 12.5 picomoles of 2-005, 50 picomoles of the nested library of target-specific primers, 5 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP), 1 unit of Phusion DNA polymerase, 5 μl of 5× Phusion buffer, 1× SYBR green, and 0.5 micromole of ammonium sulfate were combined in a final volume of 25 μl. As Figure 13 depicted in part (F) of, the mixture was then thermocycled to amplify selected regions of the first exponential amplification product. In this example, 98 °C for 1 minute, 15 cycles of 98 °C for 5 seconds, 60 °C for 5 minutes, and 72 °C for 30 seconds, then 72 °C for 2 minutes. The amplification product can then be purified by any suitable method (e.g., using magnetic beads) and will be suitable for next-generation sequencing based on compatible techniques. As Figure 13 depicted in part (G) of, in this example, the second exponential amplification product was first bead-purified and the size distribution was determined for generating 150 bp paired-end reads with a MiSeq.

[0550] Results

[0551] The combination of the first exponential amplification and the second exponential amplification can successfully amplify the target region of the modified target polynucleotide and generate a targeted amplicon library. Inclusion of SYBR green in the second exponential amplification reaction allows monitoring of the amplification rate of the final library product. Melting curve analysis shows that the overall method can produce products of substantially higher molecular weight relative to a no-template control. Evaluating the final complete sequencing library using the Agilent Bioanalyzer High Sensitivity Kit (product ID 5067-4626), we were able to demonstrate that the final product of library preparation has a size distribution equivalent to that expected given the distribution of the input material. Using this library to generate sequencing data as part of a pooled run with the MiSeq, we generated slightly more than 3.6 million reads. Mapping the reads to the reference sample using bwa resulted in a mapping rate of 98%. Examination of the insert sizes of the mapped paired-end reads revealed an insert distribution from 26 bp (filtering out inserts of 25 bp or less) up to almost 400 bp. As Figure 13 depicted in part (H), there is a peak insert size of approximately 100 bp. As Figure 13 depicted in part (I), the efficiency of each target-specific primer was examined by counting the number of reads mapping to each of the primer sites in the sequencing results. This revealed uniform coverage across the target site. As depicted in part (J), due to the incorporation of the UIDs, we were able to generate "barcode families" and count the number of families for each target site. This revealed a maximum count of just over 1000 barcode families.

[0552] Conclusion

[0553] By combining the first ATO reaction of fragmented gDNA, a process of two rounds of exponential amplification using a target-specific primer library and a second target-specific nested library, we have successfully validated this technical method for use in the generation of strand-specific targeted amplicon-based sequencing libraries. Successful sequencing of the final library product on an Illumina MiSeq sequencer confirmed that the final library product contains the components we have designed in the correct order necessary for sequencing. Examination of the data revealed successful multiplex amplification of more than 200 different target regions with similar levels of overall primer enrichment. Comparison of the number of barcode families for each of the primer sites revealed very little variation across all sites, indicating little target-sequence-based bias in the ATO reaction.

[0554] Example 3

[0555] Use deoxyribonucleic acid (DNA) as a target polynucleotide for incorporation into a promoter of an RNA polymerase to drive the amplification of the DNA into RNA using, for example but not limited to, one or more adapter template oligonucleotides, the one or more adapter template oligonucleotides containing a sequence compatible with the RNA polymerase promoter and capable of serving as an RNA polymerase promoter, and a hairpin generated from the nucleotide sequence.

[0556] Materials

[0557] Target polynucleotide, human gDNA (BIO - 35025)

[0558] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0559] Adapter Template Oligonucleotide (ATO) 1 - 012 (Table 1)

[0560] TAQ DNA Polymerase (NEB, M0273L)

[0561] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0562] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0563] dNTP (NEB, N0447s)

[0564] Enzyme (NEB, M5505S)

[0565] T7 - Scribe Standard RNA IVT Kit (CELLSCRIPT, C - AS3107)

[0566] Methods

[0567] Annealing of one or more adapter template oligonucleotides to deoxyribonucleic acid

[0568] As in Example 1, except that the ATO contains one or more sequences compatible with the RNA polymerase promoter and capable of serving as an RNA polymerase promoter (for example but not limited to, T7 promoter sequence, T3 promoter sequence, or SP6 promoter sequence) (in this example, ATO 1 - 012 containing the T7 promoter).

[0569] Extension of the target deoxyribonucleic acid by using the target deoxyribonucleic acid as a primer and the adapter template oligonucleotide as a template

[0570] As in Example 1.

[0571] In vitro transcription using RNA polymerase

[0572] The purified or unpurified target deoxyribonucleic acid extension (modified polynucleotide) product is combined with a suitable buffer, suitable NTPs, other suitable additives (such as but not limited to, DTT), and a suitable RNA polymerase (such as but not limited to, T7 RNA polymerase if a T7 RNA polymerase promoter has been used) at a suitable combination of time and temperature to promote the activity of the RNA polymerase. In this example, 5 μl of purified modified target polynucleotide and a commercial kit (CELLSCRIPT TM T7-Scribe TM Standard RNA IVT Kit) were used according to the manufacturer's instructions and incubated at 37 °C for 18 h in a final volume of 20 μl. The resulting RNA was quantified using high-sensitivity Qubit3 reagent, measuring a total of 107 ng / μl, resulting in a total amount of 2035 ng of RNA produced by the 18-hour overnight incubation. The RNA can then form the input material for any suitable RNA-based downstream application (such as but not limited to, RNA next-generation sequencing, cDNA synthesis, in situ hybridization, qPCR, or any other suitable downstream process).

[0573] Results

[0574] In the first ATO reaction used to generate the modified target polynucleotide, an ATO containing a sequence designed to act as a promoter for RNA polymerase when double-stranded was used. Since the product of this first reaction was used to generate an in vitro transcription reaction that yielded a total of 2 μg of RNA, it can be determined that the first reaction was successful.

[0575] Conclusion

[0576] The nucleotide sequence designed to act as an RNA polymerase promoter can be successfully incorporated into the design of the ATO. After the first ATO extension, a portion of the modified target polynucleotide will be double-stranded and contain the RNA polymerase promoter. This double-stranded RNA polymerase promoter can then be used to amplify the target polynucleotide into RNA (rather than DNA). This has the potential to rapidly and robustly amplify small amounts of DNA into RNA to a large extent, and the RNA can then be used as a template for any one of many downstream applications, including RNA-Seq and cDNA synthesis.

[0577] Example 4

[0578] Using deoxyribonucleic acid (DNA) as a target polynucleotide for incorporation into a promoter of an RNA polymerase to drive the amplification of DNA into RNA using, for example but not limited to, one or more adaptor template oligonucleotides and a second oligonucleotide complementary to ATO, the one or more adaptor template oligonucleotides containing a sequence compatible with and capable of serving as an RNA polymerase promoter and a hairpin generated from a nucleotide sequence.

[0579] Materials

[0580] Target polynucleotide, human gDNA (BIO-35025)

[0581] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0582] Adaptor Template Oligonucleotide (ATO) 1-019, Adaptor Template Oligonucleotide (ATO) 1-020 (Table 1)

[0583] TAQ DNA Polymerase (NEB, M0273L)

[0584] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0585] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0586] dNTP (NEB, N0447s)

[0587] Enzyme (NEB, M5505S)

[0588] Agencourt AMPure XP (Beckman Coulter, A63881)

[0589] T7-Scribe Standard RNA IVT Kit (CELLSCRIPT, C-AS3107)

[0590] Methods

[0591] Annealing of one or more adaptor template oligonucleotides to deoxyribonucleic acid

[0592] As in Example 3, except for using ATO 1-019.

[0593] Extension of the target deoxyribonucleic acid by using the target deoxyribonucleic acid as a primer and one or more adaptor template oligonucleotides as templates

[0594] As in Example 1.

[0595] Degradation of one or more adaptor template oligonucleotides

[0596] As in Example 1.

[0597] Generate double-stranded RNA polymerase promoter

[0598] Combine the purified or unpurified target deoxyribonucleic acid extension product with a sequence complementary to one or more conserved sequences initially on one or more ATOs and now present on the target DNA molecule. In this example, 15 μl of purified modified target polynucleotide in 20 μl final volume of H2O, 100 picomoles of 1-020. Heat the mixture at a suitable temperature (in this example, 95 °C) for a suitable time necessary to denature the double-stranded nucleic acid into single strands (in this example, 2 minutes). Then cool the mixture to allow formation of a double-stranded complex between the modified target polynucleotide and one or more target deoxyribonucleic acid extension complementary oligonucleotides (in this example, room temperature (25 °C) for at least 5 minutes).

[0599] In vitro transcription using RNA polymerase

[0600] As in Example 3.

[0601] Results

[0602] Use an ATO containing a sequence designed to act as a promoter for RNA polymerase when double-stranded in the first ATO reaction for generating the modified target polynucleotide. Since the product of this first reaction is used for the in vitro transcription reaction to generate RNA, it can be determined that the first reaction was successful.

