Library building method for direct sequencing of single-stranded nucleic acid

Abstract

The invention provides a library building method for direct sequencing of single-stranded nucleic acid, the library building method comprises providing a to-be-detected sequence fragment, and a preparation method of the to-be-detected sequence fragment comprises cutting a double-stranded molecular sequence formed by complementary pairing of internal bases of the single-stranded nucleic acid by using transposase so as to form the to-be-detected sequence fragment. Compared with an existing library building method of RNA direct sequencing, the library building method has the advantages that a pairing structure is formed by complementary bases of RNA or single-stranded DNA, and a transposase compound can be used for shearing and connecting the pairing structure. This is an attempt which has not been made before, and the library building efficiency is greatly improved through the library building mode.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of a prior application with the patent application number 202410177526.0, titled "A Library Construction Method for Direct Sequencing of Single - stranded Nucleic Acids", filed with the China National Intellectual Property Administration on February 8, 2024. The entire text of the prior application is incorporated into the present invention by reference. Technical field

[0003] The present invention relates to the field of gene sequencing technology, and particularly to a library construction method for direct sequencing of single - stranded nucleic acids. Background art

[0004] Nanopore gene sequencing technology is a new generation of transformative sequencing technology. In nanopore sequencing, nanopore proteins are fixed on an electrically insulating monolayer biomimetic membrane, which divides the electrolyte solution into two parts. When a constant voltage is applied across the biomimetic membrane, electrolyte ions pass through the nanopore and generate an electric current. Since nucleic acids are negatively charged, they can pass through the nanopore under the drive of the electric field force. Due to the different volumes and charged properties of different nucleotides, the current across the membrane changes, and thus the nucleic acid sequence can be determined by recording the current changes. Compared with other sequencing technologies, the bio - nanopore sequencing technology has the characteristics of being portable, real - time, having extremely long read lengths, and being able to detect epigenetic modification information on nucleic acids, becoming the future development direction of nucleic acid sequencing technology.

[0005] RNA plays an important role in gene expression in organisms. Sequencing of RNA can help us understand important information such as gene expression levels, alternative splicing, and transcriptional regulation. Compared with DNA, RNA usually exists in a single - stranded form and has various base modifications, and more than a hundred RNA modifications have been identified. Among human pathogenic viruses, RNA viruses account for more than 80% of the total, and most severe viral infectious diseases are caused by RNA viruses. Therefore, it is of great significance to establish a rapid and high - throughput RNA gene sequencing technology platform. Traditional RNA sequencing methods such as transcriptome sequencing (RNA - Seq) usually require reverse transcription and amplification steps, which may introduce biases and errors and have a relatively long processing time. In addition, after RNA is reverse - transcribed and amplified, its base modification information is also lost. Nanopore sequencing is a type of direct sequencing technology that can not only achieve DNA sequencing but also directly determine RNA sequences. Currently, Oxford Nanopore Technologies has developed an RNA direct sequencing solution and successfully achieved direct sequencing of RNA. However, in the library construction process of this method, there is still a step of reverse - transcribing RNA, and the sequencing accuracy is not high. Therefore, developing a simpler and more efficient RNA direct sequencing method will greatly promote the related development of RNA sequencing research.

[0006] Since the discovery of Tn5 transposase in Escherichia coli, it has been widely used to fragment and label double-stranded DNA and RNA / DNA hybrid strands. The transposase can insert transposon DNA into the target sequence while fragmenting the target DNA, thereby generating DNA fragments that can be directly amplified by PCR for library construction. Compared with traditional methods, the process of DNA fragmentation and adapter ligation is combined, greatly shortening the library construction time and improving the library construction efficiency. Therefore, Tn5 has been applied to various high-throughput sequencing methods. To explore how Tn5 acts on different nucleic acid substrates, researchers used double-stranded DNA, single-stranded DNA, RNA strands, and DNA / RNA hybrid strands as substrates for experiments and found that Tn5 can fragment single-stranded DNA but has little effect on single-stranded RNA (see: Zhang et al., Genome Research, 33:412–426, Tn5 tagments and transposes oligos to single-stranded DNA for strand-specific RNA sequencing). Summary of the Invention

[0007] The present invention unexpectedly discovers that by using the base complementary pairing of single-stranded RNA or single-stranded DNA itself to form a paired structure, the transposase can randomly cleave single-stranded RNA or single-stranded DNA to form RNA or DNA fragments, and then perform sequencing. For RNA, since the reverse transcription step is omitted and the modification characteristics existing on the RNA are directly detected and recorded, the accuracy and detection efficiency of sequencing are greatly improved. For some single-stranded DNA viruses in nature, their structures are more complex. The present invention provides a new method for single-stranded DNA sequencing, which has higher detection accuracy and efficiency.

[0008] The first aspect of the present invention provides a method for labeling and fragmenting single-stranded nucleic acids, including using a transposase to cleave the double-stranded molecular sequence formed by the internal base complementary pairing of the single-stranded nucleic acid to complete the fragmentation and labeling of the single-stranded nucleic acid.

[0009] In an embodiment of the present invention, the single-stranded nucleic acid includes single-stranded DNA (ssDNA) and / or single-stranded RNA (ssRNA).

[0010] In a preferred embodiment of the present invention, the single-stranded nucleic acid is single-stranded RNA (ssRNA).

[0011] In an embodiment of the present invention, the single-stranded RNA includes mRNA and inhibitory ssRNA (such as antisense ssRNA, siRNA or miRNA), tRNA, and nucleic acid sequences from single-stranded RNA viruses.

[0012] In one embodiment of the present invention, the ssRNA (such as mRNA) includes any modified ssRNA.

[0013] In one embodiment of the present invention, the modifications include 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), phosphorothioate (PS) bond substitution, m6A, etc.

