Generation of double-stranded DNA templates for single-molecule sequencing

Abstract

The present invention is a novel nucleic acid sequencing method, which involves preparing a library of double-stranded target nucleic acid containing linear partially single-stranded adapters and sequencing the library.

Description

Field of the Invention

[0001] The present invention relates to the field of nucleic acid analysis, and more particularly, to preparing templates for nucleic acid sequencing. Background of the Invention

[0003] Single molecule nucleic acid sequencing including nanopore sequencing typically utilizes circular templates. See U.S. Patent Nos. 7,302,146 and 8,153,375. Linear nucleic acids are converted into circular forms for amplification and subsequent detection and quantification, see U.S. Patent No. RE44265. For long target molecules where a linear conformation is preferred, the circular template configuration is not ideal. The present invention is a method for generating a library of linear target sequences using a novel adapter design and sequencing. The method has multiple advantages described in detail below. SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention is a method for sequencing each strand of a double-stranded target nucleic acid separately, comprising the following steps: in a reaction mixture, a double-stranded target nucleic acid is joined to an adapter to form an attached target nucleic acid, wherein the adapter comprises a shorter chain and a longer chain, which anneal together to form a double-stranded and single-stranded region, wherein the single-stranded region comprises only one unpaired chain, and wherein the single-stranded region comprises a primer binding site; a primer is annealed to the primer binding site in each adapter; the primer is extended, thereby sequencing each strand of the attached target nucleic acid. It can be joined to the adapter by connection. The adapter can include at least one barcode. The adapter can include at least one modified nucleotide in the double-stranded region. The modified nucleotide can be a 5'-phosphorylated terminal nucleotide, a thiophosphate group, or can modify the melting temperature of the double-stranded region of the adapter. In some embodiments, the method further comprises a target enrichment step, for example, by capture via a target-specific probe bound to a solid support or by amplification with a target-specific primer. In some embodiments, the method further comprises the steps of contacting the reaction mixture with a DNA damage-specific cleavage agent selected from a glycosylase and an endonuclease, thereby cleaving the damaged DNA. In some embodiments, the target nucleic acid comprises a concatemer of two or more copies of the target sequence. In some embodiments, the method further comprises the step of constructing a consensus sequence of the target nucleic acid from the sequence of the concatemer.

[0006] In another embodiment, the present invention is a method for preparing a nucleic acid library from a double-stranded target nucleic acid in a sample, the method comprising: in a reaction mixture, joining the double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises a shorter chain and a longer chain, which anneal together to form double-stranded and single-stranded regions, wherein the single-stranded region comprises only one single strand, and wherein the single-stranded region comprises a primer binding site.

[0007] In yet another embodiment, the present invention is a method for determining the sequence of a target nucleic acid library in a sample, the method comprising the following steps: forming a library of single-stranded circular target nucleic acids as described in the preceding paragraphs; annealing a primer to the primer binding site in each connected target nucleic acid, extending the primer, thereby sequencing each chain of the target nucleic acid in the library.

[0008] In yet another embodiment, the present invention is a method for sequencing a double-stranded target nucleic acid, which comprises the following steps: in a reaction mixture, amplifying the target nucleic acid with a pair of target-specific primers, thereby forming an amplicon; joining the amplicon to an adaptor to form a connected target nucleic acid, wherein the adaptor comprises a shorter chain and a longer chain, which anneal together to form double-stranded and single-stranded regions, wherein the single-stranded region comprises only one single strand, and wherein the single-stranded region comprises a primer binding site; annealing a sequencing primer to the primer binding site; extending the primer, thereby sequencing the target nucleic acid.

[0009] In yet another embodiment, the present invention is a method for preparing a library of target nucleic acids, comprising the following steps: in a reaction mixture, amplifying the target nucleic acid with a pair of target-specific primers, thereby forming an amplicon; joining the amplicon to an adapter to form an attached target nucleic acid, wherein the adapter comprises an annealed shorter chain and a longer chain, wherein the single-stranded region comprises only one single strand and further comprises a primer binding site.

[0010] In yet another embodiment, the present invention is a method for determining the sequence of a library of target nucleic acids in a sample, the method comprising the following steps: forming a library of linked target nucleic acids as described in the preceding paragraphs; annealing primers to the primer binding sites in each linked target nucleic acid; and extending the primers, thereby sequencing each target nucleic acid in the library. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An example of a novel adaptor is also represented by Seq. Id. No: 1 +2.