[0603] Conclusions

[0604] The nucleotide sequence designed to act as an RNA polymerase promoter can be successfully incorporated into the design of ATO. After the first ATO extension, the ATO is digested and removed, and the complementary oligonucleotide is hybridized with the modified target polynucleotide. A portion of the modified target polynucleotide will be double-stranded and contain the RNA polymerase promoter. Then, this double-stranded RNA polymerase promoter can be used to amplify the target polynucleotide into RNA (instead of DNA). This has the potential to quickly and robustly amplify a small amount of DNA into RNA to a large extent, and the RNA can then be used as a template for any of many downstream applications, including RNA-Seq and cDNA synthesis.

[0605] Example 5

[0606] Use deoxyribonucleic acid (DNA) as a target polynucleotide for incorporation into a promoter of an RNA polymerase to generate amplification of DNA to RNA using, for example but not limited to, one or more adaptor template oligonucleotides having a hairpin (stem-loop) structure, the hairpin (stem-loop) structure being generated by two short stem regions that are partially complementary or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence.

[0607] Materials

[0608] Target polynucleotide, human gDNA (BIO-35025)

[0609] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0610] Adaptor Template Oligonucleotide (ATO) 1-021 (Table 1)

[0611] TAQ DNA Polymerase (NEB, M0273L)

[0612] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0613] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0614] dNTP (NEB, N0447s)

[0615] High-Fidelity DNA Polymerase (NEB, M0530S)

[0616] Phusion Buffer (NEB, M0530S)

[0617] Enzyme (NEB, M5505S)

[0618] S1 Nuclease (ThermoFisher, EN0321)

[0619] Agencourt AMPure XP (Beckman Coulter, A63881)

[0620] T7-Scribe Standard RNA IVT Kit (CELLSCRIPT, C-AS3107)

[0621] Methods

[0622] One or more adaptor template oligonucleotides are annealed to deoxyribonucleic acid

[0623] As in Example 3, in addition to the ATO having a sequence designed to act as an RNA polymerase promoter, it also contains a hairpin, which is generated by two short regions that are partially complementary or fully complementary and are separated by a random sequence, a specifically designed sequence, or a specifically selected sequence (1-021).

[0624] Target DNA extension by using the target deoxyribonucleic acid as a primer and one or more adapter template oligonucleotides as templates

[0625] As in Example 3.

[0626] Generate a double-stranded target DNA extension product containing a double-stranded RNA polymerase promoter

[0627] Combine the modified target polynucleotide reaction mixture with one or more additional thermostable DNA polymerases (with or without proofreading activity) (such as, but not limited to, Taq DNA polymerase, or Phusion) mixed with one or more suitable buffers and suitable dNTPs. In this example, 20 μl of the modified target polynucleotide reaction in a final volume of 30 μl, 2 units of Phusion DNA polymerase, 6 μl of 5×Phusion buffer, and 5 nanomoles of each dNT (dATP / dTTP / dCTP / dGTP). Heat the mixture at a suitable temperature (in this example, 95 °C) for a suitable time (in this example, 2 minutes), or heat the mixture at other temperatures or times that can cause the double-stranded nucleic acid to denature into single strands. Then cool the sample to allow the complementary regions within the target DNA extension product and one or more ATO oligonucleotides (such as, but not limited to, the nucleotide sequence that forms a hairpin due to the inclusion of two partially complementary or fully complementary regions) to anneal to themselves (in this example, at room temperature (25 °C) for 5 minutes). The 3' region of the modified target DNA extension product will form a small hairpin and anneal to itself. Then, raise the temperature to a suitable temperature necessary to fully extend the 3' end for a suitable time. The 3' end will act as a primer to initiate the extension and formation of a fully double-stranded or partially double-stranded target DNA extension product with a hairpin that separates the two complementary strands (in this example, 72 °C for 10 minutes).

[0628] Optional degradation of one or more adapter template oligonucleotides

[0629] As in Example 1.

[0630] In vitro transcription using RNA polymerase

[0631] As in Example 3.

[0632] Results

[0633] In a first ATO reaction for generating a modified target polynucleotide, an ATO containing a sequence designed to act as a promoter for RNA polymerase when double-stranded and a sequence that will form an internal hairpin is used. Since the product of this first reaction is used in an in vitro transcription reaction to generate RNA, it can be determined that the first reaction was successful.

[0634] Conclusion

[0635] A nucleotide sequence designed to act as an RNA polymerase promoter can be successfully incorporated into the design of an ATO in combination with an internal hairpin and an extension sequence. After the first ATO extension, we showed that after digestion and removal of the ATO, the complementary regions were able to reanneal and extend to produce a double-stranded modified target polynucleotide with a hairpin. This double-stranded RNA polymerase promoter can then be used to amplify the target polynucleotide into RNA (rather than DNA). This has the potential to rapidly and robustly amplify small amounts of DNA into RNA to a large extent, and the RNA can then be used as a template for any of a number of downstream applications, including RNA-Seq and cDNA synthesis.

[0636] Example 6

[0637] Using deoxyribonucleic acid (DNA) as the target polynucleotide, for generating a next-generation sequencing library (e.g., but not limited to, compatible with an Illumina next-generation sequencer) using one or more adapter template oligonucleotides (ATO) with hairpins, the hairpins being generated by two short regions that are partially or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence.

[0638] Materials

[0639] Target polynucleotide, human gDNA (BIO-35025)

[0640] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0641] Adapter Template Oligonucleotide (ATO) 1-013, Adapter Template Oligonucleotide (ATO) 1-022 (Table 1)

[0642] TAQ DNA Polymerase (NEB, M0273L)

[0643] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0644] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0645] dNTP (NEB, N0447s)

[0646] High-fidelity DNA polymerase (NEB, M0530S)

[0647] Phusion buffer (NEB, M0530S)

[0648] Enzyme (NEB, M5505S)

[0649] S1 nuclease (ThermoFisher, EN0321)

[0650] Agencourt AMPure XP (Beckman Coulter, A63881)

[0651] Primer 2-002, Primer 2-003 (Table 2)

[0652] Method

[0653] Adapter template oligonucleotides are annealed to deoxyribonucleic acid

[0654] As in Example 1, except that the ATO also has a sequence designed to act as a hairpin, the hairpin being generated by two short regions that are partially or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence (1-013).

[0655] Target deoxyribonucleic acid extension by using the target deoxyribonucleic acid as a primer and one or more adapter template oligonucleotides as templates

[0656] As in Example 1.

[0657] Generate double-stranded modified target polynucleotide

[0658] As in Example 5.

[0659] Degradation of adapter template oligonucleotides and hairpin degradation

[0660] As in Example 5, plus S1 nuclease. This may require an additional inactivation step, where S1 is inactivated by adding EDTA and incubating at 70 °C for 10 minutes or incubating at a similar temperature and time that causes inactivation of S1 nuclease.

[0661] Single extension or linear amplification of double-stranded modified target polynucleotide to generate the first CS

[0662] As in Example 1.

[0663] Second adapter template oligonucleotides are annealed to the first CS

[0664] As in Example 1, except that the ATO also has a sequence designed to act as a hairpin, the hairpin being generated from two short regions that are partially or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence (1-022).

[0665] The first CS extension using the first CS as a primer and the second adaptor template oligonucleotide as a template to form a modified first CS

[0666] As in Example 1.

[0667] Generate a double-stranded modified target polynucleotide

[0668] As in Example 5.

[0669] Degradation of the adaptor template oligonucleotide and hairpin degradation

[0670] Same as above.

[0671] Exponential amplification of the modified first CS by PCR

[0672] As in Example 1.

[0673] Results

[0674] Using this example, we can show that the self-annealing and extension of the modified target polynucleotide and the modified first CS produced a template for final exponential amplification, which produced a complete whole-genome next-generation sequencing library.

[0675] Conclusion

[0676] By combining the first ATO reaction of fragmented gDNA, the self-annealing and extension process to generate double-stranded DNA, one extension or linear amplification to generate multiple first complementary strands, the second ATO reaction using the linear amplification product as the target polynucleotide, and subsequent global amplification, we have successfully verified the use of this technical method in the generation of sequencing libraries.

[0677] Example 7

[0678] Using deoxyribonucleic acid (DNA) as the target polynucleotide for generating a next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using (e.g., but not limited to) one or more adaptor template oligonucleotides with hairpins and double-stranded or partially double-stranded common adaptors, the hairpins being generated from two short regions that are partially or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence.