[0014] ssDNA can be synthesized using in vitro methods. The in vitro methods covered for synthesizing ssDNA include methods that include transcribing a DNA template encoding a promoter operably linked to a nucleotide sequence to produce an RNA transcript; synthesizing an ssDNA / RNA duplex by reverse transcription of the RNA transcript; and degrading the RNA from the ssDNA / RNA duplex using an RNA-degrading enzyme to produce ssDNA. Optionally, the method may further comprise purifying the ssDNA. Purification of ssDNA can be by gel purification or other DNA purification protocols well known in the art. Promoters suitable for in vitro transcription of DNA templates are known in the art. Examples of suitable promoters include, but are not limited to, T7, T3, and SP6 promoters. Preferably, the promoter comprises a T7 promoter and transcription is carried out using T7 RNA polymerase. The DNA can be derived from pathogen DNA, such as single-stranded DNA viruses, etc.

[0015] Synthesis of the ssDNA / RNA duplex can be carried out using an RNA-dependent DNA polymerase such as reverse transcriptase. Suitable reverse transcriptase proteins for use in the present invention can be obtained from various retroviruses, including but not limited to Moloney Murine Leukemia virus, Human Immunodeficiency virus, Simian Immunodeficiency virus, and retrotransposons isolated from various yeast and bacterial species. Preferably, the reverse transcriptase is Moloney Murine Leukemia virus.

[0016] Other in vitro methods can also be used to generate the ssDNA of the present invention. Suitable methods include: asymmetric PCR (see, for example, U.S. Patent 8,735,067), PCR using two oligonucleotide primers with one primer present at limiting concentration (see, for example, U.S. Patent 5,066,584), and generating ssDNA from a dsDNA molecule using a "nickase" and / or a restriction endonuclease (see, for example, LsODN Preparation Kit - Biodynamics Laboratory Inc., Tokyo, Japan).

[0017] In one embodiment of the present invention, the single-stranded DNA and / or single-stranded RNA contains a self-complementary sequence that allows a portion of the single-stranded DNA and / or single-stranded RNA to fold and pair with itself to form a double-stranded molecular sequence.

[0018] In one embodiment of the present invention, the self-complementary sequences of the single-stranded nucleic acids bind to each other through hydrogen bonds to form a double-stranded molecular sequence. For example, in DNA, A and T, C and G are complementary. In RNA, A and U, C and G are complementary.

[0019] In one embodiment of the present invention, the double-stranded molecular sequence has local stability. These double-stranded molecular sequences are usually composed of some specific nucleotide sequences, such as guanine-guanine (G-G) and adenine-adenine (A-A).

[0020] In one embodiment of the present invention, the double-stranded molecular sequence is located at any position of the single-stranded DNA and / or single-stranded RNA, and can be located at the 3' end, 5' end or any intermediate position.

[0021] In one embodiment of the present invention, the double-stranded molecular sequences are located at multiple randomly distributed positions.

[0022] In one embodiment of the present invention, the internal bases can be adjacent bases or non-adjacent bases.

[0023] In one embodiment of the present invention, the transposase includes but is not limited to Tn transposases (such as Tn3, Tn5 or its functional derivatives (such as the mutants reported in CN106754811A), Tn7, Tn10, Tn552, Tn903), MuA transposase or its functional derivatives or Vibhar transposase.

[0024] In some embodiments, the transposase is Tn5 transposase, and the Tn5 transposase contains a sequence having at least 80%, 90%, 95% or 99% homology with SEQ ID NO:7; further preferably, it contains a mutation at at least one of positions 54 and 372; for example, the mutant includes mutations at a total of one or two positions at positions 54 and / or 372 of SEQ ID NO:7. Such as E54 single mutation, L372 single mutation or E54+L372 mutation; preferably E54K single mutation, L372P single mutation or E54K+L372P mutation.

[0025] In some embodiments, it further includes a mutation of at least one of R26, R62, E110, D156, P242, E345;

[0026] The mutated amino acid is one or more of neutral or basic amino acids, which affects the binding to nucleic acids and makes the binding stronger.

[0027] Preferably, it is one or more of valine (V), arginine (R), tyrosine (Y), isoleucine (I), glutamic acid (E), tryptophan (W), proline (P), lysine (K), alanine (A); more preferably, it is one or more of tryptophan (W), proline (P), lysine (K), alanine (A).

[0028] Preferably, it further includes the mutation of at least one of R26, R62, E110, D156, P242, E345; more preferably, it further includes the mutation of at least one of R26W, R62P, E110K, D156K, P242A, E345K.

[0029] Or a sequence having at least 80% or 90% or 95% or 99% homology with the above.

[0030] More preferably, it is the transposase mutant shown in SEQ ID NO:13.

[0031] In some embodiments, the MuA transposase comprises a sequence having at least 80% or 90% or 95% or 99% homology with SEQ ID NO:14; more preferably, it comprises mutations at at least one of positions 179, 233, 254, 464, 487, 539, 594; for example, it includes mutations at one, two, three, four, five, six or seven of positions 179, 233, 254, 464, 487, 539, 594 in SEQ ID NO:14.

[0032] The mutated amino acid is one or more of neutral or basic amino acids, which affects the binding to nucleic acids and makes the binding stronger. Preferably, it is at least one of lysine (K), valine (V), arginine (R), tyrosine (Y), isoleucine (I), glutamic acid (E); more preferably, it is at least one of valine (V), arginine (R), tyrosine (Y), isoleucine (I).

[0033] Preferably, it is at least one of E233V, Q254R, F464Y, M487I, Q539R, Q594R, E179V.

[0034] The mutated amino acid is one or more of neutral or basic amino acids, which affects the binding to nucleic acid and makes the binding stronger. Preferably, it is at least one of lysine (K), valine (V), arginine (R), tyrosine (Y), isoleucine (I), and glutamic acid (E); more preferably, it is at least one of lysine (K), arginine (R), and glutamic acid (E).

