Structure to prevent nucleic acid template threading through nanopore during sequencing

Abstract

The present invention relates to the formation of nucleic acid templates, including control templates, for sequencing using nanopore-based methods, wherein the novel structures disclosed herein are restricted or prevented from threading into the nanopore during sequencing.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid sequencing. More specifically, this invention relates to the field of forming libraries of nucleic acid targets for sequencing. Background Technology

[0002] Nucleic acid sequencing using biological nanopores and solid nanopores is a rapidly growing field; see Ameur et al. (2019) Single molecule sequencing: towards clinical applications, Trends Biotech., 37:72. In some methods, the nucleic acid template passes through a biological nanopore (US10337060) or a solid nanopore (US10288599, US20180038001, US10364507) or a tunnel junction between two electrodes (PCT / EP2019 / 066199 and US20180217083). In other methods, the template does not pass through the nanopore but through a detectable portion (e.g., a label or tag) (US8461854). Methods exist to prevent template nucleic acids from passing through nanopores or to modulate the rate at which template nucleic acids pass through nanopores. For example, a “speed bump” is a complementary oligonucleotide placed outside the pore (US10400278). tRNAs with a trilobal structure can attach to a template and interact with "braking" proteins to regulate the rate at which the template is inserted into the pore (US10131944). Hairpins and loops can be present in primers, or complementary probes can completely prevent perforation in non-nanopore sequencing methods (US9605309). Translationases such as helicases can also be used to regulate the perforation rate (US20180201993). In solid-state sequencing, template nucleic acids pass through pores in a thin solid layer. This perforation can be regulated by magnetic beads that "stretch" the nucleic acid strand (US20190317040).

[0003] Innovative and cost-effective methods are needed to control or prevent nucleic acids from passing through biological or solid nanopores during sequencing. Ideally, these methods would add a minimal number of steps or components to complex sequencing workflows. Summary of the Invention

[0004] This invention relates to the use of a self-initiating double hairpin adaptor. It is particularly advantageous for nanopore sequencing, as the 5' hairpin prevents displaced strands from entering the nanopore. The invention also includes a method for preparing libraries for nucleic acid sequencing and control nucleic acid molecules for nanopore sequencing.

[0005] The target nucleic acid is ligated to a novel double hairpin adaptor, such that both the 5' and 3' ends of the resulting nucleic acid construct contain hairpin structures (at one or both ends). The 3' hairpin has an extendable end, which acts as a sequencing primer for the first strand. The second strand is displaced during primer extension, but both the first and second strands retain their hairpins, thus preventing penetration into the nanopore.

[0006] In some embodiments, the present invention is an adaptor for a nucleic acid library comprising a first strand and a second strand, wherein: the first strand has a 5' portion and a 3' portion, wherein the 5' portion forms a stem-loop structure having a loop and a stem containing the 5' end of the first strand, and the 3' portion contains a sequence complementary to the second strand; the second strand has a 5' portion and a 3' portion, wherein the 3' portion forms a stem-loop structure having a loop and a stem containing the 3' end of the second strand, and the 5' portion contains a sequence complementary to the first strand; and the first and second strands form a double helix via the 3' portion of the first strand and the 5' portion of the second strand. The 3' portion of the second strand may be extendable by a nucleic acid polymerase. The loop-forming region may be at least 4, 5, 6, and up to 20 or more nucleotides long. The adaptor may contain one or more molecular barcodes selected from Sample ID (SID) and Unique Molecular Identifier (UID). For example, the SID can be located outside the double-stranded structure formed by the 3' portion of the first chain and the 5' portion of the second chain, or the UID can be located inside the double-stranded structure formed by the 3' portion of the first chain and the 5' portion of the second chain. The SID and UID can include predefined sequences, or the UID can be a random sequence.

[0007] In some embodiments, the present invention is a method for preparing a nucleic acid library, comprising: attaching a plurality of adaptors to a plurality of double-stranded nucleic acids in a sample, each adaptor comprising a first strand having a 5' portion and a 3' portion, wherein the 5' portion forms a stem-loop structure having a loop and a stem containing a 5' end of the first strand, and the 3' portion containing a sequence complementary to a second strand; a second strand having a 5' portion and a 3' portion, wherein the 3' portion forms a stem-loop structure having a loop and a stem containing an extendable 3' end of the second strand, and the 5' portion containing a sequence complementary to the first strand; and the first and second strands forming a double-stranded structure via the 3' portion of the first strand and the 5' portion of the second strand. The adaptors may be attached by connecting the double-stranded structure formed by the 3' portion of the first strand and the 5' portion of the second strand to one or both ends of the double-stranded nucleic acid. In some embodiments, prior to attachment, the plurality of nucleic acids are pretreated to form blunt ends at one or both ends of each nucleic acid. Multiple nucleic acids can be further preprocessed to add one or more non-templated nucleotides to one or both ends of a strand of each nucleic acid. In some embodiments, the duplex formed by the 3' portion of the first strand and the 5' portion of the second strand has single-stranded overhangs of one or more nucleotides. The adaptor may contain one or more molecular barcodes, such as a sample barcode (SID) and a unique molecular identifier barcode (UID). In some embodiments, the number of UIDs in the multiple adaptors may exceed the number of nucleic acids in the multiple nucleic acids.

[0008] In some embodiments, the present invention is a method for sequencing nucleic acids in a sample, the method comprising forming a nucleic acid library as described herein and sequencing the nucleic acids by a sequencing-by-synthesis method comprising extending the extendable 3' end of the second strand of an extender. The sequencing-by-synthesis method may include detection using nanopores.