[0013] Figure 2 For example Figure 1Method for sequencing a single long target sequence using the adapters shown in .

[0014] Figure 3 Example of use Figure 1 or Figure 4 Concatemer strategy for short target sequences of the adaptors shown in FIG.

[0015] Figure 4 Examples of non-nucleotide linkers Figure 1 A new type of connector. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition

[0018] The following definitions assist in understanding the present disclosure.

[0019] The term "sample" refers to any composition containing or assuming to contain a target nucleic acid. This includes samples of tissues or fluids separated from individuals, for example, skin, plasma, serum, spinal fluid, lymph, synovial fluid, urine, tears, blood cells, organs and tumors, and also refers to samples of in vitro cultures established from cells taken from individuals, including formalin-fixed paraffin-embedded tissues (FFPET) and nucleic acids isolated therefrom. Samples may also include cell-free materials, such as cell-free blood fractions containing cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

[0020] The term "nucleic acid" refers to a polymer of nucleotides (e.g., natural and unnatural ribonucleotides and deoxyribonucleotides), including DNA, RNA, and subclasses thereof, such as cDNA, mRNA, etc. Nucleic acids can be single-stranded or double-stranded, and typically contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs may have other bonds. Nucleic acids may include naturally occurring bases (adenosine, guanosine, cytosine, uracil, and thymidine) as well as unnatural bases. Some examples of unnatural bases include, for example, those described in Seela et al. , (1999) Helv. Chim. Acta 82: 1640. The unnatural bases may have specific functions, for example, increasing the stability of nucleic acid duplexes, inhibiting nuclease digestion, or blocking primer extension or chain polymerization.

[0021] The terms "polynucleotide" and "oligonucleotide" are used interchangeably. A polynucleotide is a single-stranded or double-stranded nucleic acid. Oligonucleotide is a term sometimes used to describe shorter polynucleotides. Oligonucleotides are prepared by any suitable method known in the art, for example, by methods involving direct chemical synthesis, as described in Narang et al. ( 1979) Meth. Enzymol.68:90-99; Brown et al. (1979) Meth. Enzymol. 68:109-151; Beaucage et al. ( 1981) Tetrahedron Lett. 22:1859-1862; Matteucci et al. ( 1981) J. Am. Chem. Soc. 103:3185-3191.

[0022] The term "primer" refers to a single-stranded oligonucleotide that hybridizes to a sequence in a target nucleic acid (a "primer binding site") and is capable of serving as a point of initiation of synthesis along a complementary strand of the nucleic acid under conditions suitable for such synthesis.

[0023] The term "adapter" means a nucleotide sequence that can be added to another sequence in order to introduce additional properties into that sequence. An adaptor is typically an oligonucleotide that can be single-stranded or double-stranded, or can have both single-stranded and double-stranded portions.

[0024] The term "connection" refers to a condensation reaction that joins two nucleic acid chains, wherein the 5'-phosphate group of one molecule reacts with the 3'-hydroxyl group of another molecule. Connection is generally an enzymatic reaction catalyzed by a ligase or topoisomerase. Connection can join two single strands to produce a single-stranded molecule. Connection can also join two chains (each chain belongs to a double-stranded molecule), thus joining two double-stranded molecules. Connection can also join the two chains of a double-stranded molecule to the two chains of another double-stranded molecule, thus joining two double-stranded molecules. Connection can also join the two ends of the chain in the double-stranded molecule, thus repairing the nick in the double-stranded molecule.

[0025] The term "barcode" refers to a nucleic acid sequence that can be detected and identified. The barcode can be incorporated into a variety of nucleic acids. The barcode is long enough, such as 2, 5, 20 nucleotides, so that in a sample, the nucleic acids incorporated with the barcode can be distinguished or grouped according to the barcode.

[0026] The term "multiple identifier" or "MID" refers to a barcode that identifies the source of a target nucleic acid (e.g., the sample from which the nucleic acid was derived). All or substantially all target nucleic acids from the same sample will share the same MID. Target nucleic acids from different sources or samples can be mixed and sequenced simultaneously. Using the MID, sequence reads can be assigned to each sample from which the target nucleic acid originated.

[0027] The term "unique molecular identifier" or "UID" refers to a barcode that identifies the nucleic acid to which it is attached. All or substantially all target nucleic acids from the same sample will have different UIDs. All or substantially all progeny (e.g., amplicons) derived from the same original target nucleic acid will share the same UID.