[0679] Materials

[0680] Target polynucleotide, human gDNA (BIO-35025)

[0681] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0682] Adapter Template Oligonucleotide (ATO) 1 - 022 (Table 1)

[0683] TAQ DNA Polymerase (NEB, M0273L)

[0684] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0685] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0686] dNTP (NEB, N0447s)

[0687] High - Fidelity DNA Polymerase (NEB, M0530S)

[0688] Phusion Buffer (NEB, M0530S)

[0689] Enzyme (NEB, M5505S)

[0690] T4 DNA Ligase (NEB, M0202S)

[0691] Method

[0692] One or more adapter template oligonucleotides anneal to deoxyribonucleic acid

[0693] As in Example 1, except that the ATO also has a sequence designed to act as a hairpin, the hairpin is generated by two short regions that are partially or fully complementary and are separated by a random sequence, a specifically designed sequence, or a specifically selected sequence (1 - 022). The target polynucleotide is extended by using the target polynucleotide as a primer and the adapter template oligonucleotide as a template

[0694] As in Example 1.

[0695] Generate double - stranded modified target polynucleotide

[0696] As in Example 5.

[0697] Ligation of one or more double - stranded adapters to the ends of double - stranded target deoxyribonucleic acid extension products

[0698] Mix the purified or unpurified double-stranded modified target polynucleotide with the following: an appropriate amount of blunt-ended or 3’T nucleotide overhanging fully double-stranded or partially double-stranded adaptors with or without a hairpin, an appropriate amount of one or more ligases (e.g., but not limited to, T4 DNA ligase), one or more suitable buffers, and one or more any other necessary reagents. In this example, 15 μl of the purified double-stranded modified target polynucleotide, 50 nanomoles of the adaptor, 100 units of T4 DNA ligase, and 2 μl of 10× ligase buffer in a final volume of 20 μl. Incubate the mixture at a suitable temperature and for a suitable time to allow complete ligation to the ends of all double-stranded target nucleic acid extension products (in this example, 25 °C for 30 minutes, or other suitable temperature and time that can promote enzyme activity).

[0699] Optional degradation of one or more adaptor template oligonucleotides and hairpin degradation

[0700] As in Example 6.

[0701] Exponential amplification of the ligated extension products by PCR

[0702] As in Example 1.

[0703] Results

[0704] Using this example, we can show that the operation of generating double-stranded first CS by self-annealing and extension of the modified target polynucleotide and the operation of ligating the adaptor to the available ends successfully produced a template for final exponential amplification, which produced a complete whole-genome next-generation sequencing library.

[0705] Conclusion

[0706] By combining the first ATO reaction of fragmented gDNA, the process of self-annealing and extension to generate double-stranded DNA, the ligation of double-stranded adaptors to the exposed ends to generate a second double-stranded DNA product, and subsequent global amplification, we have successfully verified the use of this technical method in the generation of sequencing libraries.

[0707] Example 8

[0708] Use deoxyribonucleic acid (DNA) as the target polynucleotide to generate a "PCR"-free next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more adaptor template oligonucleotides with a hairpin and double-stranded or partially double-stranded common adaptors. The hairpin is generated by two short regions that are partially complementary or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence.

[0709] Materials

[0710] Target polynucleotide, human gDNA (BIO-35025)

[0711] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0712] Adapter template oligonucleotide (ATO) 1-023 (Table 1)

[0713] TAQ DNA polymerase (NEB, M0273L)

[0714] TAQ DNA polymerase buffer (NEB, M0273L)

[0715] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0716] dNTP (NEB, N0447s)

[0717] High-fidelity DNA polymerase (NEB, M0530S)

[0718] Phusion buffer (NEB, M0530S)

[0719] Enzyme (NEB, M5505S)

[0720] Agencourt AMPure XP (Beckman Coulter, A63881)

[0721] T4 DNA ligase (NEB, M0202S)

[0722] Method

[0723] Annealing of one or more adapter template oligonucleotides to deoxyribonucleic acid

[0724] As in Example 1, except that the ATO has a sequence that forms a hairpin due to the inclusion of two partially complementary or fully complementary regions, which includes a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence (1-023).

[0725] Target polynucleotide extension by using the target polynucleotide as a primer and one or more adapter template oligonucleotides as templates

[0726] As in Example 1.

[0727] Generating a double-stranded target deoxyribonucleic acid extension product

[0728] As in Example 5.

[0729] Annealing of one or more adaptors and end ligation to the double-stranded target deoxyribonucleic acid extension product

[0730] As in Example 7.

[0731] Optional degradation of one or more adaptor template oligonucleotides and hairpin degradation

[0732] As in Example 6.

[0733] Results

[0734] Using this example, we can show that the operations of self-annealing and extension of the modified target polynucleotide to generate the double-stranded first CS and the operation of ligating the adaptor to the available ends have successfully produced a template for next-generation sequencing without 'index PCR'.

[0735] Conclusion

[0736] By combining the first ATO reaction using fragmented gDNA, the self-annealing and extension process to generate double-stranded DNA, and the ligation of double-stranded adaptors to the exposed ends to generate the second double-stranded DNA product, we have successfully verified this technical method for next-generation sequencing without 'index PCR'.

[0737] Example 9

[0738] Using deoxyribonucleic acid (DNA) as the target polynucleotide for generating a next-generation sequencing library without PCR (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more adaptor template oligonucleotides having a hairpin (stem-loop structure where the loop includes a chemical spacer (e.g., but not limited to, C3 spacer, C18 spacer)) and double-stranded or partially double-stranded common adaptors.

[0739] Materials

[0740] Target polynucleotide, human gDNA (BIO-35025)

[0741] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0742] Adaptor template oligonucleotide (ATO) 1-023 (Table 1)

[0743] TAQ DNA polymerase (NEB, M0273L)

[0744] TAQ DNA polymerase buffer (NEB, M0273L)

[0745] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0746] dNTP (NEB, N0447s)

[0747] High-Fidelity DNA Polymerase (NEB, M0530S)

[0748] Phusion Buffer (NEB, M0530S)

[0749] Enzyme (NEB, M5505S)

[0750] T4 DNA Ligase (NEB, M0202S)

[0751] Agencourt AMPure XP (Beckman Coulter, A63881)

[0752] Primer 2-006 (Table 2)

[0753] Method

[0754] Adapter Template Oligonucleotide Annealed to Deoxyribonucleic Acid

[0755] As in Example 8, except that the hairpin (1-024) in ATO was generated via a chemical spacer arm.

[0756] Target Polynucleotide Extension by Using the Target Polynucleotide as a Primer and the Adapter Template Oligonucleotide as a Template

[0757] As in Example 1.

[0758] Degradation of Adapter Template Oligonucleotide

[0759] As in Example 1.

[0760] Linear Extension to Generate Double-Stranded Modified Target Polynucleotide

[0761] Combine the purified target nucleic acid extension product with the following: an oligonucleotide primer complementary to the conserved sequence now present at the 3' end of the modified target DNA molecule, a polymerase (such as but not limited to, Phusion DNA polymerase), one or more suitable buffers, and suitable dNTPs. In this example, 50 picomoles of primer (2-006) complementary to the conserved sequence now present on the target DNA molecule, 2 units of Phusion DNA polymerase, 6 μl of 5× Phusion DNA polymerase buffer, 12 nanomoles of each dNT (dATP / dTTP / dCTP / dGTP), and 15 μl of purified modified target polynucleotide in a final volume of 30 μl. Incubate the mixture (in this example, at 60 - 72 °C for 30 minutes), or incubate the mixture at other suitable temperatures and times that can promote enzyme activity to extend one or more oligonucleotides to create blunt-ended or A-tailed double-stranded DNA products. Then, the product can be purified using any suitable purification method.

[0762] Ligation of one or more common adaptors to the ends of the double-stranded target deoxyribonucleic acid extension product

[0763] As in Example 8.

[0764] Results

[0765] Using this example, we can show that templates for PCR-free next-generation sequencing can be successfully generated using the first ATO reaction, one round of extension, and ligation of common adaptors.

[0766] Conclusion

[0767] By combining the first ATO reaction on fragmented genomic DNA, the first ATO reaction, removal of ATO, annealing of oligonucleotides complementary to the universal sequence, one linear extension, and then ligation of double-stranded adaptors to the 5' end of the modified target polynucleotide, we have successfully validated this technical method for 'PCR'-free next-generation sequencing.

[0768] Example 10

[0769] Use deoxyribonucleic acid (DNA) as the target polynucleotide to generate a next-generation sequencing library (such as but not limited to, compatible with Illumina next-generation sequencers) using one or more single-stranded adaptor template oligonucleotides.

[0770] Materials

[0771] Target polynucleotide, human gDNA (BIO-35025)

[0772] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0773] Adapter Template Oligonucleotide (ATO) 1-025, Adapter Template Oligonucleotide (ATO) 1-026 (Table 1)

[0774] TAQ DNA polymerase (NEB, M0273L)

[0775] TAQ DNA polymerase buffer (NEB, M0273L)

[0776] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0777] dNTP (NEB, N0447s)

[0778] High-fidelity DNA polymerase (NEB, M0530S)

[0779] Phusion buffer (NEB, M0530S)

[0780] Enzyme (NEB, M5505S)

[0781] Agencourt AMPure XP (Beckman Coulter, A63881)

[0782] Primer 2-002, Primer 2-003 (Table 2)

[0783] Method

[0784] Annealing of adapter template oligonucleotide into deoxyribonucleic acid

[0785] As in Example 1, except that the ATO is single-stranded (1-025).