[0009] In some embodiments, the present invention is a control nucleic acid for sequencing reactions, comprising a first strand and a second strand complementary to the first strand, and two ends, wherein at least one of the two ends comprises: a 3' protrusion forming a stem-loop structure, wherein the 3' end of the protrusion is extendable by a nucleic acid polymerase; and a 5' end, which, upon transposition via the extended 3' end, forms a stem-loop structure. The loop-forming region of the loop formed by the 3' end or the 5' end is at least 4, 5, 6, and up to 20 or more nucleotides long.

[0010] In some embodiments, the present invention is a method for sequencing a nucleic acid library, comprising contacting the library with a control nucleic acid as described herein, and sequencing the nucleic acid library by means of a method including detection using nanopores. Attached Figure Description

[0011] Figure 1 This is a diagram of a sequencing adaptor with two hairpin loops.

[0012] Figure 2 A diagram of a sequencing adaptor with a single hairpin loop. Detailed Implementation

[0013] definition

[0014] Unless otherwise defined, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See Sambrook et al., Molecular Cloning, A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press (2012).

[0015] The following definitions are provided to facilitate understanding of this disclosure.

[0016] The term "adaptor" refers to a nucleotide sequence that can be added to another sequence to impart additional elements and properties to that other sequence. These additional elements include, but are not limited to: barcodes, primer binding sites, capture moieties, tags, and secondary structures.

[0017] The term "barcode" refers to a nucleic acid sequence that can be detected and identified. Barcodes are typically more than two and up to about 50 nucleotides long. Barcodes are designed to have at least a minimum number of differences from other barcodes in a population. A barcode can be unique to each molecule in a sample, or unique to the sample and shared by multiple molecules in the sample. The terms "multiple identifier," "MID," or "sample barcode" refer to a barcode that identifies a sample or the source of the sample. In this regard, all or substantially all MID-barcoded polynucleotides from a single source or sample will share the same MID sequence; while all or substantially all (e.g., at least 90% or 99%) MID-barcoded polynucleotides from different sources or samples will have different MID barcode sequences. Polynucleotides from different sources with different MIDs can be mixed and sequenced in parallel while preserving the sample information encoded in the MID barcode. The term "unique molecular identifier" or "UID" refers to a barcode that identifies the polynucleotide to which it is attached. Typically, all or substantially all (e.g., at least 90% or 99%) of the UID barcodes in a polynucleotide mixture are unique.

[0018] The term "DNA polymerase" refers to an enzyme that performs template-guided synthesis of polynucleotides from deoxyribonucleotides. DNA polymerases include prokaryotic PolI, PolII, PolIII, PolIV, and PolV; eukaryotic DNA polymerases; archaea DNA polymerases; telomerases; and reverse transcriptases. The term "thermostable polymerase" refers to an enzyme that is heat-resistant and retains sufficient activity to perform subsequent polynucleotide elongation reactions after being exposed to high temperatures for the time required to denature double-stranded nucleic acids, without irreversible denaturation (inactivation). In some embodiments, the following thermostable polymerases may be used: *Thermococcus litoralis* (Vent, GenBank: AAA72101), *Pyrococcus furiosus* (Pfu, GenBank: D12983, BAA02362), *Thermococcus woesii*, *Pyrococcus* GB-D (DeepVent, GenBank: AAA67131), *Thermococcus kodakaraensis* KODI (KOD, GenBank: BD175553, BAA06142; *Thermococcus* sp. strain KOD (Pfx, GenBank: AAE68738)), *Thermococcus gorgonarius* (Tgo, Pdb: 4699806), and *Sulfolobus*. *Solanum solataricus* (GenBank: NC002754, P26811), *Aeropyrum pernix* (GenBank: BAA81109), *Archaeglobusfulgidus* (GenBank: 029753), *Pyrobaculumaerophilum* (GenBank: AAL63952), *Pyrodictium occultum* (GenBank: BAA07579, BAA07580), Thermococcus 9°Nm (GenBank: AAA88769, Q56366), Thermococcus fumica (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus sp. GE8 (GenBank: CAC12850), Thermococcus sp.JDF-3 (GenBank: AX135456, WO0132887), *Pyrococcus* sp. TY (GenBank: CAA73475), *Pyrococcus abyssi* (GenBank: P77916), *Pyrococcus glycovorans* (GenBank: CAC12849), *Pyrococcus horikoshii* (GenBank: NP143776), *Pyrococcus* sp. GE23 (GenBank: CAA90887), *Pyrococcus* sp. ST700 (GenBank: CAC12847), deep-sea thermococcus Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregateans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), and Thermococcus siculi (GenBank: DD259857).1) *Thermococcus thioreducens*, *Thermococcus onnurineus* NA1, *Sulfolobus acidocaldarium*, *Sulfolobus tokodaii*, *Pyrobaculum calidifontis*, *Pyrobaculus slandicum* (GenBank: AAF27815), *Methanococcus jannaschii* (GenBank: Q58295), *Desulfococcus* (TOK), *Desulfurococcus*, *Pyrolobium* Thermophilic bacteria (e.g., *Pyrodictium*, *Staphylothermus*, *Vulcanisaetta*, *Methanococcus* (GenBank: P52025) and other archaea B polymerases, such as GenBank AAC62712, P956901, BAAA07579), and thermophilic bacteria (e.g., *Aspergillus flavus*, *Monascus ruber*, *Streptococcus thermophilus*, *lacteus*, *rubens*, *aquaticus*, *Bacillus thermophilus*) of the genus *Thermus* (e.g., *Aspergillus flavus*, *Monascus ruber*, *Streptococcus thermophilus*, *lacteus*, *rubens*, *aquaticus*, *Bacillus thermophilus*). Thermotogamaritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9°N-7, T4, T7, phi29, Pyrococcus furiosus, P. Abyssi, Thermococcus Gorgonarius, Thermococcus Litoralis, Thermococcus zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, Thermococcus zilligii, Thermococcus sp.9N-7 polymerase. In some cases, the nucleic acid (e.g., DNA or RNA) polymerase may be a modified, naturally occurring type A polymerase. Further embodiments of the invention generally relate to a method in which, for example in primer extension, end-modification (e.g., terminal transferase, degradation, or completion) or amplification reactions, the modified type A polymerase may be selected from any species of any of the following genera: *Meiothermus*, *Thermotoga*, or *Thermomicrobium*. Another embodiment of the invention generally pertains to a method in which, for example in primer extension, end-modification (e.g., terminal transferase, degradation, or completion) or amplification reactions, the polymerase may be isolated from any of the following: *Thermus aquaticus* (Taq), *Thermus thermophilus*, *Thermus caldophilus*, or *Thermus filiformis*. Further embodiments of the invention generally encompass a method in which, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or completion), or amplification reaction, the modified type A polymerase can be isolated from *Bacillus stearothermophilus*, *Sphaerobacter thermophilus*, *Dictoglomus thermophilum*, or *Escherichia coli*. In other embodiments, the invention generally relates to a method in which, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or completion), or amplification reaction, the modified type A polymerase can be a mutant Taq-E507K polymerase. Another embodiment of the invention generally pertains to a method in which a thermostable polymerase can be used to induce the amplification of target nucleic acids.