[0035] Preferably, it further includes at least one mutation of L106K, W110R, G136E, Q186E, I189K, L209R, A222K, Q229E, Q230R, N496R, V517R, or A561E.

[0036] More preferably, it is the transposase mutant shown in SEQ ID NO:15.

[0037] In one embodiment of the present invention, the transposase exists in the form of a transposase complex, and the transposase complex includes a transposase and a transposase recognition sequence.

[0038] In one embodiment of the present invention, the transposase recognition sequence optionally contains a ligation end recognized by a nucleic acid ligase, an aptamer, an antibody, biotin, digoxin or digoxigenin, a bioorthogonal reaction group, a methylated fragment, a fluorescently labeled fragment, a capture fragment, an adaptor sequence, a primer sequence, a promoter sequence, or a tag sequence, so that the fragmented nucleic acid molecules are applicable to various detection or analysis occasions.

[0039] In one embodiment of the present invention, the transposase recognition sequence is the Tn5-Me or MuA-Me sequence.

[0040] In one embodiment of the present invention, the transposase complex randomly binds to any position of the double-stranded molecular sequence, and then cuts the single-stranded nucleic acid into multiple single-stranded fragments and introduces the transposase recognition sequence into the single-stranded fragments.

[0041] In one embodiment of the present invention, the cleavage site is located at any position of the double-stranded molecular sequence.

[0042] In one embodiment of the present invention, the reaction conditions for cleavage are: reaction temperature 15 - 55°C, reaction time 15 - 60 min. In a preferred embodiment of the present invention, the reaction temperature is 37°C.

[0043] In the second aspect of the present invention, a library construction method for direct sequencing of single-stranded nucleic acid is provided, including providing a sequence fragment to be detected; the preparation method of the sequence fragment to be detected is the method of the first aspect.

[0044] In one embodiment of the present invention, the library construction method includes:

[0045] 1) Fragment the single-stranded nucleic acid using the method of the first aspect of the present invention and introduce a ligation end;

[0046] 2) Ligate the fragmented single-stranded nucleic acid to a sequencing adapter complex to obtain a sequencing library.

[0047] In one embodiment of the present invention, the sequencing adapter complex is an adapter complex for nanopore sequencing;

[0048] In one embodiment of the present invention, the sequencing adapter complex includes a region for ligation of the nucleic acid fragment to be analyzed; preferably, the region contains a complementary sequence for helicase binding and ligation of double-stranded DNA fragments.

[0049] In one embodiment of the present invention, the transposase recognition sequence and the region for ligation of the nucleic acid fragment to be analyzed have complementary sticky ends, and the ligation is achieved by a nucleic acid ligase; preferably, the ligation is achieved by T4 DNA ligase.

[0050] The present invention designs the sticky ends of the transposase recognition sequence such that the transfer strand in the transposase recognition sequence is ligated to the sequencing adapter complex to avoid the gap repair step; preferably, the transfer strand in the transposase recognition sequence and the complementary sequence in the region for ligation of the nucleic acid fragment to be analyzed in the sequencing adapter complex have complementary overhangs (such as CAC and GTG).

[0051] In the third aspect of the present invention, a sequencing library prepared by the method of the second aspect of the present invention is provided.

[0052] In the fourth aspect of the present invention, a sequencing method is provided, which includes the step of fragmenting and labeling a single-stranded nucleic acid using the method of the first aspect of the present invention, or includes the step of constructing a sequencing library using the method of the second aspect of the present invention, or includes the step of sequencing the sequencing library of the third aspect of the present invention.

[0053] In the fifth aspect of the present invention, a composition for constructing a single-stranded nucleic acid nanopore sequencing library is provided, and the composition includes:

[0054] 1) Transposase and transposase recognition sequence;

[0055] 2) Sequencing adapter complex, which includes a region for ligation of the nucleic acid fragment to be analyzed;

[0056] 3) The transposase recognition sequence and the region for ligation of the nucleic acid fragment to be analyzed have complementary sticky ends;

[0057] 4) Nucleic acid ligase.

[0058] In the sixth aspect of the present invention, a transposase mutant is provided, including

[0059] Tn5 transposase mutant, the Tn5 transposase mutant comprising a sequence having at least 80% or 90% or 95% or 99% homology with SEQ ID NO:7; and having one, two or three mutation sites of E54K, L372P, E345K; further preferably, comprising the amino acid sequence shown in SEQ ID NO:13

[0060] or

[0061] MuA transposase, the MuA transposase comprising a sequence having at least 80% or 90% or 95% or 99% homology with SEQ ID NO:14; and having one, two or more mutation sites of E179V, E233V, Q254R, F464Y, M487I, Q539R or Q594R; further preferably, comprising the amino acid sequence shown in SEQ ID NO:15.

[0062] The present invention also provides a nucleic acid sequence, the nucleic acid sequence being: 1) a sequence encoding the above transposase mutant; or 2) a sequence complementary to 1).

[0063] The present invention also provides a biological material, the biological material expressing the above transposase mutant or comprising the above nucleic acid sequence.

[0064] The present invention also provides the use of the above transposase mutant, the above nucleic acid sequence or the above biological material in single-stranded nucleic acid molecule fragmentation or sequencing library construction.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] (1) Compared with the existing library construction method for direct RNA sequencing, the present invention utilizes the base complementarity of RNA or single-stranded DNA itself to form a paired structure, enabling the transposase complex to shear and ligate it. This is an attempt that has never been made before, and this library construction method greatly improves the library construction efficiency.

[0067] (2) In the present invention, the helicase moves directionally along the 5'- to 3'-end of the RNA strand, and the pore-passing speed of the RNA strand is fast and uniform, reaching 300 nt / s.

[0068] (3) From the perspective of the library construction process, the library construction of the present invention takes a short time and is easy to operate.