[0028] The terms "universal primer" and "universal priming binding site" or "universal priming site" refer to primers and primer binding sites that are present in (usually, added in vitro to) different target nucleic acids. The universal priming site is added to a variety of target nucleic acids using adapters or using target-specific (non-universal) primers with a universal priming site in the 5'-portion. The universal primer can bind to the universal priming site and guide primer extension from the universal priming site.

[0029] More generally, the term "universal" refers to a nucleic acid molecule (e.g., a primer or other oligonucleotide) that can be added to any target nucleic acid and performs its function independently of the target nucleic acid sequence. A universal molecule can perform its function by hybridizing with a complement (e.g., a universal primer hybridizing to a universal primer binding site or a universal cyclizing oligonucleotide hybridizing to a universal primer sequence).

[0030] As used herein, the term "target sequence", "target nucleic acid" or "target" refers to a portion of a nucleic acid sequence in a sample to be detected or analyzed. The term target includes all variants of the target sequence, for example, one or more mutant variants and wild-type variants.

[0031] The term "amplification" refers to the process of preparing additional copies of a target nucleic acid. Amplification can have more than one cycle, for example, multiple cycles of exponential amplification. Amplification can have only one cycle (preparing a single copy of a target nucleic acid). The copy can have additional sequences, such as those present in the primers used for amplification. Amplification can also produce only one strand of several copies (linear amplification) or preferably one strand (asymmetric PCR).

[0032] The term "sequencing" refers to any method of determining the sequence of nucleotides in a target nucleic acid.

[0033] The present invention is a method for generating double-stranded sequence templates suitable for single-molecule long-read sequencing. Compared with existing library preparation methods, such as those utilizing circular templates (see US Pat. Nos. 7,302,146 and 8,153,375), the methods of the present invention are particularly advantageous for long amplicons.

[0034] The present invention includes detecting target nucleic acid in sample. In some embodiments, the sample is derived from a subject or patient. In some embodiments, the sample may include fragments of solid tissue or solid tumors derived from a subject or patient, such as by biopsy. The sample may also include body fluids (e.g., urine, sputum, serum, plasma or lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid and / or fecal samples). The sample may include whole blood or blood fractions in which tumor cells may be present. In some embodiments, the sample, especially the liquid sample, may include cell-free material, such as cell-free DNA or RNA, including cell-free tumor DNA or tumor RNA. The present invention is particularly suitable for analyzing rare and small amounts of targets. In some embodiments, the sample is a cell-free sample, such as a cell-free blood-derived sample, in which cell-free tumor DNA or tumor RNA is present. In other embodiments, the sample is a culture sample containing or suspected of containing an infectious agent or a nucleic acid derived from an infectious agent, such as a culture or culture supernatant. In some embodiments, the infectious agent is a bacterium, a protozoa, a virus, or a mycoplasma.

[0035] Target nucleic acid is a target nucleic acid that may be present in a sample. In some embodiments, the target nucleic acid is a gene or a gene fragment. In other embodiments, the target nucleic acid contains genetic variants, for example, polymorphisms, including single nucleotide polymorphisms or variants (SNP or SNV), or causes genetic rearrangements such as gene fusions. In some embodiments, the target nucleic acid comprises a biomarker. In other embodiments, the target nucleic acid is peculiar to a particular organism, for example, helps to identify pathogenic organisms, or peculiar to pathogenic organisms, for example, drug sensitivity or drug resistance. In other other embodiments, the target nucleic acid is peculiar to a human subject, for example, defines the unique HLA or KIR genotype of a subject, HLA or KIR sequences. In other other embodiments, all sequences in a sample are target nucleic acids, for example, in shotgun genome sequencing.

[0036] In one embodiment of the invention, a double-stranded target nucleic acid is converted to a template configuration of the invention. In some embodiments, the target nucleic acid exists in single-stranded form in nature (e.g., RNA, including mRNA, microRNA, viral RNA; or single-stranded viral DNA). The single-stranded target nucleic acid is converted to a double-stranded form to achieve the additional steps of the claimed method.

[0037] Longer target nucleic acids can be fragmented, although in some applications longer target nucleic acids may be desired to achieve longer reads. The present invention is particularly advantageous for longer nucleic acid targets.

[0038] In some embodiments, the present invention includes a target enrichment step. The enrichment can be carried out by capturing the target sequence via one or more target-specific probes. The nucleic acid in the sample can be denatured and contacted with a single-stranded target-specific probe. The probe can include a ligand of an affinity capture portion so that after forming a hybrid complex, they are captured by providing an affinity capture portion. In some embodiments, the affinity capture portion is avidin or streptavidin, and the ligand is biotin. In some embodiments, the portion is combined with a solid support. As described in further detail below, the solid support can include superparamagnetic spherical polymer particles, such as DYNABEADS™ magnetic beads or magnetic glass particles.