[0019] The term "hairpin" refers to a secondary structure formed by a single-stranded nucleic acid containing at least one double-stranded region ("stem"), and the region forming the stem is interrupted by a single-stranded region "loop". The stem and loop regions do not have defined dimensions or relative dimensions.

[0020] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. Unless otherwise specified, the term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologous gene sequences, SNPs, and complementary sequences, as well as explicitly stated sequences.

[0021] The term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction. Typically, primers comprise fewer than about 100 nucleotides, and preferably fewer than about 30 nucleotides. Target-specific primers hybridize specifically with the target polynucleotide under hybridization conditions. Such hybridization conditions may include, but are not limited to, isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4), 50 mM KCl, 2 mM MgSO4, 0.1% [unclear - possibly a specific solution or solution]. Hybridization is performed at temperatures ranging from approximately 40°C to approximately 70°C (pH 8.8 at 20-25°C). In addition to the target binding region, primers may have an additional region, typically located in the 5' portion. This additional region may include a universal primer binding site or a barcode.

[0022] The term "sample" refers to any biological sample that includes nucleic acid molecules, typically DNA or RNA. A sample can be a tissue, cells, or extract thereof, or it can be a purified sample of nucleic acid molecules. The term "sample" also refers to any composition that contains or is presumed to contain a target nucleic acid. The use of the term "sample" does not necessarily imply the presence of a target sequence in the nucleic acid molecules present in the sample. The sample can include tissues or fluids isolated from an individual, such as skin, plasma, serum, cerebrospinal fluid, lymph, synovial fluid, urine, tears, blood cells, organs, and tumors, as well as samples from in vitro cultures established from cells taken from an individual, including formalin-fixed paraffin-embedded tissue (FFPET) and nucleic acids isolated from it. Samples can also include cell-free materials, such as cell-free blood fractions containing cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). Samples can be collected from non-human subjects or from the environment.

[0023] The term "target" or "target nucleic acid" refers to the target nucleic acid in a sample. A sample may contain multiple targets and multiple copies of each target.

[0024] The term "universal primer" refers to a primer that can hybridize with a universal primer binding site. A universal primer binding site can be a natural or artificial sequence that is typically added to a target sequence in a non-target-specific manner.

[0025] Sequencing nucleic acids using biological or solid-state nanopores is a rapidly developing field with numerous technological solutions available (see Ameur et al. (2019) Single molecule sequencing: towards clinical applications, Trends Biotech., 37:72). A common problem in nanopore sequencing is controlling the translocation (perforation) of nucleic acids through the pore. Some workflows only involve controlling the rate at which nucleic acids move through the opening. Solutions include using helicases (US20180201993) or magnetic particles (US20190317040) to slow down or modulate the translocation rate. Structures can be attached to the pore, such as “speed band” complementary oligonucleotides (US10400278). Other structures can be attached to template molecules; for example, tRNA structures attached to the template interact with “braking” proteins in the pore complex (US10131944) or hairpins present on the sequencing primers (US9605309).

[0026] Some nanopore sequencing technologies do not involve strand translocation through the nanopore. In such technologies, complete prevention of translocation (perforation) is required. This invention addresses the control and prevention of translocation through the nanopore.

[0027] Besides controlling the translocation of the strand to be sequenced, another distinct issue is the handling of the complementary strand in the case of double-stranded templates. When the double-stranded nucleic acid template unwinds during sequencing-by-synthesis (SBS) reactions, non-sequencing strands must be prevented from passing through the nanopores in both perforated and non-perforated nanopore sequencing embodiments.

[0028] This invention includes methods and compositions relating to the formation of nucleic acid templates for sequencing and nucleic acid template libraries for sequencing. These templates and libraries are applicable to any sequencing method, but offer unique advantages for nanopore sequencing.