[0069] (4) The library construction method for direct RNA sequencing of the present invention realizes the direct sequencing of RNA or single-stranded DNA, and can directly detect and record the modification characteristics existing on the RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 is a schematic diagram of the present invention: Figure 1ACartoon schematic diagram of single-stranded nucleic acid labeling and fragmentation; Figure 1B Cartoon schematic diagram of single-stranded nucleic acid library construction; The transposase complex recognizes and cleaves the local double-stranded region of single-stranded nucleic acid, and ligates short DNA fragments to the fragment to be tested. Subsequently, the sequencing adapter complex is ligated to the DNA fragment through ligase to complete the library construction of the sample to be tested;

[0071] Figure 2 Gel diagram of the cleavage effect of Tn5 transposase complex on single-stranded RNA;

[0072] Figure 3 For Figure 2 Pore signal diagram after library construction of single-stranded RNA in

[0073] Figure 4 Pore signal diagram after cleavage and library construction of PhiX174 virion DNA single-stranded DNA by Tn5 transposase complex;

[0074] Figure 5 Gel diagram of the cleavage effect of MuA transposase complex on single-stranded RNA;

[0075] Figure 6 For Figure 5 Pore signal diagram after library construction of single-stranded RNA in Specific embodiments

[0076] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only illustrative and explanatory of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0077] Technical terms:

[0078] Tn5 transposase complex

[0079] A homodimer formed by Tn5 enzyme and the transposase recognition sequence (including the ME sequence: CTGTCTCTTATACACATCT and other labeling molecules or fragments) can insert the transposase recognition sequence into the target nucleic acid sequence while cleaving the DNA double-stranded sequence, and at the same time introduce labeling molecules or fragments. The application of Tn5 transposase in genomic research can be referred to: Li N, Jin K, Bai Y, et al. Tn5 Transposase Applied in Genomics Research[J]. International Journal of Molecular Sciences, 2020, 21(21): 8329.

[0080] The term "transposase recognition sequence" or "transposon end" refers to a double-stranded nucleic acid molecule of a nucleotide sequence that is necessary for forming a functional complex with a transposase in a transposition reaction. In the present invention, "transposon end" and "transposase recognition sequence" have the same meaning and are interchangeably used. The transposon end forms a "transposase complex" with a transposase that recognizes and binds to the transposon end, and this complex is capable of inserting or transposing the transposon end into a double-stranded nucleic acid molecule incubated with it in an in vitro transposition reaction, or into the double-stranded structure of a single-stranded nucleic acid molecule as described in the present invention. The transposon end contains two complementary sequences consisting of a "transferred transposon end sequence" and a "non-transferred transposon end sequence". The nucleic acid strand containing the transferred transposon end sequence is called the "transfer strand". The nucleic acid strand containing the non-transferred transposon end sequence is called the "non-transfer strand". In an in vitro transposition reaction, the 3'-end of the transfer strand joins or transfers to a target nucleic acid molecule (such as a DNA molecule, an RNA molecule). In an in vitro transposition reaction, the 5'-end of the transposon end sequence complementary to the transferred transposon end sequence (i.e., the non-transferred transposon end sequence) does not join or transfer to the target nucleic acid molecule, forming a 9-bp gap.

[0081] Preferably, in the present invention, the transposase includes:

[0082] The amino acid sequence (M-Tn5) of the Tn5 transposase mutant (E54K, L372P, E345K) SEQ ID NO:13:

[0083] MVTFMITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEKPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALW

[0084] Expression and purification of Tn5 transposase mutants

[0085] The recombinant plasmid pET28a containing the Tn5 mutant was transformed into the Escherichia coli expression host BL21(DE3) by heat shock method. During the induction expression process, first, the host bacteria containing the expression plasmid were cultured overnight at 37 °C in LB medium supplemented with kanamycin resistance, then amplified at 37 °C at a ratio of 1:100. When the OD(600) value reached 0.4 - 0.6, the culture was stopped and cooled at 4 °C for 1 hour. Subsequently, isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM was added to induce expression at 16 °C for 12 - 16 h. Then, the bacteria were collected by centrifugation at 4 °C and 15,000 rpm, and the cells were disrupted by a high-pressure homogenizer at 4 °C. Subsequently, the supernatant was collected by centrifugation at 4 °C, and the target protein was separated and purified step by step through nickel column, heparin column, and molecular sieve, and finally a large amount of high-purity Tn5 transposase mutant protein was obtained.

[0086] Amino acid sequence of Escherichia phage MuA transposase mutants (truncated 1 - 76, E179V, E233V, Q254R, F464Y, M487I, Q539R, Q594R) (M-MuA) SEQ ID NO:15:

[0087] IARPTLEAHDYDREALWSKWDNASDSQRRLAEKWLPAVQAADEMLNQGISTKTAFATVAGHYQVSASTLRDKYYQVQKFAKPDWAAALVDGRGASRRNVHKSVFDEDAWQFLIADYLRPEKPAFRKCYERLELAAREHGWSIPSRATAFRRIQQLDVAMVVACREGEHALMHLIPAQRRTVEHLDAMQWINGDGYLHNVFVRWFNGDVIRPKTWFWQDVKTRKILGWRCDVSENIDSIRLSFMDVVTRYGIPEDFHITIDNTRGAANKWLTGGAPNRYRFKVKEDDPKGLFLLMGAKMHWTSVVAGKGWGQAKPVERAFGVGGLEEYVDKHPALAGAYTGPNPQAKPDNYGDRAVDAELFLKTLAEGVAMFNARTGRETEMCGGKLSYDDVFEREYARTIVRKPTEEQKRILLLPAEAVNVSRKGEFTLKVGGSLKGAKNVYYNMALMNAGVKKVVVRFDPQRLHSTVYCYTLDGRFICEAECLAPVAFNDAAAGREYRRRQKQLKSATKAAIKAQKRMDALEVAELLPQIAEPAAPESRIVGIFRPSGNTERVKNQERDDEYETERDEYLNHSLDILEQNRRKKAI

