Sequencing element and application thereof

Abstract

The invention provides a sequencing element and application thereof. The invention provides a sequencing element which comprises any of the following items: (I), a transposition complex and a primer group; the transposon complex comprises a transposase and a transposon; and / or (II) a ligase, a primer group and a nucleic acid linker molecule. According to the sequence screening scheme provided by the invention, the preference problem of the transposase recognition sequence can be comprehensively evaluated, and help is provided for selecting a more proper transposase recognition sequence. A large number of accurate tag sequences which can be rapidly used for library construction and sequencing are screened out at a time, so that the sequence screening flux is greatly improved, the operation process is simplified, the time is saved, and the screening cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biological sequencing, and in particular to sequencing elements and applications thereof. Background Art

[0002] With the rapid development of sequencing technology, pathogen detection, and the widespread application of a large number of DNA sequencing technologies in disease diagnosis, the requirements for sequencing throughput are increasing. As a sequencing technology for COVID-19 nucleic acid detection and subsequent support for vaccine development, there are higher requirements for sequencing throughput at this time. In order to obtain nucleic acid sequences of more sample viruses at the same time and faster, track the mutation process, and assist in vaccine development, we mix a large number of samples together for library construction and sequencing, which requires adding an identifiable label sequence (barcode) to each sample to facilitate the analysis of subsequent sequencing data.

[0003] Transposase is a type of enzyme that is encoded by a transposon and recognizes specific sequences at both ends of the transposon. It binds to the ends of the transposon and performs transposition functions by cutting and pasting and reintegrating. In the era of rapid development of sequencing technology, the application of transposase has made greater progress in the library preparation technology for next-generation sequencing (NGS). Transposase uses its own cutting and connecting function to insert a specific adapter sequence into double-stranded DNA, achieving a major breakthrough in combining the four steps of interruption, end repair, adding "A", and adding adapters in the library construction process into one step, greatly reducing the time for library construction, simplifying the experimental operation process, reducing the cost of library construction, and also reducing the requirements for the starting amount of the sample to be tested. Currently, the transposases used for library construction include Mu transposase, Mos1 transposase, Vibrio harvey transposase, Tn5 transposase, etc. Among them, Tn5 transposase is the most widely used. In January 2011, Illumina acquired Epicentre Biotechnology and integrated its EZ-Tn5 patented product into the Nextera series of commercial library construction kits. The Tn5 transposon consists of a central region and two flanks, each of which includes a 19bp outside end (OE) and inside end (IE) terminal sequence, where OE is the recognition and binding site of the transposase. Although Tn5 insertion is random, stable, and the insertion site is easy to confirm, analysis of the sequencing results found that the first 9bp of the Tn5 insertion position still has a preference. The modification of some amino acid sites of the Tn5 transposase reduced the insertion preference, but it has not been completely eliminated. This technical solution uses Tn5 transposase as an example to provide a method for screening the recognition and binding sites of transposases, in order to improve the problem of insertion position preference. This technical solution can also be used for other sequence screening, such as screening of sample tag sequences in mixed library construction or mixed sequencing.

[0004] Ligase is a widely used tool enzyme in molecular biology experiments. In 1967, DNA ligase was discovered in Escherichia coli. The enzyme uses the energy generated by the hydrolysis of ATP or NAD to catalyze the 5-phosphate of a DNA chain and the 3-hydroxyl of another DNA chain to form a phosphodiester bond, connecting the cuts in the DNA chain. Currently, the most widely used enzyme is T4 DNA ligase, which has the function of connecting sticky ends and blunt ends. It is an indispensable enzyme in the construction of many types of libraries.

[0005] Previous technologies have all studied the function of the Tn5 transposase recognition sequence through a series of complex methods such as plasmid construction, bacterial transfection, and DNA extraction.