[0786] Target polynucleotide extension by using the target polynucleotide as a primer and one or more adapter template oligonucleotides as templates

[0787] As in Example 1.

[0788] Degradation of one or more adapter template oligonucleotides

[0789] As in Example 1.

[0790] Linear amplification of the modified target polynucleotide

[0791] As in Example 1.

[0792] Annealing of the second adapter template oligonucleotide into the first complementary strand

[0793] As in Example 1, except that the ATO is single-stranded (1-026).

[0794] Extending the first complementary strand by using the first complementary strand as a primer and the second adaptor template oligonucleotide as a template to form a modified first complementary strand

[0795] As in Example 1.

[0796] Degradation of one or more second adaptor template oligonucleotides

[0797] As in Example 1.

[0798] Exponential amplification of the second adaptor template extension product by PCR

[0799] As in Example 1.

[0800] Results

[0801] Using single-stranded ATO, we could demonstrate the successful completion of genomic next-generation sequencing libraries (similar to that detailed in Example 1).

[0802] Conclusion

[0803] By combining the first ATO reaction with fragmented gDNA, the linear amplification process to generate multiple first complementary strands, the second ATO reaction using the linear amplification product as the target polynucleotide, and subsequent global amplification, we have successfully validated this technical method for use in the generation of sequencing libraries.

[0804] Example 11

[0805] Using deoxyribonucleic acid (DNA) as the target polynucleotide for generating a next-generation sequencing library (e.g., but not limited to, compatible with an Illumina next-generation sequencer) using one or more adaptor template oligonucleotides (e.g., but not limited to) that are split into two complementary strands.

[0806] Materials

[0807] Target polynucleotide, human gDNA (BIO-35025)

[0808] DNA fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0809] Adaptor template oligonucleotide (ATO) 1-027, adaptor template oligonucleotide (ATO) 1-028, adaptor template oligonucleotide (ATO) 1-029, adaptor template oligonucleotide (ATO) 1-030 (Table 1)

[0810] TAQ DNA Polymerase (NEB, M0273L)

[0811] TAQ DNA Polymerase Buffer (NEB, M0273L)

[0812] DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210L, M0212L)

[0813] dNTP (NEB, N0447s)

[0814] High-Fidelity DNA Polymerase (NEB, M0530S)

[0815] Phusion Buffer (NEB, M0530S)

[0816] Enzyme (NEB, M5505S)

[0817] Agencourt AMPure XP (Beckman Coulter, A63881)

[0818] T4 DNA Ligase (NEB, M0202S)

[0819] Primer 2-002, Primer 2-003 (Table 2)

[0820] Method

[0821] Adapter Template Oligonucleotide Annealed into Deoxyribonucleic Acid

[0822] As in Example 1, except that ATO is an equimolar mixture of two single-stranded oligonucleotides (1-027, 1-028).

[0823] Target Deoxyribonucleic Acid Extension and Ligation by Using Target Deoxyribonucleic Acid as Primer and One or More Adapter Template Oligonucleotides as Template

[0824] As in Example 1, in addition, the reaction contains DNA ligase. If the 3'-end of the target polynucleotide hybridizes to the ATO and is located directly adjacent to the phosphorylated 5'-end of the upper strand of the ATO, the 3'-end of the target polynucleotide is ligated to the 5'-end upper strand of the ATO without extension. If the 3'-end of the target polynucleotide hybridizes to the ATO and is located at a certain distance from the phosphorylated 5'-end of the upper strand of the ATO, the 3'-end of the target polynucleotide is extended by DNA polymerase and ligated to the 5'-end upper strand of the ATO. In this example, the reaction contains the extension components as shown in Example 1, and additionally 100 units of T4 DNA ligase and 2.5 μl of 10× DNA ligase buffer in a final volume of 25 μl. Incubate the mixture at a suitable temperature and for a suitable time to allow ligation or extension-ligation between the 3'-end of the target polynucleotide or its extension product and one or more adaptor template complementary oligonucleotides (in this example, 25 °C for 30 minutes, or other suitable temperature and time that can promote enzyme activity). One or more adaptor template complementary oligonucleotides are the 5'-ends of the upper single strand of the ATO or the stem part of the stem structure.

[0825] Degradation of one or more adaptor template oligonucleotides

[0826] As in Example 1.

[0827] Optional linear amplification of the modified target polynucleotide

[0828] As in Example 1.

[0829] The second adaptor template oligonucleotide is optionally annealed to the first complementary strand

[0830] As in Example 1, except that the ATO is an equimolar mixture of two single-stranded oligonucleotides (1-029, 1-030).

[0831] Optional extension of the first complementary strand using the first complementary strand as a primer and the second adaptor template oligonucleotide as a template to form a modified first complementary strand, and ligation as shown in the previous steps in this example

[0832] As in Example 1 and above.

[0833] Degradation of one or more second adaptor template oligonucleotides

[0834] As in Example 1.

[0835] Exponential amplification of the second adaptor template extension product by PCR

[0836] As in Example 1.

[0837] Results

[0838] Using ATOs that are separated into two complementary strands, we can demonstrate the successful completion of genomic next-generation sequencing libraries (similar to those detailed in Example 1).

[0839] Conclusion

[0840] By combining the first ATO reaction with fragmented gDNA, the linear amplification process to generate multiple first complementary strands, the second ATO reaction using the linear amplification product as the target polynucleotide, and subsequent global amplification, we have successfully verified the use of this technical method in the generation of sequencing libraries.

[0841] Example 12

[0842] Using deoxyribonucleic acid (DNA) as the target polynucleotide, for generating a targeted amplicon next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more target-specific adapter template oligonucleotides (ATOs) having a hairpin (stem-loop), where the loop includes a chemical spacer (e.g., but not limited to, a C3 spacer, a C18 spacer), and where the 3' end of the ATO includes a target-specific sequence. The 3' target-specific sequence is used to direct hybridization between the target polynucleotide and the ATO. The ATO can include the usual 3' random sequence between the 3' target-specific sequence and the 5' universal sequence as described in other examples, or can include a short 3' random sequence (since this 3' random sequence can only act as a UID and not as a template for annealing), or can not include any 3' random sequence. The 5' universal sequence can include an RNA polymerase promoter sequence.

[0843] Materials

[0844] Target polynucleotide, human gDNA (BIO-35025)

[0845] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0846] Multiple adapter template oligonucleotides (ATOs)

[0847] TAQ DNA polymerase (NEB, M0273L)

[0848] TAQ DNA polymerase buffer (NEB, M0273L)

[0849] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0850] dNTP (NEB, N0447s)

[0851] High-fidelity DNA polymerase (NEB, M0530S)

[0852] Phusion buffer (NEB, M0530S)

[0853] Enzyme (NEB, M5505S)

[0854] Ammonium sulfate (SIGMA, A4418)

[0855] Agencourt AMPure XP (Beckman Coulter, A63881)

[0856] Primer 2-002, Primer 2-003 (Table 2)

[0857] Method

[0858] Anneal the adaptor template oligonucleotide library into deoxyribonucleic acid

[0859] As in Example 1, except that the ATO is a library of 200 different ATOs with target-specific sequences at the 3'-end.

[0860] Target DNA extension by using the target deoxyribonucleic acid as a primer and one or more adaptor template oligonucleotides as templates

[0861] As in Example 1. Since the 3'-end of the target polynucleotide can be an overhang when hybridized to its ATO, the 3'-exonuclease of the DNA polymerase trims the overhang before extension occurs.

[0862] Degradation of one or more adaptor template oligonucleotides

[0863] As in Example 1.

[0864] First exponential amplification of the adaptor template extension product by PCR

[0865] As in Example 1.

[0866] Second exponential amplification of the first exponential amplification product by PCR for producing the final sequencing library

[0867] As shown in Example 1.

[0868] Results

[0869] By using a library of ATOs with 3'-target-specific sequences, we could demonstrate the successful completion of a targeted amplicon next-generation sequencing library (similar to that detailed in Example 2).

[0870] Conclusions

[0871] By combining the first ATO reaction using fragmented gDNA and an ATO library, and a process of two rounds of target-specific amplification, we have successfully validated the use of this technical method for the generation of targeted next-generation sequencing libraries.

[0872] Example 13

[0873] In a single round of PCR, deoxyribonucleic acid (DNA) is used as the target polynucleotide for generating a targeted amplicon next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more target-specific adapter template oligonucleotides (ATO) having a hairpin (stem-loop), wherein the loop includes a chemical spacer arm (e.g., but not limited to, a C3 spacer arm, a C18 spacer arm).