[0029] In one embodiment, the present invention is a novel adaptor comprising a double-stranded portion for linking to a nucleic acid to be sequenced. The adaptor further includes a novel feature of at least one stem-loop or hairpin structure at one end opposite the double-stranded portion. In some embodiments, the adaptor has a single stem-loop structure. In other embodiments, the adaptor has two stem-loop structures. The adaptor retains an extendable 3' end, which can be used as a primer, such as a sequencing primer or an amplification primer. The adaptor may include any features useful in a sequencing adaptor, including barcodes, primer binding sites, or capture portions, for example, for isolating or purifying adapted nucleic acids. For example, the double-stranded portion of the adaptor may contain a unique molecular barcode (UID) to uniquely label adapted nucleic acids; or contain a multiple sample barcode (SID) or (MID) to identically label all nucleic acids from the same source in a sample. The adaptor may contain a capture portion, such as biotin, for capturing adapted nucleic acids and separating them from non-adapted nucleic acids. A particular advantage of the adaptor is the stem-loop or hairpin structure formed by at least one end of each strand of the adapted nucleic acid. The size of the loop is sufficient to inhibit or prevent chain translocation through the nanopore during sequencing. Depending on the nanopore used, the length of the loop-forming sequence can be 4, 5, 6, and up to 20 or more nucleotides to form a loop of sufficient size to prevent or inhibit translocation. Those skilled in the art can determine the length of the loop-forming sequence experimentally or empirically given the sequence of the nanopore-forming protein and its secondary, tertiary, or quaternary structure.

[0030] In one embodiment, the present invention is a method for preparing adapted nucleic acid libraries for sequencing. Novel adaptors are attached to one or both ends of each sample nucleic acid. The library can be purified or isolated from unused adaptors and unadapted sample nucleic acids.

[0031] In another embodiment, the present invention is a control molecule for sequencing a sample nucleic acid library. The control molecule has a novel structure at its ends as described herein. Specifically, the control nucleic acid molecule comprises a first strand and a second strand complementary to the first strand. The control molecule further comprises two ends, at least one of which includes: a 3' protrusion forming a stem-loop structure, wherein the 3' end of the protrusion is extendable by a nucleic acid polymerase, and a recessed 5' end, which, upon translocation through the extended 3' end, also forms a stem-loop structure. The size of the loop in the stem-loop structure on both strands is sufficient to inhibit or prevent strand translocation through the nanopore during sequencing.

[0032] This invention includes the simultaneous isolation and sequencing of target nucleic acids in a sample. In some embodiments, the sample is obtained from a subject or patient. In some embodiments, the sample may include fragments of solid tissue or solid tumors obtained, for example, by biopsy from the subject or patient. The sample may also include bodily fluids (e.g., urine, sputum, serum, plasma or lymph, saliva, phlegm, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid, or fecal samples). The sample may include whole blood or a blood fraction in which normal cells or tumor cells may be present. In some embodiments, the sample, particularly a liquid sample, may include cell-free material, such as cell-free DNA or RNA, including cell-free tumor DNA or tumor RNA. In some embodiments, the sample is a cell-free sample, for example, a cell-free blood-derived sample containing cell-free tumor DNA or tumor RNA. In other embodiments, the sample is a culture sample, for example, a culture or a culture supernatant containing or suspected to contain nucleic acids derived from cells in the culture or from an infectious agent present in the culture. In some embodiments, the source of infection is bacteria, protozoa, viruses, or mycoplasma.

[0033] Target nucleic acids are the target nucleic acids that may be present in a sample. Each target is characterized by its nucleic acid sequence. This invention is capable of detecting one or more RNA or DNA targets. In some embodiments, the DNA target nucleic acid is a gene or gene segment (including exons and introns) or an intergenic region, and the RNA target nucleic acid is a transcript or part of a transcript to which a target-specific primer hybridizes. In some embodiments, the target nucleic acid includes a locus of a genetic variant, such as a polymorphism, including a single nucleotide polymorphism or variant (SNP of a SNV), or a gene rearrangement that results in, for example, gene fusion. In some embodiments, the target nucleic acid includes a biomarker, i.e., a gene, a variant of which is associated with a disease or condition. For example, the target nucleic acid may be selected from combinations of disease-related biomarkers described in U.S. Patent Application Ser. 14 / 774,518, filed September 10, 2015. Such combinations are available as the AVENIOctDNA Analysis Kit (Roche Sequencing Solutions, Pleasanton, CA). In other embodiments, the target nucleic acid is characterized by a specific organism and facilitates organism identification, or it is characterized by pathogenic organisms, such as drug sensitivity or resistance. In other embodiments, the target nucleic acid is characterized by a unique feature of a human subject, for example, a combination of HLA or KIR sequences defining the subject's unique HLA or KIR genotype. In other embodiments, the target nucleic acid is a somatic cell sequence, such as a rearranged immune sequence representing an immunoglobulin (including IgG, IgM, and IgA immunoglobulins) or a T-cell receptor (TCR) sequence. In yet another application, the target is a fetal sequence present in the mother's blood, including fetal sequences characteristic of fetal diseases or conditions or pregnancy-related maternal conditions.

[0034] In some embodiments, the target nucleic acid is RNA (including mRNA, microRNA, viral RNA). In other embodiments, the target nucleic acid is DNA comprising cellular DNA or cell-free DNA (cfDNA) comprising circulating tumor DNA (ctDNA). The target nucleic acid may be present in short or long forms. Longer target nucleic acids may be fragmented. In some embodiments, the target nucleic acid is naturally fragmented, for example, comprising circulating cell-free DNA (cfDNA) or chemically degraded DNA, such as that found in chemically preserved or ancient samples.