[0006] For the screening of sample tag sequences, various rule parameters are set by computer, such as the principle of base balance, etc., to output sequences that may be used as tags, artificially synthesize sequences, and then conduct rounds of library construction experiments. After the library quality control indicators such as uniform library yield and uniform inserted fragments are passed, the library sequencing data is evaluated to ensure that each sample can be accurately distinguished and the amount of data under each sample is uniform, and finally included in the tag sequence for use. This screening method is often not enough for one round of library construction and sequencing, and usually requires several rounds of screening and confirmation, which consumes a lot of time and labor costs.

[0007] In the prior art, screening transposase recognition sequences requires a complex experimental process, which is time-consuming, costly, and may not necessarily produce good results. It is difficult to accurately determine the recognition sequence of the transposase, and it cannot be quickly used in technical solutions such as library construction. The method of screening tag sequences requires a long experimental process, layer-by-layer analysis and screening, and the operation is complex, time-consuming, costly, and has low throughput. In the current era of high-throughput sequencing, it is impossible to screen a large number of tag sequences at one time, and it is difficult to quickly apply them to library construction and sequencing. Summary of the invention

[0008] In view of this, the present invention provides a sequencing element and its application. The sequence screening scheme proposed by the present invention can comprehensively evaluate the preference of the transposase recognition sequence and provide assistance for selecting a more suitable transposase recognition sequence. By screening a large number of accurate tag sequences that can be quickly used for library construction and sequencing at one time, the throughput of sequence screening is greatly improved, the operation process is simplified, time is saved, and the screening cost is reduced.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a sequencing element, comprising any of the following:

[0011] (I), a transposition complex and a primer set; the transposition complex comprises a transposase and a transposon; and / or

[0012] (II), ligase, primer set and nucleic acid adaptor molecule;

[0013] The transposition complex includes one or more of transposition complex 1, transposition complex 2 or transposition complex 3;

[0014] The transposon includes one or more of transposon 1, transposon 2, transposon 3 or transposon 4;

[0015] The transposition complex 1 includes the transposase and the transposon 1;

[0016] The transposon 1 includes transposon 1-F and transposon 1-R;

[0017] The transposon 1-F comprises a primer binding fragment, a tag fragment, a connection fragment and a binding fragment of the transposon 1-R;

[0018] The primer binding fragment is identical to the upstream primer sequence of the primer set;

[0019] The transposition complex 2 includes the transposase and the transposon 2;

[0020] The transposon 2 is prepared by reacting the vector connection fragment and the extended primer fragment;

[0021] The vector connection fragment includes a transposase recognition fragment to be screened, a complementary fragment of the extended primer fragment, and a fragment containing A;

[0022] The extended primer fragment is identical to the downstream primer sequence of the primer set;

[0023] The transposition complex 3 includes the transposase, the transposon 3 and the transposon 4;

[0024] The transposon 3 is prepared by reacting transposon 3-F and transposon 1-R;

[0025] The transposon 4 is prepared by reacting transposon 4-F and transposon 1-R;

[0026] The transposon 3-F comprises a primer binding fragment, a tag fragment to be screened, a connection fragment and a binding fragment of the transposon 1-R; the primer binding fragment is identical to the upstream primer sequence of the primer set;

[0027] The transposon 4-F includes an extended primer segment and a binding segment of the transposon 1-R;

[0028] The nucleic acid linker molecule includes a linker-top and a linker-bottom;

[0029] The adapter-top comprises a primer binding fragment, a tag fragment to be screened and a connection fragment; the primer binding fragment is identical to the upstream primer sequence of the primer set;

[0030] The adapter-bottom includes a complementary segment of the connection segment and a complementary segment of the extension primer segment.