[0039] In some embodiments of the present invention, the adapter molecule is connected to the target nucleic acid. The connection can be a flat end connection or a more effective sticky end connection. By including "end repair" of chain filling, that is, extending the 3'-end by DNA polymerase to eliminate the 5'-overhang, the target nucleic acid can be made flat-ended. In some embodiments, by adding a single nucleotide to the 3'-end of the adapter and adding a single complementary nucleotide to the 3'-end of the target nucleic acid, for example, by DNA polymerase or terminal transferase, the flat-ended nucleic acid can be made sticky. In other embodiments, the adapter and the target nucleic acid can obtain sticky ends (overhangs) by digestion with restriction endonucleases. The restriction enzyme recognition site can be inherent or engineered into the sequence. In some embodiments, other enzymatic steps may be required to complete the connection. In some embodiments, polynucleotide kinases can be used to add 5'-phosphate to the target nucleic acid molecule and the adapter molecule.

[0040] In some embodiments, using Figure 1 The adapter shown in . The adapter has a single-stranded and double-stranded region, wherein the single-stranded region comprises a primer binding site (e.g., a sequencing primer binding site). In order to enable the connection of the adapter to the double-stranded target nucleic acid, the 5'-end of the adapter can be phosphorylated. In order to prevent the degradation of the shorter chain of the adapter by polymerase extension of the sequencing primer bound to the sequencing primer binding site, the 3'-end of the shorter chain of the adapter can contain modified nucleotides at or near the 3'-end of the shorter chain. In some embodiments, the modified nucleotides are phosphorothioate nucleotides.

[0041] In some embodiments, the double-stranded region of the adaptor comprises a region that modifies (e.g., increases) the melting temperature (T m). In some embodiments, the modified nucleotide is a modified pyrimidine, such as methyl-dC or propynyl-dU. In some embodiments, the modified nucleotide is a modified purine, such as G-clamp.

[0042] In some embodiments, the primer binding site in the single-stranded region of the adaptor comprises a modified nucleotide that modifies (e.g., increases) the melting temperature (T) of a primer bound to the primer binding site. m ). In some embodiments, the modified nucleotide is a modified pyrimidine, such as methyl-dC or propynyl-dU. In some embodiments, the modified nucleotide is a modified purine, such as G-clamp.

[0043] Figure 1 Examples of sequences and structures of linear adapters are illustrated. The adapter includes a short double-stranded portion with a 5'-phosphate and a single 3'-T overhang to facilitate connection to the target insert. The adapter includes a long arm with a phosphorothioate modification (asterisk) to avoid digestion by the 5'-3' exonuclease activity of a DNA polymerase. The long arm further includes a binding site for a sequencing primer to enable the polymerase to bind to the target insert and perform sequencing.

[0044] Figure 2 For example Figure 1 Figure 2. Sequencing of a single long target sequence using the adapter shown in Figure 2. Linear adapters are ligated at both ends of the template, with arrows showing the direction of sequencing into the template. Reads from multiple wells will be used to obtain a consensus sequence (intermolecular consensus sequence).

[0045] Figure 3 The concatemer strategy of short target sequences is illustrated. Before concatenation, an adaptor with a unique molecular ID (UID) is connected to each target sequence. The reads from multiple holes can be used and the consensus sequence is obtained using the UID in each read.

[0046] Figure 4 The sequence and structure of an adaptor with an 18-carbon non-nucleotide linker (HEG) are illustrated.

[0047] In some embodiments, the adapter molecule is an artificial sequence synthesized in vitro. In other embodiments, the adapter molecule is a naturally occurring sequence synthesized in vitro. In yet other embodiments, the adapter molecule is an isolated naturally occurring molecule.

[0048] In some embodiments, the method includes a step of amplifying the target nucleic acid prior to linking the adaptor. The amplification can be by exponential polymerase chain reaction (PCR), linear amplification of only one strand, or any other method utilizing oligonucleotide primers. PCR Strategies (MA Innis, DH Gelfand, and JJ Sninsky, eds., 1995, Academic Press, San Diego, CA) Chapter 14; PCR Protocols: A Guide to Methods and Applications Various PCR conditions are described in (MA Innis, DH Gelfand, JJ Sninsky, and TJ White, eds., Academic Press, NY, 1990).