[0035] In some embodiments, the present invention includes a nucleic acid isolation step. Generally, any method for extracting isolated nucleic acids containing DNA or RNA can be used. Genomic DNA or RNA can be extracted from tissues, cells, and liquid biopsy samples (including blood or plasma samples) using solution-based or solid-phase nucleic acid extraction techniques. Nucleic acid extraction may include detergent-based cell lysis, nucleoprotein denaturation, and optionally, removal of contaminants. Extraction of nucleic acids from preserved samples may also include a dewaxing step. Solution-based nucleic acid extraction methods may include salting out, or methods using organic solvents or dissociation agents. Solid-phase nucleic acid extraction methods may include, but are not limited to, silica resin methods, anion exchange methods, or magnetic glass particles and paramagnetic beads (KAPA pure beads, Roche Sequencing Solutions, Pleasanton, CA) or AMPure beads (Beckman Coulter, Brea, CA).

[0036] Typical extraction methods involve lysing tissue material and cells present in the sample. Nucleic acids released from the lysed cells can bind to a solid support (beads or particles) present in solution, a column, or a membrane, where the nucleic acids can undergo one or more washing steps to remove contaminants, including proteins, lipids, and their fragments, from the sample. Finally, the bound nucleic acids can be released from the solid support, column, or membrane and stored in the appropriate buffer until ready for further processing. Because DNA and RNA must be separated, nucleases cannot be used, and care should be taken to inhibit any nuclease activity during purification.

[0037] In some embodiments, the input DNA or input RNA needs to be fragmented. In such embodiments, RNA can be fragmented by a combination of heat and metal ions, such as magnesium. In some embodiments, the sample is heated to 85°–94°C for 1–6 minutes in the presence of magnesium (KAPARNA HyperPrep Kit, KAPA Biosciences, Wilmington, MAS). DNA can be fragmented by physical means, such as sonication, using available instruments (Covaris, Woburn, MA), or by enzymatic means (KAPA Fragmentation Enzyme Kit, KAPA Biosciences).

[0038] In some embodiments, the isolated nucleic acids are treated with DNA repair enzymes. In some embodiments, the DNA repair enzymes include DNA polymerases having 5'-3' polymerase activity and 3'-5' single-stranded exonuclease activity, polynucleotide kinases that add 5' phosphate to dsDNA molecules, and DNA polymerases that add a single dA base to the 3' end of dsDNA molecules. End repair / A-tailing kits are available, for example, from Kapa library preparation kits including KAPAHyperPrep and KAPAHyperPlus (Kapa Biosciences, Wilmington, MA).

[0039] In some embodiments, DNA repair enzymes target damaged bases in isolated nucleic acids. In some embodiments, the sample nucleic acid is partially damaged DNA from a preserved sample, such as a formalin-fixed paraffin-embedded (FFPET) sample. Deamination and oxidation of bases can lead to erroneous base reads during sequencing. In some embodiments, the damaged DNA is treated with uracil N-DNA glycosylase (UNG / UDG) and / or 8-oxoguanine DNA glycosylase.

[0040] In some embodiments, the invention includes an amplification step. Isolated nucleic acids can be amplified prior to further processing. This step may involve linear or exponential amplification. Amplification can be isothermal or involve thermal cycling. In some embodiments, amplification is exponential and involves PCR. In some embodiments, gene-specific primers are used for amplification. In other embodiments, a universal primer binding site is added to the target nucleic acid, for example, by ligating an adaptor that includes a universal primer binding site. All nucleic acids ligated to the adaptor have the same universal primer binding site and can be amplified using the same set of primers. The number of amplification cycles using universal primers can be low, but can also be 10, 20, or up to about 30 or more cycles, depending on the amount of product required for subsequent steps. Because PCR using universal primers reduces sequence bias, there is no need to limit the number of amplification cycles to avoid amplification bias.

[0041] In some embodiments, the present invention utilizes an adaptor nucleic acid comprising two strands having the structure described herein. In some embodiments, the adaptor molecule is an artificially synthesized sequence in vitro. In other embodiments, the adaptor molecule is a naturally occurring sequence synthesized in vitro. In other embodiments, the adaptor molecule is an isolated naturally occurring molecule or an isolated non-naturally occurring molecule.

[0042] The double-stranded ends of a hairpin adaptor can be attached to a double-stranded nucleic acid molecule. The adaptor can be attached at one or both ends of the double-stranded molecule.

[0043] The adaptor oligonucleotide may have a protrusion or a blunt end for ligation to the target nucleic acid. In some embodiments, the novel adaptors described herein include blunt ends to which blunt-end ligation of the target nucleic acid may be applied. The target nucleic acid may be blunt-ended or may be made blunt-ended by enzymatic treatment (e.g., “end repair”). In other embodiments, the blunt-ended DNA undergoes A-tailing, wherein a single A nucleotide is added to the 3' end of one or both blunt ends. The adaptors described herein are prepared with a single T nucleotide extending from the blunt end to facilitate ligation between the nucleic acid and the adaptor. Commercially available kits for performing adaptor ligation include the AVENIOct DNA Library Preparation Kit, or the KAPA HyperPrep and HyperPlus Kit (Roche Sequencing Solutions, Pleasanton, CA). In some embodiments, the adaptor-ligated DNA may be separated from excess adaptor and unligated DNA.