[0088] Expression and purification of MuA transposase mutants

[0089] The pBAD recombinant plasmid containing the MuA mutant was transformed into the Escherichia coli expression host BL21(DE3) by heat shock. During the induction expression process, first, the host bacteria containing the expression plasmid were cultured overnight at 37 °C in LB medium supplemented with Ampicillin resistance, and then amplified at 37 °C at a ratio of 1:100. The culture was stopped when the OD(600) value reached 0.4 - 0.6 and cooled at 4 °C for 1 hour. Subsequently, arabinose was added to a final concentration of 0.006%, and induction expression was carried out at 28 °C for 4 h. Then, the bacteria were collected by centrifugation at 4 °C and 15,000 rpm, and the cells were disrupted by a high-pressure cell disrupter at 4 °C. Subsequently, the supernatant was collected by centrifugation at 4 °C, and the target protein was separated and purified through a nickel column, a heparin column, and a molecular sieve, etc. Finally, a large amount of high-purity MuA transposase mutant protein was obtained.

[0090] Labeling and fragmentation

[0091] In the present invention, "tagging and fragmenting" has its ordinary meaning in the art, that is, while fragmenting a nucleic acid molecule, a labeling molecule such as an antibody, a fluorescent fragment or a tag fragment is introduced; when a tag fragment is introduced, it has the same meaning as the common expression "tagmenting" in the art, that is, a tag sequence for NGS sequencing such as P5 or P7, or other complex adapters such as Y-adapters are introduced through a transposase recognition sequence.

[0092] Single-stranded DNA and / or single-stranded RNA

[0093] In one embodiment of the present invention, the length of the single-stranded DNA and / or single-stranded RNA usually varies between 6 bases and 30,000 bases, preferably 10 to 12,000, particularly 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000 or 300 to 5,000 bases. In one embodiment, the single-stranded DNA and / or single-stranded RNA has a length of at least 2,700 bases (such as at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, at least 5,000 bases). The single-stranded DNA and / or single-stranded RNA used herein has at least 3,500 bases (such as at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500 bases), preferably at most 15,000, such as at most 14,000, at most 13,000 or at most 12,000 bases of single-stranded DNA and / or single-stranded RNA.

[0094] In one embodiment of the present invention, the ssDNA can be phage virus DNA, including but not limited to Debruijin DNA, PhiX174 virion DNA.

[0095] In one embodiment of the present invention, the single-stranded RNA includes mRNA and inhibitory ssRNA (such as antisense ssRNA, siRNA or miRNA), tRNA and nucleic acid sequences from single-stranded RNA viruses.

[0096] In one embodiment of the present invention, single-stranded RNA viruses include positive-strand RNA viruses (+ssRNA), negative-strand RNA viruses (-ssRNA) and retroviruses. The +ssRNA viruses include picornaviruses (rhinovirus, poliovirus, hepatitis A virus), coronaviruses (MERS), flaviviruses (yellow fever, Japanese encephalitis, dengue fever, West Nile virus, Zika virus), alphaviruses (chikungunya) and hepatitis C virus, etc. The (-ssRNA) includes Bornaviruses, paramyxoviruses (measles, mumps, parainfluenza, respiratory syncytial virus (separated from paramyxoviruses in 2016), henipavirus), rhabdoviruses (rabies virus), filoviruses (Ebola virus, Marburg virus). The retroviruses include human immunodeficiency virus (HIV), murine leukemia virus (MLV), etc.

[0097] The term "mRNA" means "messenger RNA" and refers to a "transcript" that can be produced by using a DNA template and can encode a peptide or protein. Generally, mRNA contains a 5′-UTR, a protein-coding region and a 3′-UTR. In the context of the present invention, mRNA is preferably produced from a DNA template by in vitro transcription. As described above, in vitro transcription methods are known to those skilled in the art, and a variety of commercially available in vitro transcription kits are available.

[0098] The term "modification" includes any modification of RNA (preferably ssRNA, such as mRNA) that is not naturally present in the RNA.

[0099] Nanopore sequencing adapter complex

[0100] The nanopore sequencing adapter complex comprises a sequencing adapter and a helicase. A typical nanopore sequencing adapter contains four segments: the first segment is a single-stranded nucleotide region for guiding the nucleic acid to be tested into the nanopore, the second segment is a polynucleotide-binding protein (nucleic acid-binding protein or rate-limiting protein) binding region, the third segment is a linker region that blocks the progress of the polynucleotide-binding protein, and the fourth segment is a region for ligating the target polynucleotide (such as the sticky end used in the present invention). The fourth segment usually contains a sequence complementary to the adapter for helicase binding and ligation of double-stranded nucleic acid sequences.

[0101] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0102] Example 1 Sequencing of Single-stranded Nucleic Acid RNA

[0103] 1. Preparation of Adapter Complex

[0104] 1) Design and synthesize adapter sequence (SEQ ID NO:1), sequence 2 (SEQ ID NO:2) and sequence 3 ((SEQ ID NO:3)).

[0105] 2) Annealing of Adapter Sequence

[0106] Dissolve the adapter sequence, sequence 2, and sequence 3 in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) respectively to prepare stock solution 1, stock solution 2, and stock solution 3 with a concentration of 100 μM. Mix stock solution 1, stock solution 2, and stock solution 3 in a ratio of 1:1.1:1.1, and perform annealing treatment to obtain annealed adapters. Annealing program: lower the temperature from 95 °C to 22 °C at -2 °C per minute.