[0874] Materials

[0875] Target polynucleotide, human gDNA (BIO-35025)

[0876] DNA fragmentation enzyme, KAPA Fragmentation Kit (Roche, 7962517001)

[0877] Adapter template oligonucleotide (ATO) 1-014, adapter template oligonucleotide (ATO) 1-015, adapter template oligonucleotide (ATO) 1-016, adapter template oligonucleotide (ATO) 1-017 (Table 1)

[0878] TAQ DNA polymerase (NEB, M0273L)

[0879] TAQ DNA polymerase buffer (NEB, M0273L)

[0880] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0881] dNTP (NEB, N0447s)

[0882] High-fidelity DNA polymerase (NEB, M0530S)

[0883] Phusion buffer (NEB, M0530S)

[0884] Enzyme (NEB, M5505S)

[0885] Primer 2-003 (Table 2)

[0886] Agencourt AMPure XP (Beckman Coulter, A63881)

[0887] Ammonium sulfate (SIGMA, A4418)

[0888] SYBR TM Green I nucleic acid gel stain (Invitrogen, S7563)

[0889] A target-specific primer library having the following 5'-tail: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CACGAC GCT CTT CCG ATC T

[0890] Method

[0891] Annealing of one or more adaptor template oligonucleotide libraries to deoxyribonucleic acid

[0892] As in Example 2, except using a library of each ATO (50 nanomoles) of 1-014, 1-015, 1-016, and 1-017.

[0893] Target polynucleotide extension by using the target polynucleotide as a primer and one or more adaptor template oligonucleotides as templates

[0894] As in Example 2.

[0895] Degradation of one or more adaptor template oligonucleotides

[0896] As in Example 2.

[0897] Exponential amplification of the linear extension products by PCR for generating the final sequencing library

[0898] Combine the purified modified target polynucleotide with the following: a universal primer (having sequence similarity to one or more conserved sequences introduced by one or more ATOs and designed to bind to one or more conserved sequences introduced by one or more ATOs, which contains sequences compatible with and necessary for next-generation sequencing technologies (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence)), a primer pool designed to target regions of DNA proximal to the ATO oligonucleotide library (which also targets regions of DNA containing the mutation of interest and also contains sequences compatible with and necessary for compatibility with next-generation sequencing technologies (e.g., but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence)), a proofreading polymerase (e.g., but not limited to, Phusion DNA polymerase, Q5), one or more suitable buffers, suitable dNTPs, and other suitable or necessary additives (e.g., but not limited to, DMSO, betaine, and ammonium sulfate). In this example, combine 20 μl of the purified modified target polynucleotide, 25 picomoles of 2-003, a total of 100 picomoles of the target-specific primer pool, 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP), 1 unit of Phusion DNA polymerase, 10 μl of 5× Phusion buffer, 1× SYBRgreen, and 1 micromole of ammonium sulfate in a final volume of 50 μl. Then, thermocycler the mixture to amplify the selected regions, in this example, 98 °C for 1 minute, 15 cycles of 98 °C for 5 seconds, 60 °C for 5 minutes, and 72 °C for 30 seconds, then 72 °C for 2 minutes. Then, the amplification products can be purified by any suitable method (e.g., using magnetic beads) and will be suitable for next-generation sequencing based on compatible technologies.

[0899] Results

[0900] Using a single round of exponential amplification (instead of two rounds as shown in Example 2), we were able to generate a target amplicon next-generation sequencing library.

[0901] Conclusions

[0902] By combining the first ATO reaction of fragmented gDNA, the process of single-round exponential amplification using a target-specific primer pool and a universal primer, we successfully validated the use of this technical method for the generation of strand-specific targeted amplicon-based sequencing libraries.

[0903] Example 14

[0904] Using ribonucleic acid (RNA) as a target polynucleotide for generating a next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more adaptor template oligonucleotides having a hairpin.

[0905] Materials

[0906] Adaptor template oligonucleotide (ATO) 1-014, adaptor template oligonucleotide (ATO) 1-015, adaptor template oligonucleotide (ATO) 1-016, adaptor template oligonucleotide (ATO) 1-017, adaptor template oligonucleotide (ATO) 1-018

[0907] TAQ DNA polymerase (NEB, M0273L)

[0908] TAQ DNA polymerase buffer (NEB, M0273L)

[0909] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0910] dNTP (NEB, N0447s)

[0911] High-fidelity DNA polymerase (NEB, M0530S)

[0912] Phusion buffer (NEB, M0530S)

[0913] Enzyme (NEB, M5505S)

[0914] AMV reverse transcriptase (NEB, M0277S)

[0915] Thermostable phosphatase (Antarctic Phosphatase) (NEB, M0289S)

[0916] Agencourt AMPure XP (Beckman Coulter, A63881)

[0917] Primers 2-001, 2-002, 2-003 (Table 2)

[0918] Methods

[0919] Annealing one or more adaptor template oligonucleotides to ribonucleic acid

[0920] As in Example 1, except that 25 ng of fragmented messenger RNA was used as the target polynucleotide.

[0921] Target ribonucleic acid extension by using the target ribonucleic acid as a primer and one or more adaptor template oligonucleotides as templates

[0922] As in Example 1, additionally ensure that the selected DNA polymerase can use RNA primers and optionally additional 5 units of thermosensitive phosphatase.

[0923] Degradation of one or more adaptor template oligonucleotides

[0924] As in Example 1, additionally ensure that any digestion / removal step does not non-specifically degrade RNA.

[0925] One-time extension or linear amplification of the modified target polynucleotide by reverse transcriptase

[0926] Combine the purified or unpurified modified target polynucleotide with a primer complementary to the conserved (universal) sequence now present at the 3'-end of the target RNA molecule, reverse transcriptase (e.g., but not limited to, M-MuLV reverse transcriptase, or AMV reverse transcriptase), one or more suitable buffers, and suitable dNTPs. In this example, mix the purified modified target polynucleotide with 50 nanomoles of 2-001, 10 units of AMV reverse transcriptase, 2 μl of 10× AMV buffer, and 10 nanomoles of each dNT (dATP / dTTP / dCTP / dGTP). Then, incubate the mixture to produce cDNA (first CS) (in this example, at 42 °C for 30 minutes). Then, the reverse transcription product or linear amplification product can be purified by any suitable method (e.g., using magnetic beads).

[0927] Annealing of one or more second adaptor template oligonucleotides to the first complementary strand

[0928] As in Example 1.

[0929] Extension of the first complementary strand by using the first complementary strand as a primer and the second adaptor template oligonucleotide as a template to form a modified first complementary strand

[0930] As in Example 1.

[0931] Degradation of one or more second adaptor template oligonucleotides

[0932] As in Example 1.

[0933] Exponential amplification of the second adaptor template extension product by PCR

[0934] As in Example 1.

[0935] Results

[0936] Using RNA as the target polynucleotide and using a method similar to the method in Example 1, we have been able to generate RNA sequencing libraries containing UIDs for assessing read - sequence repeats, low - frequency mutation detection, and have been able to improve molecular counting for more precise quantification.

[0937] Conclusion

[0938] By combining the first ATO reaction using fragmented messenger RNA, the process of linear amplification using reverse transcriptase to generate multiple first complementary strands, the second ATO reaction using the first complementary strand as the target polynucleotide, and subsequent global amplification, we have successfully verified the use of this technical method in the generation of RNA sequencing libraries.

[0939] Example 15

[0940] Using deoxyribonucleic acid (DNA) as the target polynucleotide, for generating next - generation sequencing libraries (e.g., but not limited to, compatible with Illumina next - generation sequencers) using one or more RNA adapter - template oligonucleotides having hairpins (generated using chemical spacers (e.g., but not limited to, C3 spacer, C18 spacer)).

[0941] Materials

[0942] Target polynucleotide, human gDNA (BIO - 35025)

[0943] DNA fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[0944] Adapter - template oligonucleotide (ATO) 1 - 031, Adapter - template oligonucleotide (ATO) 1 - 032

[0945] dNTP (NEB, N0447s)

[0946] High - fidelity DNA polymerase (NEB, M0530S)

[0947] Phusion buffer (NEB, M0530S)

[0948] Enzyme (NEB, M5505S)

[0949] Primer 2 - 001, Primer 2 - 002, Primer 2 - 003 (Table 2)

[0950] AMV reverse transcriptase (NEB, M0277S)

[0951] Ribonuclease H (NEB, M0297S)

[0952] Agencourt AMPure XP (Beckman Coulter, A63881)

[0953] Method

[0954] Adapter template oligonucleotide annealing to deoxyribonucleic acid

[0955] As in Example 1, except for using 200 picomoles of RNA ATO (1 - 031).

[0956] Target deoxyribonucleic acid extension by using the target deoxyribonucleic acid as a primer and one or more RNA adapter template oligonucleotides as templates

[0957] As in Example 1, except for using 10 units of AMV reverse transcriptase and 2 μl of 10×AMV buffer, with an extension temperature of 42°C for 30 minutes.

[0958] Degradation of adapter template oligonucleotide

[0959] Selectively degrade the adapter template oligonucleotide by adding ribonuclease (such as but not limited to, ribonuclease H, ribonuclease HII, ribonuclease A, ribonuclease T1), and then incubating at 37°C for 30 minutes or incubating at other suitable temperatures or times that cause degradation of one or more RNA - ATOs. In this example, 10 units of ribonuclease H are mixed with 20 modified target polynucleotides and incubated at 37°C for 30 minutes, and inactivated by incubating at 65°C for 20 minutes. Any suitable purification method can be used to optionally remove one or more degraded ATOs.