[0044] In some embodiments, the adapter contains additional features, such as a barcode, an amplification primer binding site, or a sequencing primer binding site.

[0045] Figure 1 A novel adaptor connected to the nucleic acid of interest is illustrated. (Reference) Figure 1 The adaptor comprises a first strand (the top strand, "adaptor oligonucleotide 1") and a second strand (the bottom strand, "adaptor oligonucleotide 2"). The adaptor contains a double-stranded hybridization region between the first and second adaptor strands ("adaptor oligonucleotides"). This region can be ligated to a target nucleic acid ("librium insert"). Each strand also contains a hairpin region with a stem and loop. Reference Figure 1 The adaptor illustrated herein has one end connected to the target nucleic acid, and the remaining ends are an unconnected 5' end and an unconnected 3' end. The first (top) strand contains a non-extendable 5' end located within the double-stranded stem portion of the top stem-loop structure. The second (bottom) strand contains a 3' end located within the double-stranded stem portion of the bottom stem-loop structure. The 3' end is extendable and can serve as a primer for replicating the bottom strand of the target nucleic acid without requiring a separate primer. In this embodiment, the 3' end of the adaptor is a self-starting region. This self-starting region serves as a sequencing primer or an amplification primer.

[0046] In some embodiments, the nucleic acids linked to the adaptor are sequenced after adaptor ligation. In other embodiments, the target nucleic acids linked to the adaptor are amplified before sequencing. Each strand of the replication strand contains components capable of folding into... Figure 1 The adaptor sequence of the double hairpin structure is shown. The size of the loop is sufficient to suppress or prevent translocation of either strand through the nanopore during sequencing. The length of the loop-forming region can be designed to be at least 3, 4, 5, 6, and up to 20 or more nucleotides long, depending on the nanopore used in sequencing.

[0047] Figure 2 Different embodiments of novel adaptors linked to nucleic acids of interest are illustrated. (Reference) Figure 2 The adaptor comprises a first strand (top strand, "adaptor oligonucleotide 1") and a second strand (bottom strand, "adaptor oligonucleotide 2"). The adaptor contains a double-stranded hybridization region between the first and second adaptor strands ("adaptor oligonucleotides"). This region can be ligated to a target nucleic acid ("library insert"). The first (top) strand contains a hairpin region with a stem and loop. The second (bottom) strand contains a capture portion. Figure 2 In the embodiment shown, the capture portion is present in a third oligonucleotide that hybridizes with the 3' end of the bottom linker strand.

[0048] The capture portion can be any portion capable of specifically interacting with another capture molecule. Capture portion-capture molecule pairs include avidin-biotin, antigen-antibody, magnetic (paramagnetic) particle-magnet, or oligonucleotide-complementary oligonucleotide. The capture molecule can be bound to a solid support such that any nucleic acid on which the capture portion is located is captured on the solid support and separated from the remainder of the sample or reaction mixture. In some embodiments, the capture molecule includes a capture portion for a second capture molecule. For example, the capture portion can be an oligonucleotide complementary to the capture oligonucleotide (capture molecule). The capture oligonucleotide can be biotinylated and captured on streptavidin magnetic beads.

[0049] In some embodiments, the adaptor-linked nucleic acids are enriched by capturing the captured portion and separating the target nucleic acid linked by the adaptor from the unlinked nucleic acids in the sample.

[0050] In some embodiments, the third oligonucleotide that hybridizes with the 3' end of the bottom linker strand ( Figure 2 It is used as a sequencing primer or amplification primer. In some embodiments, the extension product of the third oligonucleotide is captured by a capture moiety. The capture of the extension product separates the extension product from the unligated sample nucleic acid and optionally from the target nucleic acid strand that does not have a capture moiety.

[0051] In some embodiments, the stem portion of the adaptor includes a modified nucleotide that increases the melting temperature of the captured oligonucleotide, such as 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutynyl-2'-deoxyuridine, 8-aza-7-deazaguanidine, ribonucleotide, 2'O-methylribonucleotide, or locked nucleic acid. On the other hand, the captured oligonucleotide is modified to inhibit digestion by nucleases (e.g., phosphate-thionucleotides).

[0052] In some embodiments, the present invention relates to an amplification step, such as an amplification step prior to ligating a novel adaptor described herein. Primers may be target-specific. Target-specific primers include at least a portion complementary to the target. If additional sequences, such as barcodes or second primer-binding sites, are present, they are typically located in the 5' portion of the primer. The target may be a gene sequence (coding or non-coding) or a regulatory sequence present in RNA, such as an enhancer or promoter. The target may also be an intergenetic sequence. In other embodiments, primers are universal, for example, capable of amplifying all nucleic acids in a sample regardless of the target sequence. Universal primers are annealed to universal primer-binding sites of nucleic acids added to the sample by extending primers having universal primer-binding sites or by ligating adaptors (including adaptors having novel structures described herein).

[0053] In some embodiments, the present invention utilizes barcodes. Detection of individual molecules typically requires molecular barcodes, such as those described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. A unique molecular barcode is a short, artificial sequence that is typically added to each molecule in a patient sample during the initial steps of an in vitro procedure. The barcode identifies the molecule and its progeny. The unique molecular barcode (UID) has a variety of uses. Barcodes allow tracking of each individual nucleic acid molecule in a sample to assess, for example, the presence and quantity of circulating tumor DNA (ctDNA) molecules in a patient's blood, in order to detect and monitor cancer without a biopsy (Newman, A., et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi:10.1038 / nm.3519).