[0031] In some embodiments of the present invention, in the above sequencing element, the primer binding segment has:

[0032] (7) the nucleotide sequence shown in SEQ ID NO: 1; or

[0033] (8) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or

[0034] (9) a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (7) or (8); and / or

[0035] The connecting fragment has:

[0036] (10), the nucleotide sequence shown in SEQ ID NO: 2; or

[0037] (11) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or

[0038] (12) a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (10) or (11); and / or

[0039] The tag fragment has:

[0040] (13), the nucleotide sequence shown in SEQ ID NO: 3; or

[0041] (14) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13), and having the same or similar function as the nucleotide sequence shown in (13); or

[0042] (15) a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (13) or (14); and / or

[0043] The transposon 1-R has:

[0044] (16), the nucleotide sequence shown in SEQ ID NO: 4; or

[0045] (17) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (16), and having the same or similar function as the nucleotide sequence shown in (16); or

[0046] (18), a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (16) or (17); and / or

[0047] The vector connection fragment has:

[0048] (19), the nucleotide sequence shown in SEQ ID NO: 5; or

[0049] (20) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (19), and having the same or similar function as the nucleotide sequence shown in (19); or

[0050] (21), a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (19) or (20); and / or

[0051] The extension primer fragment has:

[0052] (22), the nucleotide sequence shown in SEQ ID NO: 6; or

[0053] (23) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (22), and having the same or similar function as the nucleotide sequence shown in (22); or

[0054] (24), a nucleotide sequence having at least 40% identity with the nucleotide sequence shown in (22) or (23); and / or

[0055] The fragment containing A has:

[0056] (25), the nucleotide sequence shown in SEQ ID NO:7; or

[0057] (26) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (25), and having the same or similar function as the nucleotide sequence shown in (25); or

[0058] (27) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (25) or (26).

[0059] In some embodiments of the present invention, the sequence of SEQ ID NO: 1 is: CGATCCTTGGTGATCCGTCAGTGC.

[0060] In some embodiments of the present invention, the sequence of SEQ ID NO: 2 is: TTGTCTTCCT.

[0061] In some embodiments of the present invention, the sequence of SEQ ID NO: 3 is: CGGATTGCCG.

[0062] In some embodiments of the invention, the sequence of SEQ ID NO:4 is: pho-CTGTCTCTTATACACATCT.

[0063] In some embodiments of the present invention, the sequence of SEQ ID NO:5 is: p-NNNNNNNNN NNNNNNNNNNTCTGCTGAGTCGAGAACGTCTCdUdUCGAAAAAA-biotin.

[0064] In some embodiments of the invention, the sequence of SEQ ID NO:6 is: GAGACGTTCT CGACTCAGCAGA.

[0065] In some embodiments of the invention, the sequence of SEQ ID NO:7 is: dUdUCGAAAA AA.

[0066] The present invention also provides the use of the above sequencing element in library construction and / or sequencing.

[0067] The present invention also provides the use of the above sequencing element in screening tag fragments and / or transposase recognition fragments.

[0068] The present invention also provides a method for preparing the transposon in the above sequencing element, comprising the following steps:

[0069] S1: taking the transposon 1-F and the transposon 1-R, mixing them, and amplifying them to obtain the transposon 1; and / or

[0070] S2: taking the vector connection fragment and the extension primer fragment, mixing them, and amplifying them to obtain the transposon 2; and / or

[0071] S3: Mix the transposon 3-F and the transposon 1-R, and amplify them to obtain the transposon 3; and / or

[0072] S4: taking the transposon 4-F and the transposon 1-R, mixing them, and amplifying them to obtain the transposon 4;

[0073] S1 to S4 are in no particular order.

[0074] The present invention also provides a method for preparing the transposition complex in the above sequencing element, comprising the following steps:

[0075] S1: embedding the transposase into the transposon 1 to obtain the transposition complex 1; and / or

[0076] S2: embedding the transposase 2 into the transposon 2 to obtain the transposition complex 2; and / or

[0077] S3: mixing the transposase, the transposon 3 and the transposon 4 to obtain the transposition complex 3;

[0078] S1 to S3 are in no particular order.