[0960] Linear amplification of modified target polynucleotide

[0961] As in Example 1.

[0962] Second adapter template oligonucleotide annealing to the first complementary strand

[0963] As in Example 1, except for using 200 picomoles of RNA ATO (1 - 032).

[0964] First complementary strand extension by using the first complementary strand as a primer and the second adapter template oligonucleotide as a template to form a modified first complementary strand

[0965] As in Example 1, except for using 10 units of AMV reverse transcriptase and 2 μl of 10×AMV buffer, with an extension temperature of 42°C for 30 minutes.

[0966] Degradation of one or more second adapter template oligonucleotides

[0967] As in the previous step of "Degradation of adapter template oligonucleotides".

[0968] Exponential amplification of the second adapter template extension product by PCR

[0969] As in Example 1.

[0970] Results

[0971] Using RNA ATO and a method similar to that in Example 1, we have been able to generate a sequencing library containing UIDs for evaluating read repeats and low-frequency mutation detection.

[0972] Conclusion

[0973] By combining the use of RNA ATO and the first ATO reaction of fragmented gDNA, the linear amplification process of generating multiple first complementary strands, using the second RNA ATO (linear amplification product) as the second ATO reaction of the target polynucleotide and subsequent global amplification, we have successfully verified the use of this technical method in the generation of sequencing libraries.

[0974] Example 16

[0975] Using ribonucleic acid (RNA) as the target polynucleotide for determining the transcription start site using one or more adapter template oligonucleotides (such as but not limited to) having a hairpin (generated using a chemical spacer arm (such as but not limited to, C3 spacer arm, C18 spacer arm)).

[0976] Materials

[0977] Adapter template oligonucleotide (ATO) 1-014

[0978] TAQ DNA polymerase (NEB, M0273L)

[0979] TAQ DNA polymerase buffer (NEB, M0273L)

[0980] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[0981] dNTP (NEB, N0447s)

[0982] High-fidelity DNA polymerase (NEB, M0530S)

[0983] Phusion buffer (NEB, M0530S)

[0984] Enzyme (NEB, M5505S)

[0985] Agencourt AMPure XP (Beckman Coulter, A63881)

[0986] Primer 2-003 (Table 2)

[0987] AMV Reverse Transcriptase (NEB, M0277S)

[0988] Method

[0989] Target-specific reverse transcription to generate cDNA

[0990] An appropriate amount of fragmented or unfragmented ribonucleic acid (RNA) of any length is mixed in a suitable buffer with an appropriate amount of one or more gene-specific primers targeting regions of RNA (such as, but not limited to, messenger RNA, microRNA, ribosomal RNA, or non-coding RNA) for which the transcription start site is undefined. In this example, 100 ng of mRNA is combined with 10 picomoles of target-specific primer, 10 units of AMV Reverse Transcriptase, 2 μl of 10× AMV buffer, and 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) in a final volume of 20 μl. The mixture is heated at a suitable temperature (in this example, 65 °C) for a suitable time (in this example, 2 minutes), or the mixture is heated at other temperatures or times that can cause double-stranded nucleic acids to denature into single strands. Subsequently, the nucleic acid is cooled with the target-specific primer (in this example, cooled for 5 minutes to 25 °C, or other suitable temperatures or times that can promote annealing and reverse transcription between the nucleic acid and one or more gene-specific primers). Then, the mixture is incubated to generate cDNA (in this example, 42 °C for 30 minutes). Then, the cDNA can be purified by any suitable method (such as using magnetic beads).

[0991] Adapter template oligonucleotides anneal to cDNA

[0992] As in Example 1.

[0993] cDNA extension by using cDNA as primer and one or more adapter template oligonucleotides as template

[0994] As in Example 1.

[0995] Degradation of one or more adapter template oligonucleotides

[0996] As in Example 1.

[0997] Exponential amplification of target complementary DNA extension products by PCR for generating a final sequencing library

[0998] The modified cDNA is combined with: a universal primer complementary to a conserved sequence introduced by one or more ATOs, a second primer designed to target a region of the mRNA close to the initial gene-specific primer, a DNA polymerase (such as but not limited to, Taq DNA polymerase, or Phusion), one or more suitable buffers, suitable dNTPs, and other suitable or necessary additives (such as but not limited to, DMSO, betaine, and ammonium sulfate). In this example, 5 μl of cDNA in a final volume of 50 μl, 10 picomoles of 2-003, 10 picomoles of the target-specific primer, 2 units of Phusion DNA polymerase, 10 μl of 5×phusion DNA polymerase buffer, and 10 nanomoles of each dNT (dATP / dTTP / dCTP / dGTP). Then, the mixture is thermocycled to exponentially amplify the target nucleic acid extension products. In this example, 98 °C for 30 seconds, 25× cycles of 98 °C for 5 seconds, 60 °C for 1 minute, and 72 °C for 1 minute, then 72 °C for 2 minutes. Then, the amplification products can be purified by any suitable method (such as using magnetic beads). Then, the purified products can be used for downstream processes (such as gel electrophoresis to identify the major products, cloning and sequencing, or directly used in a compatible cloning system and sequenced after transformation and colony selection).

[0999] Exponential amplification of target complementary DNA extension products by PCR for generating a final sequencing library

[1000] This can be carried out together with or instead of the previous step. The method is as in the previous step, except that the target-specific primer requires a 5' tail, and the 5' tail contains sequences compatible with and necessary for next-generation sequencing technologies (such as but not limited to, a P5 adapter sequence or a P7 adapter sequence compatible with Illumina next-generation sequencers, a patient / sample index sequence, an Illumina or custom read 1 or read 2 sequence). The amplification products can be purified by any suitable method (such as using magnetic beads) and will be suitable for next-generation sequencing based on compatible technologies.

[1001] Results

[1002] Using gene-specific primers, we successfully generated cDNA that was used as a template for the ATO reaction. When used with two additional primers, its products generated a library that could be sequenced on an Illumina machine, and the results revealed the transcription start sites of the selected transcripts.

[1003] Conclusion

[1004] By generating primer-binding gene-specific cDNA near the 5' of the mRNA, we were able to successfully adapt the technique to identify the transcription start sites of selected genes. Given the fact that only a small number of reads are needed to identify all possible transcription start sites, the entire sequencing run (even on a small-output machine) is not necessary. The method of generating products compatible with next-generation sequencing is preferably used as a low incorporation in another library that requires greater depth, or as a multiplex amplification of a large number of gene-specific primers to identify a large number of transcription start sites.

[1005] Example 17

[1006] Using a mixture of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as the target polynucleotide, for generating a combined DNA and RNA next-generation sequencing library (e.g., but not limited to, compatible with an Illumina next-generation sequencer) using one or more adapter-template oligonucleotides having a hairpin (generated using a chemical spacer (e.g., but not limited to, a C3 spacer, a C18 spacer)).

[1007] Materials

[1008] Target polynucleotide, human gDNA (BIO-35025)

[1009] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[1010] Adapter-Template Oligonucleotide (ATO) 1-014, Adapter-Template Oligonucleotide (ATO) 1-015, Adapter-Template Oligonucleotide (ATO) 1-016, Adapter-Template Oligonucleotide (ATO) 1-017, Adapter-Template Oligonucleotide (ATO) 1-018 (Table 1)

[1011] TAQ DNA Polymerase (NEB, M0273L)

[1012] TAQ DNA Polymerase Buffer (NEB, M0273L)

[1013] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[1014] dNTP (NEB, N0447s)

[1015] High-Fidelity DNA Polymerase (NEB, M0530S)

[1016] Phusion Buffer (NEB, M0530S)

[1017] Enzyme (NEB, M5505S)

[1018] Primers 2-001, 2-002, 2-003 (Table 2)

[1019] Agencourt AMPure XP (Beckman Coulter, A63881)

[1020] Ammonium sulfate (SIGMA, A4418)

[1021] AMV Reverse Transcriptase (NEB, M0277S)

[1022] Method

[1023] Annealing of one or more adaptor template oligonucleotides to nucleic acids

[1024] As in Example 1, except that the target polynucleotide is a mixture of RNA and DNA.

[1025] Target nucleic acid extension by using the target nucleic acid as a primer and one or more adaptor template oligonucleotides as a template

[1026] As in Example 1, plus ensuring that the polymerase can use RNA primers.

[1027] Degradation of one or more adaptor template oligonucleotides

[1028] As in Example 1.

[1029] Single extension or linear amplification of adaptor template extension products

[1030] As in Example 1, plus 10 units of AMV Reverse Transcriptase.

[1031] Annealing of one or more second adaptor template oligonucleotides to form a first complementary strand

[1032] As in Example 1.