[0054] Barcodes can serve as multiplexed sample IDs (MIDs) to identify the source of a sample when samples are mixed (multiplexed). Barcodes can also serve as unique molecular IDs (UIDs) to identify each original molecule and its progeny. Barcodes can also be a combination of UID and MID. In some embodiments, a single barcode is used as both UID and MID. In some embodiments, each barcode includes a predefined sequence. In other embodiments, barcodes include random sequences. In some embodiments of the invention, the barcode length is between about 4 and 20 bases, thereby adding 96 to 384 different adaptors to a human genome sample, each adaptor having a different pair of identical barcodes. Those skilled in the art will recognize that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules) and will be able to create an appropriate number of barcodes for each experiment.

[0055] Unique molecular barcoding can also be used for molecule counting and correcting sequencing errors. The entire progeny of a single target molecule is tagged with the same barcode, forming a barcode family. Sequence variations not shared by all members of a barcode family are discarded as artifacts rather than true mutations. Barcoding can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[0056] In some embodiments, the number of UIDs in the multiple adaptors may exceed the number of nucleic acids in the multiple nucleic acids.

[0057] In some embodiments, the present invention is a target nucleic acid library formed as described herein. The library comprises a double-stranded nucleic acid molecule that includes a nucleic acid target present in a raw sample. The nucleic acid molecule of the library further includes a novel adaptor as described herein at one or both ends of the target nucleic acid sequence. The nucleic acid of the library may include additional elements, such as barcodes and primer binding sites. In some embodiments, the additional elements are present in the adaptor and added to the nucleic acid of the library via adaptor ligation. In other embodiments, some or all of the additional elements are present in the amplification primers and added to the nucleic acid of the library via primer extension prior to adaptor ligation. Amplification may be linear (including only one round of extension) or exponential, such as polymerase chain reaction (PCR). In some embodiments, some of the additional elements are added via primer extension, while the remaining additional elements are added via adaptor ligation.

[0058] For example, U.S. Patent Nos. 9,476,095, 9,260,753, 8,822,150, 8,563,478, 7,741,463, 8,182,989, and 8,053,192 describe the utility of adaptors and amplification primers for introducing additional elements into a nucleic acid library to be sequenced.

[0059] In some embodiments, the invention further includes the step of enriching the desired target nucleic acid. According to the novel library formation method described herein, the desired nucleic acid can be enriched before library formation. Alternatively, enrichment can be performed after the formation of the eh library, i.e., on the molecules of the library.

[0060] In some embodiments, the method utilizes a library of target-specific oligonucleotide probes (e.g., capture probes). Enrichment can be achieved through subtraction, in which case the capture probe is complementary to a large number of unwanted sequences, including ribosomal RNA (rRNA) or highly expressed genes (e.g., globin). In the case of subtraction, unwanted sequences are captured by the capture probe and removed and discarded from the target nucleic acid mixture or nucleic acid library. For example, the capture probe may include a binding moiety that can be captured on a solid support.

[0061] In other embodiments, enrichment involves capture and retention, where the capture probe is complementary to one or more target sequences. In this case, the target sequence is captured and retained from the target nucleic acid mixture or nucleic acid library by the capture probe, while the remainder of the solution is discarded.

[0062] For enrichment, the capture probe can be free in solution or immobilized on a solid support. The probe can be generated and amplified, for example, by the method described in U.S. Patent 9,790,543. The probe may also contain a binding moiety (e.g., biotin) and be capable of being captured on a solid support (e.g., avidin or streptomycin containing a supporting material).

[0063] In some embodiments, the invention includes an intermediate purification step. For example, any unused oligonucleotides (such as excess primers and excess adaptors) are removed, for example by size selection methods selected from gel electrophoresis, affinity chromatography, and size exclusion chromatography. In some embodiments, size selection can be performed using solid-phase reversible immobilization (SPRI) from Beckman Coulter (Breja, CA). In some embodiments, the captured portion ( Figure 2 It is used to capture and separate adapter-linked nucleic acids from unlinked nucleic acids or primer extension products from the template strand.

[0064] Nucleic acids and nucleic acid libraries or their amplicones formed as described herein can be sequenced. Sequencing can be performed by any method known in the art. High-throughput single-molecule sequencing using nanopores is particularly advantageous. In some embodiments, nucleic acids and nucleic acid libraries formed as described herein are sequenced by methods involving passing through biological nanopores (US10337060) or solid nanopores (US10288599, US20180038001, US10364507). In other embodiments, sequencing involves passing a tag through a nanopore (US8461854) or any other existing or future DNA sequencing technology utilizing nanopores.

[0065] In some embodiments, the sequencing step involves sequence analysis. In some embodiments, this analysis includes a sequence alignment step. In some embodiments, alignment is used to determine a common sequence from multiple sequences (e.g., multiple sequences having the same barcode (UID)). In some embodiments, the barcode (UID) is used to determine the common sequence from multiple sequences having the same barcode (UID). In other embodiments, the barcode (UID) is used to eliminate artifacts, i.e., variations present in some, but not all, sequences having the same barcode (UID). Such artifacts originating from PCR errors or sequencing errors can be eliminated.

[0066] In some embodiments, the quantity of each sequence in a sample can be quantified by the relative number of sequences for each barcode (UID) in the sample. Each UID represents a single molecule in the original sample, and counting the different UIDs associated with each sequence variant can determine the proportion of each sequence in the original sample. Those skilled in the art will be able to determine the number of sequence reads necessary to determine the common sequences. In some embodiments, a corresponding number of reads are required for each UID (“sequence depth”) for accurate quantification results. In some embodiments, a desired depth is 5-50 reads per UID.