[0107] 3) Crosslinking of Annealed Adapter Sequence with GG-Dda Helicase

[0108] (1) Treat GG-Dda helicase (SEQ ID NO:4) with DTT (dithiothreitol, final concentration 2 mM), incubate at room temperature for 30 minutes to open the disulfide bond for subsequent protein-nucleic acid binding in the experiment;

[0109] (2) Incubate the annealed adapter sequence obtained in step 2) with the GG-Dda helicase with the disulfide bond opened at a molar ratio of 1:15 at room temperature for 30 min to allow the helicase to bind to the adapter;

[0110] (3) Use a desalting column to replace the buffer in the sample of step (2);

[0111] (4) Add N,N,N',N'-tetramethylazodicarboxamide (TMAD, final concentration 500 μM) to the sample, incubate at 35 °C for 1 h to lock the helicase on the nucleic acid strand;

[0112] (5) Add ATP (final concentration 1 mM) and incubate at room temperature for 30 min to allow the GG-Dda helicase bound to other parts of the nucleic acid strand to move and fall off under the action of ATP;

[0113] (6) Purify the sample using a molecular sieve, and collect the adapter complex that binds only one GG-Dda helicase according to the molecular weight; store it in a -20 °C refrigerator for subsequent experiments.

[0114] 2. Preparation of Tn5 transposome complex

[0115] Design and synthesize the Tn5 recognition sequences SEQ ID NO:5 and recognition sequence SEQ ID NO:6. Dissolve the two sequences separately in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare a stock solution of 100 μM. Mix the above sequences in equimolar ratio and perform annealing treatment to obtain Tn5-Me. Annealing program: Optionally, first heat up to 95 °C / 98 °C, then cool down 1-2 °C per minute until reaching 23-26 °C. Gently mix M-Tn5 transposase (SEQ ID NO:13) and Tn5-Me in equimolar ratio and incubate at room temperature for 1 hour to obtain the Tn5 transposome complex.

[0116] 3. Extraction of total RNA from Escherichia coli

[0117] Pick 3-5 single colonies of DH5α into 2-8 mL of LB antibiotic-free liquid medium and culture them in a shaker at 37 °C for 2-4 hours. Detect the OD600 of the bacterial liquid by spectrophotometer to be 0.7, and take 1.2 ml of the bacterial liquid for this RNA extraction. In this example, MolPure Bacterial RNA Kit (YE SEN) is used for RNA extraction, and the specific operation steps are carried out according to the kit instructions. Measure the concentration of the Escherichia coli RNA sample extracted this time by using nanodrop to be 940 ng / μl, A260 / A280 = 2.02; A260 / A230 = 2.09; at the same time, use Agilent 4150 to detect this sample, and the detailed quality inspection results are shown in Figure 3 . The qualified RNA sample after quality control is used for subsequent library construction.

[0118] 4. Fragmentation of Escherichia coli RNA

[0119] Gently mix the Escherichia coli RNA obtained in step 3, the M-Tn5 transposome complex in step 2, and reaction buffer (10 mM Tris-HCl, 5 mM MgCl2, 8% PEG8000, pH 7.5), and react in a PCR instrument at 16 °C or 37 °C for 15 minutes. Subsequently, immediately terminate the reaction with Stop Buffer (0.2% SDS) in a PCR instrument at 55 °C for 10 minutes, and characterize the fragmentation result by agarose gel electrophoresis, as Figure 2 shown. Finally, purify the RNA fragmented at 16 °C in Figure 2 using GeneJET PCR Purification Kit (Thermo Scientific) for subsequent ligation reaction.

[0120] 5. Ligation of the adapter complex to fragmented Escherichia coli RNA

[0121] The adapter complex from step 1 and the fragmented RNA from step 4 were first incubated at room temperature for 30 minutes and then at 4 °C for 20 minutes in ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% Polyethylene glycol (PEG 6000), pH 8.0) and T4 DNA ligase to complete the ligation reaction. Subsequently, the ligated sample was purified using AMPure XP beads, and the purified sample could be directly used for sequencing.

[0122] 6. Sequencing of Escherichia coli RNA samples on the machine

[0123] In buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 8.0), electrical signal measurements were obtained from MspA nanopores embedded in a DPhPC (Cas No.: 207131-40-6, Chinese name: Diphytanoylphosphatidylcholine) lipid bilayer.

[0124] After achieving single-pore insertion into the lipid bilayer, 2 ml of buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM HEPES, pH 8.0) was passed through the system to remove residual excess nanopores. Then the sample to be tested from step 5, ATP (final concentration 2 mM), and MgCl2 (final concentration 10 mM) were flowed into a single nanopore experimental system (total volume 100 μL) together, and signals were measured at a constant voltage of +180 mV for 1 h (including a potential 2 s voltage inversion at -180 mV).

[0125] Figure 3 It is shown that direct library construction of RNA can be achieved through the M-Tn5 transpososome complex, and this process does not require reverse transcription. Moreover, this method of directly constructing a library for RNA can more intuitively record modification characteristics such as methylation present in the RNA itself.

[0126] Example 3 Sequencing of single-stranded nucleic acid DNA of PhiX174 virion DNA

[0127] 1. Preparation of the adapter complex

[0128] It is the same as the preparation of the adapter complex in step 1 of Example 1.

[0129] 2. Preparation of the M-Tn5 transposase complex

[0130] It is consistent with the preparation of the M-Tn5 transposase complex in Step 2 of Example 1.

[0131] 3. Preparation of PhiX174 virion DNA

[0132] Gently mix PhiX174 virion DNA (NEB, product number: N3023S), the M-Tn5 transposome complex in Step 2, and the reaction buffer [10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 5 mM MgCl2, 8% PEG8000 (Cas number 25322-68-3, Sigma item number P2139), pH 7.5], and react in a 55°C PCR instrument for 10 - 12 minutes. Then immediately terminate the reaction by reacting with Stop Buffer (0.2% SDS, Chinese name sodium dodecyl sulfate) in a 55°C PCR instrument for 10 minutes. Finally, purify the fragmented DNA using the GeneJET PCR Purification Kit (Thermo Scientific, Thermo item number K0701) for subsequent ligation reactions.