[1033] First complementary strand extension by using the first complementary strand as a primer and the second adaptor template oligonucleotide as a template to form a modified first complementary strand

[1034] As in Example 1.

[1035] Degradation of one or more second adaptor template oligonucleotides

[1036] As in Example 1.

[1037] Exponential amplification of second adaptor template extension products by PCR

[1038] As in Example 1.

[1039] Results

[1040] Using a method similar to that in Example 1, we were able to combine both RNA and DNA to generate a next-generation sequencing library that contains UIDs suitable for removing PCR duplicates and correcting errors in discriminating low-frequency mutations.

[1041] Conclusions

[1042] The ability to combine DNA and RNA to generate a mixed sequencing library allows for many potential applications (especially considering the inherent nature of the technology to include UIDs for all target polynucleotides). For example, the ability to sequence both RNA and DNA can allow for comparative mutation analysis.

[1043] Example 18

[1044] Using a mixture of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as the target polynucleotide for generating a combined DNA and RNA next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using various adapter-template oligonucleotides (e.g., but not limited to) having hairpins, the hairpins being generated by two short regions that are partially or fully complementary and separated by a random sequence, a specifically designed sequence, or a specifically selected sequence, which assign different unique identifiers to RNA molecules and DNA molecules for separating the molecules after sequencing.

[1045] Materials

[1046] Target polynucleotide, human gDNA (BIO-35025)

[1047] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[1048] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[1049] Adapter-template oligonucleotide (ATO) 1-013, Adapter-template oligonucleotide (ATO) 1-033, Adapter-template oligonucleotide (ATO) 1-036 (Table 1)

[1050] dNTP (NEB, N0447s)

[1051] High-fidelity DNA polymerase (NEB, M0530S)

[1052] Phusion buffer (NEB, M0530S)

[1053] Deep VentR TM DNA polymerase (NEB, M0258S)

[1054] Tth DNA polymerase (SIGMA, 000000011480022001)

[1055] Enzyme (NEB, M5505S)

[1056] Agencourt AMPure XP (Beckman Coulter, A63881)

[1057] Primer 2-002, Primer 2-003 (Table 2)

[1058] Ammonium sulfate (SIGMA, A4418)

[1059] AMV reverse transcriptase (NEB, M0277S)

[1060] Method

[1061] Adapter template oligonucleotides annealed to nucleic acids

[1062] As in Example 1, except for using ATO 1-033.

[1063] Specific extension of target deoxyribonucleic acid by using the target deoxyribonucleic acid (instead of ribonucleic acid) as a primer and the adapter template oligonucleotide as a template

[1064] Mixing an appropriate amount of one or more DNA polymerases (capable of using DNA primers (instead of RNA primers), with or without proofreading activity) (such as but not limited to, DeepVentR TM DNA polymerase, Hot start Taq DNA polymerase, or Hot start DNA polymerase) is combined with the DNA / RNA ATO mixture. In this example, a total of 2 units of Deep VentR DNA polymerase, 2 μl of 10×Deep VentR DNA polymerase buffer, and 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) are combined with the DNA-ATO mixture to a final volume of 20 μl. In this example, in order to further promote the annealing of the target nucleic acid and one or more ATOs in the presence of one or more DNA polymerases and one or more buffers, the mixture is incubated at less than 25°C for 30 minutes, or the mixture is incubated at other suitable temperatures or times that can promote annealing between nucleic acids and one or more ATOs. In this example, in order to use the ATO sequence as a template to initiate the extension of the target polynucleotide, the incubation temperature is increased to 37°C for 30 minutes, or the incubation temperature is increased to other suitable temperatures that can promote DNA polymerase activity. The product is a modified target polynucleotide.

[1065] Generating double-stranded modified target polynucleotides

[1066] As in Example 5.

[1067] Degradation of one or more adapter template oligonucleotides

[1068] As in Example 1.

[1069] One or more second adaptor template oligonucleotides are annealed to the nucleic acid

[1070] As in Example 1, except ATO 1-036 was used.

[1071] Extension of target RNA using the target RNA as a primer and the adapter template oligonucleotide as a template

[1072] Combine a suitable amount of one or more DNA polymerases (capable of using one or more RNA primers, with or without proofreading activity) (such as, but not limited to, Escherichia coli DNA polymerase I) mixed with one or more suitable buffers and suitable dNTPs with one or more DNA / RNA ATO mixtures. In this example, a total of 2.5 units of Klenow, 2 μl of 10×Klenow buffer, and 10 nanomoles of each dNTP (dATP / dTTP / dCTP / dGTP) were combined with the DNA-ATO mixture to a final volume of 20 μl. In this example, to further promote annealing of the target nucleic acid and one or more ATOs in the presence of one or more DNA polymerases and one or more buffers, the mixture was incubated at a temperature below 25 °C for 30 minutes, or the mixture was incubated at other suitable temperatures or times capable of promoting annealing between the nucleic acid and one or more ATOs. In this example, to initiate extension of the target polynucleotide using the ATO sequence as a template, the incubation temperature was increased to 37 °C for 30 minutes, or the incubation temperature was increased to other suitable temperatures capable of promoting DNA polymerase activity. The product is a modified target polynucleotide.

[1073] Generate double-stranded target ribonucleic acid extension products

[1074] As in Example 5, except that an additional 10 units of AMV reverse transcriptase was used as the polymerase.

[1075] Degradation of one or more adapter template oligonucleotides and hairpin degradation

[1076] As in Example 5.

[1077] Single extension or linear amplification of double-stranded target deoxyribonucleic acid and one or more ribonucleic acid extension products

[1078] As in Example 1, except that 2.5 units of Tth DNA polymerase and Tth DNA polymerase buffer were used, and the extension temperature was 70 °C.

[1079] Third adapter template oligonucleotides anneal to one or more extension products or one or more linear amplification products

[1080] As in Example 1, except that ATO 1-022 was used.

[1081] Linear extension of the linear extension product by using it as a primer and one or more third adapter template oligonucleotides as templates to form a second adapter template extension product

[1082] As in Example 1.

[1083] Degradation of one or more third adapter template oligonucleotides

[1084] As in Example 1.

[1085] Exponential amplification of the second adapter template extension product by PCR

[1086] As in Example 1.

[1087] Results

[1088] Using a method similar to that in Example 1, we were able to combine both RNA and DNA to generate a next-generation sequencing library that contains UIDs suitable for removing PCR duplicates and correcting errors for discriminating low-frequency mutations.

[1089] Conclusion

[1090] The ability to combine DNA and RNA to generate a hybrid sequencing library, along with the ability to separate reads derived from DNA target polynucleotides and reads derived from RNA target polynucleotides during bioinformatics analysis, allows for many potential applications (especially considering the inherent nature of the technology that includes UIDs for all target polynucleotides). For example, the ability to sequence both RNA and DNA can allow for comparative mutation analysis. Different adapters can also be used for DNA and RNA ATOs such that they can be amplified differently to increase the DNA copies in the final sequencing library.

[1091] Example 19

[1092] Using deoxyribonucleic acid (DNA) as the target polynucleotide, for generating a template suitable for rolling circle amplification using one or more adapter template oligonucleotides having a hairpin (e.g., but not limited to, produced by two short regions that are partially complementary or fully complementary separated by a random sequence, a specifically designed sequence, or a specifically selected sequence) and a double-stranded adapter linked to the hairpin.

[1093] Materials

[1094] Target polynucleotide, human gDNA (BIO-35025)

[1095] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[1096] TAQ DNA Polymerase (NEB, M0273L)

[1097] TAQ DNA Polymerase Buffer (NEB, M0273L)

[1098] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[1099] Adapter Template Oligonucleotide (ATO) 1-013 (Table 1)

[1100] dNTP (NEB, N0447s)

[1101] High-fidelity DNA polymerase (NEB, M0530S)

[1102] Phusion buffer (NEB, M0530S)

[1103] Enzyme (NEB, M5505S)

[1104] T4 DNA ligase (NEB, M0202S)

[1105] Agencourt AMPure XP (Beckman Coulter, A63881)

[1106] Method

[1107] Annealing of one or more adapter template oligonucleotides into deoxyribonucleic acid

[1108] As in Example 1.

[1109] Target deoxyribonucleic acid extension by using the target deoxyribonucleic acid as a primer and one or more adapter template oligonucleotides as a template

[1110] As in Example 1.

[1111] Generate double-stranded target deoxyribonucleic acid extension products

[1112] As in Example 5.

[1113] Degradation of one or more adapter template oligonucleotides

[1114] As in Example 1.

[1115] Annealing of one or more double-stranded adapters and ligation to the ends of the double-stranded target deoxyribonucleic acid extension products

[1116] As in Example 5, plus the adapter forms a hairpin.

[1117] Results

[1118] Using a combination of ATO that can form a hairpin and an adapter with a hairpin, we have successfully generated circular modified target polynucleotides.

[1119] Conclusions

[1120] The ability to generate circular target polynucleotides allows rolling circle amplification, which allows the generation of very long products necessary for certain downstream processes.