[0067] Example

[0068] Example 1. Novel adaptors for nucleic acid sequencing

[0069] The sequencing adaptor consists of two oligonucleotides (adaptor oligonucleotide 1 and adaptor oligonucleotide 2) with complementary portions, enabling them to form a partially double-stranded adaptor. Adaptor oligonucleotide 1 further contains DNA sequenced at the 5' end, resulting in an intramolecular stem-loop structure under sequencing reaction conditions. The size of the loop structure can be tuned by DNA length and sequence composition to minimize the possibility of the displaced 5' end crossing the sequencing nanopore. Adaptor oligonucleotide 2 also contains a stem-loop forming structure at the 3' end and a free 3' end for binding to nucleic acid polymerase. This 3' end extends during amplification or sequencing, and the strand linked by adaptor oligonucleotide 1 is ultimately displaced by the sequencing or amplification polymerase, with the 5' loop restricting the displaced strand from crossing the sequencing nanopore.

[0070] Example 2. Formation of control nucleic acids for nanopore sequencing

[0071] First, a DNA insert containing the desired endonuclease site, DNA primer annealing site, and complementary end sequence is cloned into a plasmid vector. The plasmid is grown in host bacteria; the plasmid is then extracted and purified. The plasmid is then linearized by digestion with an endonuclease that provides a flat cut (e.g., PmeI). The linearized plasmid is further digested with a cut endonuclease (e.g., Nt.BbvCI) to provide a short, excised single-stranded fragment at the 5' end of the linearized plasmid. The plasmid is further denatured and cooled in the presence of an oligonucleotide complementary to the excised fragment. The excised fragment is hybridized to the complementary oligonucleotide, which may be biotin-labeled to allow purification using streptavidin beads to remove the hybridized excised fragment. Excision of the 5' end of the plasmid results in a secondary structure at the 3' end, such as a hairpin (stem-loop), which prevents the single-stranded end from passing through a nanopore during DNA sequencing. The nascent 5' end of this plasmid is also designed in a similar way, so that when the primer oligonucleotide annealed to the 3' end is displaced during polymerase extension, the 5' end also forms a secondary structure to prevent the displaced 5' end from passing through the nanopore.

Claims

1. An adapter for a nucleic acid library comprising a first strand and a second strand, wherein: a. The first chain has a 5' portion and a 3' portion, wherein the 5' portion forms a stem-loop structure having a loop and a stem containing the 5' end of the first chain, and the 3' portion contains a sequence complementary to the second chain; b. The second chain has a 5' portion and a 3' portion, wherein the 3' portion forms a stem-loop structure having a loop and a stem containing the 3' end of the second chain, and the 5' portion contains a sequence complementary to the first chain; and c. The first chain and the second chain form a double chain via the 3' portion of the first chain and the 5' portion of the second chain.

2. The adapter according to claim 1, wherein the 3' end of the second strand is extendable by a nucleic acid polymerase.

3. The adaptor according to claim 1, wherein one or both loop forming regions are at least 4 nucleotides long.

4. The adaptor according to claim 1, wherein one or both loop forming regions are at least 5 nucleotides long.

5. The adaptor according to claim 1, wherein one or both loop forming regions are at least 6 nucleotides long.

6. The adaptor according to claim 1, wherein one or both loop forming regions are up to 20 nucleotides long.

7. The connector of claim 1, wherein the connector comprises one or more molecular barcodes.

8. The connector according to claim 7, wherein the molecular barcode is selected from sample barcode (SID) and unique molecular identifier barcode (UID).

9. The connector of claim 8, wherein the SID is located outside the double chain formed by the 3' portion of the first chain and the 5' portion of the second chain.

10. The connector of claim 8, wherein the UID is located inside the double chain formed by the 3' portion of the first chain and the 5' portion of the second chain.

11. The connector of claim 8, wherein the SID and the UID comprise a predefined sequence or a random sequence.

12. A method for preparing a nucleic acid library, the method comprising: Multiple adaptors are attached to multiple double-stranded nucleic acids in a sample, each adaptor containing... a. A first chain having a 5' portion and a 3' portion, wherein the 5' portion forms a stem-loop structure having a loop and a stem containing the 5' end of the first chain, and the 3' portion contains a sequence complementary to the second chain; b. A second chain having a 5' portion and a 3' portion, wherein the 3' portion forms a stem-loop structure having a loop and a stem containing an extendable 3' end of the second chain, and the 5' portion contains a sequence complementary to the first chain; and c. The first chain and the second chain form a double chain via the 3' portion of the first chain and the 5' portion of the second chain.

13. The method of claim 12, wherein the adapter is attached via the double-stranded body formed by attaching the 3' portion of the first strand and the 5' portion of the second strand to one or both ends of the double-stranded nucleic acid.

14. The method of claim 12, wherein prior to attachment, the plurality of nucleic acids are pretreated to form blunt ends at one or both ends of each nucleic acid.

15. The method of claim 12, wherein the duplex formed by the 3' portion of the first chain and the 5' portion of the second chain has single-stranded overhangs of one or more nucleotides.

16. A method for sequencing nucleic acids in a sample, the method comprising forming a library of nucleic acids according to claim 12, and sequencing the library by a sequencing-by-synthesis method, the sequencing-by-synthesis method comprising extending the extendable 3' end of the second strand of the adapter.

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