[0079] The present invention also provides a method for genome fragmentation, wherein a pretreated sample is taken and enzymatically cut to obtain a fragmented genome;

[0080] The pretreatment includes one or more of a transposition reaction, an enzyme digestion reaction and / or an ultrasonic reaction;

[0081] When the pretreatment is a transposition reaction, the transposition reaction uses the transposition complex 1 and / or the transposition complex 3 in the above-mentioned sequencing element;

[0082] In the above method, the enzyme digestion and disruption uses an enzyme digestion and disruption buffer;

[0083] The enzyme digestion and disruption uses the transposition complex 2 in the above sequencing element. The enzyme digestion and disruption buffer comprises: 50 mM HEPES-KOH, 50% DMF (v / v) and 25 mM MgCl2.

[0084] In some embodiments of the present invention, in the above method, when the pretreatment is an enzyme digestion reaction and / or an ultrasonic reaction, the enzyme digestion and interruption further include the steps of: end repair, A addition, and adapter addition;

[0085] The linker addition adopts the nucleic acid linker molecule in the above-mentioned sequencing element.

[0086] The present invention also provides a method for enriching a genome, and after polymerase chain reaction of the fragmented genome obtained by the above method, an enriched genome is obtained.

[0087] The present invention also provides a method for screening target nucleic acids, based on the above sequencing elements, sequencing to obtain the target nucleic acids.

[0088] The present invention provides a method for screening label fragments, wherein a sample to be tested is fragmented, polymerized chain reaction is performed, purified, and sequenced to obtain label fragments;

[0089] The fragmentation comprises one or more of a transposition reaction, an enzyme digestion reaction and / or an ultrasonic reaction;

[0090] When the fragmentation is a transposition reaction, the transposition reaction uses the transposition complex 3 of the above-mentioned sequencing element.

[0091] In some embodiments of the present invention, the above-mentioned screening method, when the fragmentation is an enzyme cutting reaction and / or an ultrasonic reaction, after the fragmentation and before the polymerization chain reaction, it also includes the steps of end repair, adding A, and adding adapters; the adding adapters uses the nucleic acid adapter molecules in the above-mentioned sequencing elements.

[0092] The present invention provides a method for screening transposase recognition fragments, taking a first fragmented sample to be tested, second fragmenting, polymerizing chain reaction, purifying, and circularizing to obtain the transposase recognition fragments;

[0093] The first fragmentation uses the transposition complex 1 in the above sequencing element;

[0094] The second fragmentation uses the transposition complex 2 in the above-mentioned sequencing element.

[0095] The present invention provides a tag recognition fragment having:

[0096] (1), the nucleotide sequence shown in SEQ ID NO: 8; or

[0097] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or

[0098] (3) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (1) or (2).

[0099] In some embodiments of the present invention, the sequence of SEQ ID NO: 8 is: CGGATTGCCG.

[0100] The present invention provides a transposase recognition fragment having:

[0101] (4) the nucleotide sequence shown in SEQ ID NO: 9 to SEQ ID NO: 11; or

[0102] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0103] (6) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (4) or (5).

[0104] In some embodiments of the present invention, the sequence of SEQ ID NO:9 is: CTGGCTCTTAT ACATAGGT.

[0105] In some embodiments of the present invention, the sequence of SEQ ID NO: 10 is: CTGGCTCTTA TACAGAGTT.

[0106] In some embodiments of the present invention, the sequence of SEQ ID NO: 11 is: CTGGCTCTTA TACAAATGT.

[0107] The present invention also provides the use of the tag recognition fragment and / or the transposase recognition fragment in library construction and / or sequencing.

[0108] The present invention also provides the use of the tag recognition fragment and / or the transposase recognition fragment in screening tag fragments and / or transposase recognition fragments.

[0109] The present invention also provides the use of the tag recognition fragment and / or the transposase recognition fragment in detecting the preference of nucleic acid binding proteins.