[0049] In some embodiments, amplification utilizes a universal primer binding site introduced into the target sequence. In other embodiments, a gene-specific (target-specific) primer or primer pair is used. The amplified target nucleic acid is connected to an adapter as described herein.

[0050] In some embodiments, the target nucleic acid is connected in series to form a longer nucleic acid. For example, a shorter nucleic acid shorter than 1 kilobase long (e.g., 100, 200, 300 base lengths) can be connected in series to form a fragment of several thousand bases long. Before the series connection, the end of the target nucleic acid is connected to a double-stranded adapter comprising a barcode. The resulting concatemer is a molecule comprising two or more target nucleic acid sequences interrupted by an adapter sequence. If multiple copies of the same target nucleic acid are connected in series, a consensus sequence can be constructed from a single read of this concatemer.

[0051] In some embodiments, concatenation is accomplished by digesting the ends of the adapters with a type II restriction enzyme at a specific restriction enzyme recognition site. The enzyme generates sticky ends that allow multiple connected target nucleic acids to be ligated using a DNA ligase. In other embodiments, concatenation is accomplished independently of the nucleic acid sequence by the Gibson assembly method (Gibson et al., (2009), Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature Methods , 6(5): 343-348 and U.S. Patent No. 8,968,999.

[0052] In some embodiments, the present invention includes introducing a barcode into a target nucleic acid. Molecular barcodes are generally required for sequencing each molecule, such as described in, for example, 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 of each molecule added to a sample (such as a patient's sample) during the earliest step of in vitro operation. The barcode marks the molecule and its offspring. The unique molecular barcode (UID) has multiple uses. The barcode allows tracking of each single nucleic acid molecule in a sample to assess, for example, the presence and amount of circulating tumor DNA (ctDNA) molecules in the patient's blood, so as to detect and monitor cancer without biopsy. See U.S. Patent Applications 14 / 209,807 and 14 / 774,518. Unique molecular barcodes can also be used for sequencing error correction. The entire offspring of a single target molecule is labeled with the same barcode and forms a barcoded family. Variations in sequence that are not shared by all members of a barcoded family are discarded as artifacts and are not true mutations. Barcodes can also be used for positional deduplication and target quantification, since the entire family represents a single molecule in the original sample. See supra.

[0053] In some embodiments, the adapter comprises one or more barcodes. In other embodiments, the amplification primer (for example, the primer used in the amplification before the adapter is connected) comprises a barcode in the 5'-part of the primer. The barcode can be a multiple sample ID (MID) for identifying the source of the sample when the sample is mixed (multiplexed). The barcode can also serve as a unique molecule ID (UID) for identifying every kind of original molecule and its offspring. The barcode can also be a combination of UID and MID. In some embodiments, a single barcode is used as both UID and MID.

[0054] In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. The barcode can be 1-20 nucleotides long.

[0055] In some embodiments, the present invention is a method of preparing a library of linked double-stranded target nucleic acids ready for sequencing as described herein and a library produced by the method. Specifically, the library comprises a collection of linked double-stranded molecules derived from nucleic acids present in a sample. The molecules of the library comprise a target sequence joined to an adapter sequence.

[0056] In some embodiments, the present invention includes detecting the target nucleic acid in the sample by nucleic acid sequencing. A variety of nucleic acids (including all nucleic acids in the sample) can be converted into the template configuration of the present invention and sequenced. In some embodiments, nucleic acid sequencing can be performed on a library of single-stranded circular molecules.

[0057] In some embodiments, the method further includes a step of eliminating damaged or degraded targets to improve the quality and length of sequencing reads. This step may include contacting the reaction mixture with uracil DNA N-glycosylase (UNG or UDG) or AP nuclease to degrade such damaged target nucleic acids. In some embodiments, the method further includes a step of eliminating damaged or degraded targets from the library to improve the quality and length of sequencing reads. This step may include contacting the library with one or more of uracil DNA N-glycosylase (UNG or UDG), AP nuclease, and Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) to degrade such damaged target nucleic acids.

[0058] As described above, the adaptor or the target-specific primer may comprise a sequencing primer binding site that can initiate a sequencing read for each strand.