[1121] Example 20

[1122] Using deoxyribonucleic acid (DNA) as the target polynucleotide, for generating a template suitable for rolling circle amplification using one or more adaptor template oligonucleotides having hairpins (generated using a chemical spacer such as, but not limited to, a C3 spacer, a C18 spacer), at least two adaptor template oligonucleotides are required, and at least two adaptor template oligonucleotides contain complementary regions and are allowed to anneal to produce a short double-stranded DNA with an exposed 3' end to allow extension.

[1123] Materials

[1124] Target polynucleotide, human gDNA (BIO-35025)

[1125] DNA Fragmentase, KAPA Fragmentation Kit (Roche, 7962517001)

[1126] TAQ DNA Polymerase (NEB, M0273L)

[1127] TAQ DNA Polymerase Buffer (NEB, M0273L)

[1128] DNA Polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[1129] Adaptor Template Oligonucleotide (ATO) 1-034, Adaptor Template Oligonucleotide (ATO) 1-035 (Table 1)

[1130] dNTP (NEB, N0447s)

[1131] High-Fidelity DNA Polymerase (NEB, M0530S)

[1132] Phusion Buffer (NEB, M0530S)

[1133] Enzyme (NEB, M5505S)

[1134] Methods

[1135] One or more adaptor template oligonucleotides are annealed to deoxyribonucleic acid

[1136] As in Example 1, additionally using ATO.

[1137] Target DNA extension by using the target deoxyribonucleic acid as a primer and one or more adaptor template oligonucleotides as templates

[1138] As in Example 1.

[1139] Degradation of one or more adaptor template oligonucleotides

[1140] As in Example 1.

[1141] Single extension or linear amplification of the modified target polynucleotide

[1142] As in Example 1.

[1143] Annealing of one or more second adaptor template oligonucleotides to the first complementary strand

[1144] As in Example 1, plus the use of ATO.

[1145] Extension of the first complementary strand by using the first complementary strand as a primer and one or more second adaptor template oligonucleotides as templates to form a modified first complementary strand

[1146] As in Example 1.

[1147] Degradation of one or more second adaptor template oligonucleotides

[1148] As in Example 1.

[1149] Self-annealing of the second extension product to generate a double-stranded region of DNA (the 3' end of which will be used as a primer)

[1150] Heat the purified extension product in a suitable buffer at a suitable temperature (in this example, 95 °C) for a suitable time (in this example, 2 minutes), or heat the purified extension product at other temperatures or times that can cause the double-stranded nucleic acid to denature into single strands. Subsequently, cool the extension product (in this example, cool for 2 minutes to 25 °C, or other suitable temperatures or times that can promote annealing between the complementary regions present at both ends of the extension product). Now, the circular DNA molecule can be used as a template for rolling circle amplification of the circular DNA.

[1151] Results

[1152] Similar to Example 19, we have successfully generated circular products whose 3' ends can serve as initiation sites for further rolling circle amplification.

[1153] Conclusions

[1154] The ability to generate circular target polynucleotides allows rolling circle amplification, which allows the generation of very long products necessary for certain downstream processes. The absence of additional primers means that competition with self-annealing of circular DNA can be avoided.

[1155] Example 21

[1156] Use of a PCR product as a target polynucleotide for generating a single (or multiple) amplicon high-diversity next-generation sequencing library (e.g., but not limited to, compatible with Illumina next-generation sequencers) using one or more adaptor template oligonucleotides (e.g., but not limited to) having a hairpin generated using a chemical spacer (e.g., but not limited to, a C3 spacer, a C18 spacer).

[1157] Materials

[1158] Adaptor template oligonucleotide (ATO) 1-014 (Table 1)

[1159] TAQ DNA polymerase (NEB, M0273L)

[1160] TAQ DNA polymerase buffer (NEB, M0273L)

[1161] DNA polymerase I, large (Klenow) fragment (NEB, M0210L, M0212L)

[1162] dNTP (NEB, N0447s)

[1163] High-fidelity DNA polymerase (NEB, M0530S)

[1164] Phusion buffer (NEB, M0530S)

[1165] Enzyme (NEB, M5505S)

[1166] Primer 2-003 (Table 2)

[1167] Ammonium sulfate (SIGMA, A4418)

[1168] Agencourt AMPure XP (Beckman Coulter, A63881)

[1169] Methods

[1170] Annealing of one or more adaptor template oligonucleotides to a PCR product

[1171] As in Example 1, except that the target polynucleotide is a PCR product.

[1172] Target PCR product extension by using the target PCR product as a primer and one or more adaptor template oligonucleotides as templates

[1173] As in Example 1.

[1174] Degradation of one or more adaptor template oligonucleotides

[1175] As in Example 1.

[1176] Exponential amplification of the linear extension products by PCR for generating the final sequencing library

[1177] As in Example 1, except that one of the universal primers was replaced with a target-specific primer with a 5' tail, the 5' tail containing sequences compatible with and required for next-generation sequencing technologies (e.g., but not limited to, a P5 adaptor sequence or a P7 adaptor sequence compatible with an Illumina next-generation sequencer, a patient / sample index sequence, an Illumina or custom read 1 ...

Claims

1. A method for extending a population of target polynucleotides, the method comprising: (i) Incubate the target polynucleotide with an adaptor template oligonucleotide having: (a) a 3'-end with a blocker that renders the adaptor template oligonucleotide non-extendable; (b) a 3'-random sequence that hybridizes to the target polynucleotide; (c) a nucleotide sequence that renders the adaptor template oligonucleotide degradable, the nucleotide sequence including a uracil moiety; and (d) a universal sequence 5' to the random sequence, wherein the target polynucleotide hybridizes to the 3'-random sequence of the adaptor template oligonucleotide; (ii) Perform polymerase extension of the target polynucleotide using the adaptor template oligonucleotide as a template to produce an extended target polynucleotide having a 3'-universal sequence; (iii) Digest the adaptor template oligonucleotide with an enzyme having dU-glycosylase activity that recognizes the nucleotide sequence; and (iv) Generate a first complementary sequence of the modified target polynucleotide, wherein generating the first complementary sequence comprises polymerase extension from the 3'-universal sequence using the modified target polynucleotide as a template.

2. The method according to claim 1, wherein the polymerase extension that generates the first complementary sequence is any one of the following: Extension of a self-priming 3'-stem-loop structure; or Extension of a primer hybridized to the 3'-universal sequence.

3. The method according to claim 1, wherein the first complementary sequence is extended to produce an extended first complementary sequence having a 3'-universal sequence.

4. The method according to claim 1, wherein the first complementary sequence is extended using a DNA ligase to ligate an adaptor to the first complementary sequence.

5. The method according to claim 1, the method further comprising extending a primer hybridized to the first complementary sequence or the modified first complementary sequence to form a second complementary sequence, wherein the primer hybridized to the first complementary sequence or the modified first complementary sequence comprises a target-specific portion, a universal sequence, or both a 3'-target-specific sequence and a 5'-universal sequence.

6. The method according to claim 1, wherein the polymerase used in step (ii) has 3'-to-5' exonuclease activity.

7. The method according to claim 1, wherein the target polynucleotide is derived from an FFPE sample, cell-free nucleic acid, or a bisulfite-treated sample.

8. The method according to claim 2, wherein the extension of the primer hybridized to the 3'-universal sequence repeats linear amplification.

9. The method according to claim 1, wherein the adaptor template oligonucleotide comprises a 3'-random sequence of 3 to 36 'N' bases.

10. The method according to claim 1, wherein the universal sequence comprises a sequence capable of serving as an RNA polymerase promoter.

11. The method according to claim 1, wherein the adaptor template oligonucleotide comprises, in 5'-to-3' order: A 5'-stem portion, an RNA polymerase sequence, a priming site sequence, a single-stranded overhanging 3'-random sequence, and a 3'-end with a blocker.

12. The method according to claim 1, wherein the adaptor template oligonucleotide comprises a stem-loop structure, the stem-loop structure containing a non-replicable linker selected from a C3 spacer, a triethylene glycol spacer, an 18-atom hexaethylene glycol spacer, and 1’,2’-dideoxyribose.

13. The method according to claim 1, wherein the 3’ end of the adaptor template oligonucleotide is blocked by a moiety selected from the group consisting of at least one ribonucleotide, at least one deoxynucleotide, a C3 spacer, a phosphate, a dideoxynucleotide, an amino group, and a reverse deoxythymidine.

14. A method for preparing a sequencing library from a population of single-stranded nucleic acids, the method comprising: (a) Perform the method according to claim 1 to produce a first complementary sequence; (b) Generate a second complementary sequence having a 5'-universal sequence and a 3'-universal sequence, wherein the 5'-universal sequence and the 3'-universal sequence are different and non-complementary to each other; and (c) Amplify the first complementary sequence and the second complementary sequence using primers targeting the 5'-universal sequence and the 3'-universal sequence to prepare a sequencing library of double-stranded nucleic acid fragments having known universal ends with different sequences.

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