[0133] 4. Ligation of the adapter complex and fragmented DNA

[0134] Incubate the adapter complex in Step 1 and the fragmented DNA in Step 4 in the ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% Polyethylene glycol (PEG 6000, Sigma item number 81260), pH 8.0) and in the presence of T4 DNA ligase. First, incubate at room temperature for 30 minutes, and then incubate at 4°C for 20 minutes to complete the ligation reaction. Subsequently, perform magnetic bead purification of the ligation sample using AMPure XP beads, and the purified sample can be directly used for sequencing.

[0135] 5. Sequencing of single-stranded DNA samples on the machine

[0136] The sequencing method is the same as that in Example 1. Figure 4 A typical nucleic acid pore signal graph is shown. By collecting and analyzing the pore signals of this sample, it is found that this library construction method can detect and piece together the complete sequence information of PhiX174 virion DNA. A new method for sequencing DNA viruses is provided, which greatly shortens the library construction time.

[0137] Example 3 Sequencing of single-stranded nucleic acid RNA based on the M-MuA transposase system

[0138] 1. Preparation of the adapter complex

[0139] In this example, the preparation of the adapter complex is the same as the preparation of the adapter complex in step 1 of Example 1.

[0140] 2. Preparation of M-MuA transposase complex

[0141] Design and synthesize the upstream primer SEQ ID NO:11 and the downstream primer SEQ ID NO:12 of M-MuA. Take the annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to dissolve the two primers respectively to prepare a stock solution of 100 μM. Mix the upstream and downstream primers in an equimolar ratio and perform annealing treatment to obtain MuA-Me. Annealing program: Optionally, first heat up to 95 °C / 98 °C, then lower the temperature by 1-2 °C per minute until it reaches 23-26 °C. Gently mix the M-MuA transposase (SEQ ID NO:15) and MuA-Me in an equimolar ratio and incubate at 30 °C for 1 hour to obtain the MuA transpososome complex.

[0142] 3. Preparation of RNA

[0143] Using lambda DNA (NEB) as a template and SEQ ID NO:8 and SEQ ID NO:9 as primers, perform PCR amplification to obtain a 2Kb DNA sequence containing the T7 RNA Polymerase binding site. Purify the obtained DNA sequence as a template for subsequent transcription reactions. Use HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) for transcription reaction to obtain lambda 2K nt RNA. Purify the RNA using the TRIzol method; use DNaseⅠ (GenScript) to remove the DNA template in the mRNA, treat at 37 °C for 30 min, and then further purify the RNA using the TRIzol method. Obtain Lambda 2K ntRNA and store it in a -80 °C refrigerator.

[0144] 4. Fragmentation of RNA

[0145] Gently mix the RNA purified in step 3, the MuA transpososome complex in step 2, and the reaction buffer (5 mM Tris-HCl, 55 mM NaCl, 10 mM MgCl2, pH 8) and react in a 37 °C PCR instrument for 15 minutes. Immediately terminate the reaction with StopBuffer (0.2% SDS) in a 55 °C PCR instrument for 10 minutes. Characterize the fragmentation result by agarose gel electrophoresis, such as Figure 5As shown. Finally, the fragmented RNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific) for subsequent ligation reactions.

[0146] 5. Ligation of Adapter Complex with Fragmented DNA

[0147] The adapter complex in Step 1 and the fragmented DNA in Step 4 were first incubated at room temperature for 30 minutes under the conditions of ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% Polyethylene glycol (PEG 6000), pH 8.0) and T4 DNA ligase, and then incubated at 4°C for 20 minutes to complete the ligation reaction. Subsequently, the ligated sample was purified using AMPure XP beads, and the purified sample could be directly used for sequencing.

[0148] 6. Sequencing of Single-Stranded DNA Samples on the Machine

[0149] The sequencing method was the same as that in Example 1. The pore signal collection and analysis of this sample, as Figure 5 shown, found that under appropriate reaction conditions, MuA transposase could also achieve enzymatic cleavage of single-stranded RNA, thus enabling direct RNA library construction.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

[0151] Sequence Information:

[0152] SEQ ID NO:1: Adapter Sequence

[0153] SEQ ID NO:2: Sequence 2

[0154]

[0155] SEQ ID NO:3: Sequence 3

[0156]

[0157] SEQ ID NO:4: GG-Dda

[0158]

[0159] SEQ ID NO:5: Tn5 transposase recognition sequence

[0160] SEQ ID NO:6: Tn5 transposase recognition sequence

[0161] SEQ ID NO:7:

[0162]

[0163] SEQ ID NO:8: lambda DNA primer

[0164] SEQ ID NO:9: lambda DNA primer

[0165] SEQ ID NO:10: Amino acid sequence of wild-type Escherichia phage MuA transposase

[0166] SEQ ID NO:11: (MuA-Me strand 1)

[0167]

[0168] SEQ ID NO:12: (MuA-Me strand 2)

[0169]

[0170] SEQ ID NO:13: M-Tn5

[0171]

[0172] SEQ ID NO:14: Truncated sequence of wild-type Escherichia phage MuA transposase

[0173] SEQ ID NO:15: M-MuA

[0174]

Claims

1. A method for labeling and fragmenting single-stranded nucleic acids, characterized in that: Including using a transposase to cleave the double-stranded molecular sequence formed by the internal base complementary pairing of the single-stranded nucleic acid, to complete the fragmentation and labeling of the single-stranded nucleic acid.

2. The method for labeling and fragmenting single-stranded nucleic acid according to claim 1, characterized in that: The single-stranded nucleic acid includes single-stranded DNA (ssDNA) and / or single-stranded RNA (ssRNA); Preferably, the single-stranded nucleic acid is single-stranded RNA (ssRNA); Preferably, the single-stranded RNA includes mRNA, inhibitory ssRNA, tRNA, and nucleic acid sequences from single-stranded RNA viruses; Preferably, the ssRNA includes any modified ssRNA; Preferably, the modification includes 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), phosphorothioate (PS) bond substitution, m6A; Preferably, the single-stranded DNA and / or single-stranded RNA contains self-complementary sequences, which allow a part of the single-stranded DNA and / or single-stranded RNA to fold and pair with itself to form a double-stranded molecular sequence; Preferably, the self-complementary sequences of the single-stranded nucleic acid bind to each other through hydrogen bonds to form a double-stranded molecular sequence; Preferably, the double-stranded molecular sequence has local stability; Preferably, the double-stranded molecular sequence is located at any position of the single-stranded DNA and / or single-stranded RNA, and can be located at the 3' end, 5' end, or any intermediate position; Preferably, the double-stranded molecular sequences are located at multiple randomly distributed positions.