[0059] Sequencing can be performed by any method known in the art. Particularly advantageous is the high-throughput single-molecule sequencing that can read the circular target nucleic acid. The example of such technology includes utilizing the Pacific BioSciences platform (Pacific Biosciences, Menlo Park, Cal.) of SMRT or utilizing the platform of nanopore technology (such as those manufactured by Oxford Nanopore Technologies (Oxford, UK) or Roche Sequencing Solutions (Genia) (Santa Clara, Cal.) and any other currently existing or future DNA sequencing technology involving or not involving sequencing while synthesizing (sequencing by synthesis). The sequencing step can utilize platform-specific sequencing primers. The binding sites of these primers can be introduced into the method of the present invention as described herein, that is, by becoming a part of the second adapter or amplification primer.

[0060] Analysis and Error Correction

[0061] In some embodiments, the sequencing step involves sequence analysis, which includes the step of sequence alignment. In some embodiments, alignment is used to determine the consensus sequence from multiple sequences (e.g., multiple sequences with the same barcode (UID)). In some embodiments, a barcode (UID) is used to determine the consensus sequence from multiple sequences that all have the same barcode (UID). In other embodiments, a barcode (UID) is used to eliminate artifacts, that is, variations present in some but not all sequences with the same barcode (UID). Such artifacts derived from PCR errors or sequencing errors can be eliminated.

[0062] In some embodiments, by the relative number of sequences with each barcode (UID) in the quantitative sample, the number of each sequence in the quantitative sample can be determined. Each UID represents a single molecule in the original sample, and counting the different UIDs associated with each sequence variant can determine the score of each sequence in the original sample. Those skilled in the art will be able to determine the number of sequences read necessary for determining the consensus sequence. In some embodiments, the relevant number is the reading / UID ("sequence depth") necessary for accurate quantitative results. In some embodiments, the desired depth is 5-50 readings / UID.

[0063] In some embodiments, the present invention includes the step of constructing a consensus sequence based on multiple reads of the target nucleic acid. In some embodiments, the target nucleic acid of each copy is connected to an adapter and sequenced once. The consensus sequence is assembled intermolecularly by comparing the reading of several copies of the target nucleic acid that have been read once. For example, in this way, a nucleic acid of about 1 kb long or longer is sequenced. In some embodiments, several copies of the target nucleic acid are connected together (in series). Then the concatemer is connected to the adapter and sequenced once. By comparing the reading of the target nucleic acid from several copies in the concatemer, the consensus sequence is assembled in the molecule. For example, in this way, a nucleic acid less than 1 kb long is sequenced. Example

[0064] Example 1. Template preparation

[0065] The sequencing template was linearized pUC19 plasmid DNA (2.7 kb) or 500bp and 1.15kb HIV amplicons. DNA was quantitatively input using Qubit fluorescence analysis (ThermoFisher Scientific). DNA size, quality, and quantity were evaluated using a bioanalyzer (Agilent Technologies, Santa Clara, Cal.) with a DNA 12000 kit. Purification was performed using KAPA PureBeads (KAPA Biosystems, Inc., Wilmington, Mass.) according to the manufacturer's instructions.

[0066] Example 2. Library preparation and sequencing on the Pacific Biosciences RSII platform

[0067] Template DNA (1 ug plasmid or amplicon) was prepared for adapter ligation using the KAPA HyperPrep Kit library preparation. Briefly, purified linearized plasmid DNA was end-repaired and A-tailed according to the manufacturer's instructions. This step was immediately followed by adapter ligation using linear adapters or HEG adapters ( Figure 1 or Figure 4 ). In the same tube, add the master mixture and adapter, and incubate the reaction mixture at 20 ° C for 30 minutes. The ligase is inactivated by further incubation at 65 ° C for 10 min. All adapters are present at a template: adapter connection ratio of 1: 200. After connection, the final 0.8x KAPA Purebeads bead cleaning step is performed. The library yield is quantified using Qubit fluorescence analysis (LifeTechnologies), and the size of the library is checked using the Bioanalyzer HS kit (Agilent Technologies, Santa Clara, CA). The purified template of the connection is sequenced on the Pacific Biosciences RSII platform and on the Roche nanopore platform according to the manufacturer's instructions. The RSII results are shown in Table 1, which have amplicons of different sizes, and Table 2 shows the results on the Roche nanopore platform.