3. The method for labeling and fragmenting single-stranded nucleic acid according to claim 1, wherein: The internal bases can be adjacent bases or non-adjacent bases; Preferably, the transposase includes but is not limited to Tn transposase, MuA transposase, or its functional derivatives, or Vibhar transposase; Preferably, the Tn transposase includes Tn3, Tn5, or its functional derivatives, Tn7, Tn10, Tn552, Tn903; Preferably, the Tn transposase is Tn5 transposase, and the Tn5 transposase contains a sequence having at least 80%, or 90%, or 95%, or 99% homology with SEQ ID NO:7; further preferably, it contains one, two, or three of the mutation sites E54K, L372P, E345K; further preferably, it is the transposase mutant shown in SEQ ID NO:13; Preferably, the MuA transposase contains a sequence having at least 80%, or 90%, or 95%, or 99% homology with SEQ ID NO:14; further preferably, it contains one, two, or more of the mutation sites E179V, E233V, Q254R, F464Y, M487I, Q539R, or Q594R; further preferably, it is the transposase mutant shown in SEQ ID NO:15; Preferably, the transposase exists in the form of a transposase complex, and the transposase complex includes a transposase and a transposase recognition sequence; Preferably, the transposase recognition sequence optionally comprises a ligation end recognized by a nucleic acid ligase, an aptamer, an antibody, biotin, digoxin or digoxigenin, a bioorthogonal reaction group, a methylation fragment, a fluorescently labeled fragment, a capture fragment, a linker sequence, a primer sequence, a promoter sequence or a tag sequence, so that the fragmented nucleic acid molecules can be applicable to various detection or analysis occasions; Preferably, the transposase recognition sequence is a Tn5-Me or MuA-Me sequence; Preferably, the transposase complex randomly binds to any position of the double-stranded molecular sequence, and then cuts the single-stranded nucleic acid into multiple single-stranded fragments, and introduces the transposase recognition sequence into the single-stranded fragments; Preferably, the cleavage site is located at any position of the double-stranded molecular sequence; Preferably, the reaction conditions for the cleavage are: reaction temperature 15-55 °C, reaction time 15-60 min; more preferably, the reaction temperature is 37 °C.

4. A library construction method for direct sequencing of single-stranded nucleic acids, characterized in that: Comprising providing a sequence fragment to be tested; the preparation method of the sequence fragment to be tested is the method according to any one of claims 1-3; Preferably, it comprises: 1) Fragmenting the single-stranded nucleic acid and introducing the ligation end of the transposase recognition sequence by the method according to any one of claims 1-3; 2) Ligating the fragmented single-stranded nucleic acid to a sequencing adapter complex to obtain a sequencing library; Preferably, the sequencing adapter complex is an adapter complex for nanopore sequencing; Preferably, the sequencing adapter complex comprises a region for ligation of the nucleic acid fragment to be analyzed; more preferably, the region contains a complementary sequence for helicase binding and ligation of double-stranded DNA fragments; Preferably, the transposase recognition sequence and the region for ligation of the nucleic acid fragment to be analyzed have complementary sticky ends, and the ligation is achieved by a nucleic acid ligase; more preferably, the ligation is achieved by T4 DNA ligase; Preferably, the transfer strand in the transposase recognition sequence and the complementary sequence of the region for ligation of the nucleic acid fragment to be analyzed in the sequencing adapter have complementary overhangs.

5. A sequencing library prepared by the library construction method according to claim 4.

6. A sequencing method, characterized in that: The sequencing method includes the step of fragmenting and labeling the single-stranded nucleic acid by the method according to any one of claims 1-3, or includes the step of using the library construction method according to claim 4, or includes the step of sequencing the sequencing library according to claim 5.

7. A composition for constructing a single-stranded nucleic acid nanopore sequencing library, characterized in that: The composition comprises: 1) A transposase and a transposase recognition sequence; 2) A sequencing adapter complex, the sequencing adapter comprising a region for ligation of the nucleic acid fragment to be analyzed; 3) The transposase recognition sequence and the region for ligation of the nucleic acid fragment to be analyzed have complementary sticky ends; 4) A nucleic acid ligase.

8. A transposase mutant, comprising A Tn5 transposase mutant, the Tn5 transposase mutant comprising a sequence having at least 80%, 90%, 95% or 99% homology with SEQ ID NO: 7; and having one, two or three mutation sites of E54K, L372P, E345K; further preferably, comprising the amino acid sequence shown in SEQ ID NO: 13; or MuA transposase, wherein the MuA transposase comprises a sequence having at least 80%, 90%, 95% or 99% homology with SEQ ID NO: 14; and has one, two or more mutation sites of E179V, E233V, Q254R, F464Y, M487I, Q539R or Q594R; further preferably, it comprises the amino acid sequence shown in SEQ ID NO:

15.

9. Nucleic acid sequence, wherein the nucleic acid sequence is: 1) a sequence encoding the transposase mutant according to claim 8; 2) a sequence complementary to 1).

10. Biological material, wherein the biological material expresses the transposase mutant according to claim 8 or comprises the nucleic acid sequence according to claim 9.

11. Use of the transposase mutant according to claim 8, the nucleic acid sequence according to claim 9 or the biological material according to claim 10 in single-stranded nucleic acid molecule fragmentation or library construction for sequencing.

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