[0068] Table 1: RSII Results

[0069] template 500 bp amplicon with linear adapters – Repeat 1 500 bp amplicon with linear adapters – replicate 2 1.15 kb amplicon with linear adapters – Repeat 1 1.15 kb amplicon with linear adapters – replicate 2 2.7 kb amplicon with linear adapters – Repeat 1 2.7 kb amplicon with linear adapters – replicate 2

[0070] Table 2: Nanopore results

[0071] Template (# of sequencing runs) % of aligned reads in high quality regions from a single well Median run length (bp) Control (n=4) 44.88 ± 0.43 1664 ±78 Control (n=3) 29.39 ± 1.26 1769 ± 40 Control (n=3) 45.24 ± 2.96 1525 ± 79 Control (n=4) 23.63 ± 3.66 1765 ± 129 Linear adapter (n=2) 33.15 ± 0.33 2423 ± 13 Linear adapter (n=4) 34.46 ± 2.03 2043 ± 67 Linear adapter (n=8) 30.53 ± 2.18 2162 ± 147 Linear adapter (n=6) 30.66 ± 1.77 1856 ± 178 Linear adapter (n=5) 31.71 ± 0.32 2352 ± 86

[0072] Example 3. Sequencing of concatenated linear templates using novel adapters.

[0073] In this example, the starting target sequence is a 187 bp long NRAS exon 3 amplicon, which is concatenated and ligated to a novel adapter ( Figure 1 ) and sequenced. The control for this experiment was pUC2.7 kb plasmid and was used with SMRTBell TM Adaptors are connected to generate circular libraries. In order to further test the robustness of the series connection, we connected oncology amplicons of different sizes. 11 additional oncology amplicons (different sizes) are connected in series, connected to novel adapters, and sequenced on a nanopore platform. 11 amplicons are NRAS exon 3, NRAS exon 4.1, NRAS exon 4.2, KRAS exon 2, KRAS exon 3, KRAS exon 4, BRAF exon 11, PIK3CA exon 9, EGFR exon 18, EGFR exon 20, EGFR exon 21. Templates are sequenced on RSII and Roche nanopore platforms. The results from RSII instruments are shown in Table 3, and the results from Roche nanopore platforms are shown in Table 4.

[0074] Sequencing of tandem templates

[0075] Table 3: RSII Results

[0076] template One amplicon concatemer (NRAS; 180 bp) - linear adapter One amplicon concatemer (NRAS; 180 bp) - linear adapter 11 Amplicon concatemer – SMRTBell adapter (control) – replicate 1 11 Amplicon concatemer – SMRTBell adapter (control) – replicate 2 11 Amplicon concatemer – linear adapter – repeat 1 11 Amplicon concatemer – linear adapter – repeat 2

[0077] Table 4: Nanopore results

[0078] Template (# of sequencing runs) The single hole All single wells with library (DNA) #High quality single read <![CDATA[Control {Plasmid pUC2.7kb DNA with SMRTBell TM adapter, i.e., circular library (n = 4)}]]> 6233 1726 1152 <![CDATA[Control {Plasmid pUC2.7kb DNA with SMRTBell TM adapter, i.e., circular library (n = 3)}]]> 7979 2447 1582 One Amplicon Concatemer – Linear Adapter (Run #1) 5886 1175 816 One Amplicon Concatemer – Linear Adapter (Run #2) 7500 1606 909 One Amplicon Concatemer – Linear Adapter (Run #3) 6207 1927 1215 11 Amplicon Concatemers – Linear Adapters (Run #1) 6369 1476 1045 11-amplicon concatemers – linear adapters (run #2) 7086 1108 754 11-amplicon concatemers – linear adapters (run #3) 4777 1835 1378

[0079] Example 4. Construction of HEG adapter library and sequencing

[0080] In this example, linearized plasmid pUC19 (2.7 kb) was used. A novel dumbbell-shaped adapter ( Figure 4 ) was used to prepare the library. SMRTBell adapters were used as controls. 50uM of linear or HEG adapters were used during library preparation. The HEG / linear adapter-ligated libraries were incubated with biotinylated sequencing primers to anneal at 45°C for 30 seconds and cooled to 20°C at a ramp rate of 0.1°C / s. This was followed by (polymerase-and-pore) complex binding. Enrichment was achieved by using streptavidin-coupled beads to bind the biotinylated primer-complex and enrich the complex ready for sequencing. Sequencing was performed on the Roche nanopore platform. The results are shown in Table 5.

[0081] Table 5. Sequencing of HEG adapter libraries on nanopore systems

[0082] Template (# of sequencing runs) % of aligned reads in high quality regions from a single well Median run length (bp) HEG Adaptor Library #1 42.04% 2555 HEG Adaptor Library #2 33.61% 2264 HEG Adaptor Library #3 42.34% 2476 HEG Adaptor Library #4 40.78% 2478 HEG Adaptor Library #5 43.07% 2413 HEG Adaptor Library #6 41.66% 2367 Average – HEG adapter (n=6) 40.58% 2425.5 Dumbbell adapter library #1 27.28% 2219 Dumbbell adapter library #2 21.77% 2508 Dumbbell adapter library #3 16.65% 2167 Dumbbell adapter library #4 29.07% 2494 Dumbbell adapter library #5 27.35% 2569 Mean – Dumbbell adapter (n=5) 24.43% 2391.40 Sequence Listing <110> F. Hoffmann-La Roche AG Roche Diagnostics GmbH Roche Sequencing Solutions, Inc. <120> Generation of double-stranded DNA templates for single-molecule sequencing <130> P34426-WO <150> US 62 / 685,817 <151> 2018-06-15 <150> US 62 / 638,034 <151> 2018-03-02 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> DNA <213> Artificial single chain <400> 1 atctctctct actgactgtc ctcctcctcc gttttt 36 <210> 2 <211> 10 <212> DNA <213> Artificial single chain <400> 2 gagagagatt 10

Claims

1. A method for non-diagnostic purposes for separately sequencing each strand of a double-stranded target nucleic acid, comprising the following steps: a) in a reaction mixture, joining a double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises a shorter strand and a longer strand that anneal together to form a double-stranded and a single-stranded region, the single-stranded region comprising only one unpaired strand, wherein the single-stranded region comprises a primer binding site and an 18-carbon non-nucleotide linker, and wherein the adaptor has the structure b) annealing a primer to the primer binding site in each adaptor; c) extending the primers, thereby sequencing each strand of the adapted target nucleic acid by nanopore sequencing, wherein the adaptor comprises at least one barcode.

2. The method of claim 1, wherein the 5'-phosphorylated terminal nucleotide modifies the melting temperature of the double-stranded region of the adaptor.

3. The method of claim 1, further comprising a target enrichment step.

4. The method of claim 3, wherein the enrichment is by amplification with target-specific primers.

5. The method of claim 1, further comprising, before step b), contacting the reaction mixture with a DNA damage-specific cleavage agent selected from the group consisting of a glycosylase and an endonuclease, thereby cleaving the damaged DNA.

6. A method for preparing a nucleic acid library from a double-stranded target nucleic acid in a sample, the method comprising: In a reaction mixture, the double-stranded target nucleic acid is joined to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises a shorter strand and a longer strand that anneal together to form a double-stranded and single-stranded region, wherein the single-stranded region comprises only one single strand, wherein the single-stranded region comprises a primer binding site and an 18-carbon non-nucleotide linker, and wherein the adaptor has the structure wherein the adaptor comprises at least one barcode.

7. A method for non-diagnostic purposes for determining the sequence of a target nucleic acid library in a sample, the method comprising the following steps: a) forming a library of target nucleic acids by the method of claim 6; b) annealing a primer to the primer binding site in each of the adapted target nucleic acids; c) extending the primers, thereby sequencing each strand of the target nucleic acid in the library by nanopore sequencing.

8. A method for non-diagnostic purposes of sequencing a double-stranded target nucleic acid, comprising the following steps: a) amplifying a target nucleic acid using a pair of target-specific primers in a reaction mixture, thereby forming an amplicon; b) joining the amplicon to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises a shorter strand and a longer strand that anneal together to form a double-stranded and single-stranded region, wherein the single-stranded region comprises only one single strand, wherein the single-stranded region comprises a primer binding site and an 18-carbon non-nucleotide linker, and wherein the adaptor has the structure c) annealing a sequencing primer to the primer binding site; d) extending the primer, thereby sequencing the target nucleic acid by nanopore sequencing, wherein the adaptor comprises at least one barcode.

9. A method for preparing a library of target nucleic acids, comprising the following steps: a) amplifying a target nucleic acid using a pair of target-specific primers in a reaction mixture, thereby forming an amplicon; b) joining the amplicon to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises a shorter strand and a longer strand that anneal together to form a double-stranded and single-stranded region, wherein the single-stranded region comprises only one single strand and further comprises a primer binding site and an 18-carbon non-nucleotide linker, and wherein the adaptor has the structure wherein the adaptor comprises at least one barcode.

10. A method for non-diagnostic purposes for determining the sequence of a target nucleic acid library in a sample, the method comprising the following steps: a) forming a library of linked target nucleic acids by the method of claim 9; b) annealing a primer to the primer binding site in each of the adapted target nucleic acids; c) extending the primers, thereby sequencing each target nucleic acid in the library by nanopore sequencing.

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