[0110] In some embodiments of the present invention, in the above application, the nucleic acid binding protein includes: one or more of a ligase, a topoisomerase or a helicase. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0112] Figure 1 Shows the results of agarose gel electrophoresis after genomic DNA was fragmented with transposition complex 1;

[0113] Figure 2 The results of agarose gel electrophoresis of the Tn5 transposase recognition sequence screening library are shown, where: 1 and 2 are libraries of two recognition sequences respectively;

[0114] Figure 3 The results of agarose gel electrophoresis after genomic DNA was fragmented by transposition complex 3 are shown, wherein: 4-2 and 5 are libraries of two recognition sequences respectively;

[0115] Figure 4 The results of agarose gel electrophoresis of the Tn5 transposase tag sequence screening library are shown, where: 1-1 and 1-2 are two parallel libraries;

[0116] Figure 5 The results of agarose gel electrophoresis after genomic DNA was fragmented by shearing enzyme; 1 is before purification, and 2 is after purification. DETAILED DESCRIPTION

[0117] The invention discloses a sequencing element and application thereof.

[0118] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0119] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0120] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0121] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0122] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0123] The invention provides a method for preparing a transposition complex containing a 19bp transposase recognition sequence with four bases randomly arranged and combined.

[0124] The present invention also provides a method for re-fragmenting a DNA product with a linker by using a transposition complex containing a 19 bp random arrangement combination.

[0125] The present invention also provides a method for enriching a transposition complex containing a 19 bp random arrangement combination inserted with a linker DNA product.

[0126] The present invention is achieved through the following technical solutions

[0127] 1) If the transposition complex is used to fragment the genomic DNA later, a transposition complex with a known transposon sequence needs to be prepared. If the genomic DNA is fragmented by enzyme digestion or ultrasound, an adapter sequence needs to be prepared.

[0128] 2) Preparation of a transposition complex containing a 19bp transposase recognition sequence with a random combination of four bases: Design a sequence with a known adapter sequence and a 19bp random sequence, connect the sequence to a vector, and then use the known adapter sequence for extension reaction to form a complementary double strand. Finally, cut the double strand from the vector and embed it with a transposase to form a transposition complex.

[0129] 3) Genomic DNA fragmentation: Use a transposon complex with a known transposon sequence to fragment the DNA and directly add a known adapter sequence to both ends of the DNA, or use enzyme cutting to fragment the DNA and then connect the two ends of the DNA to the adapter sequence, or use ultrasonic shearing to fragment the genomic DNA and then connect the two ends of the DNA to the adapter sequence.

[0130] 4) The transposase complex containing 19 bp randomly arranged combinations is used to fragment the adapter DNA product again, and the transposase complex is mixed with the genomic DNA, incubated at 55° C. for 10 minutes on a PCR instrument to terminate the reaction.

[0131] 5) Enrichment of transposition complexes containing 19 bp randomly arranged combinations inserted into genomic DNA, using the known adapter sequence at one end of the fragmented DNA and the known adapter sequence on the transposition complex as primers, respectively, to perform polymerase chain reaction to enrich the products with transposition complex insertion.

[0132] In Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0133] The present invention will be further described below in conjunction with embodiments:

[0134] Example 1 Screening of Tn5 enzyme recognition sequence

[0135] 1) Design and synthesize the following sequence

[0136] Transposon 1-F: 5'-CGATCCTTGGTGATCCGTCAGTGCCGGATTGCCGTTGTCTTCCTAGATGTGTATAAGAGACAG-3'; (as shown in SEQ ID NO: 16)

[0137] Transposon1-R: 5′pho-CTGTCTCTTATACACATCT-3′;

[0138] Vector linker sequence (capture oligo): 5′p-NNNNNNNNNNNNNNNNNNNTCTGCTGAGTCGAGAACGTCTCdUdUCGAAAAAA-3′biotin;

[0139] Extension primer sequence (ex primer): 5′-GAGACGTTCTCGACTCAGCAGA-3′;

[0140] 2) Preparation of transposition complex 1

[0141] 20 μL of transposon 1-F and transposon 1-R with a concentration of 100 μM were mixed, placed on a PCR instrument, 70°C for 1 minute, slowly cooled to 20°C at a rate of 0.1°C / s, and reacted at 20°C for 30 minutes to finally obtain a transposon 1 with a concentration of 50 μM. 11.8 μL of 1U / μL Tn5 transposase within the shelf life was used, 3.2 μL of transposon 1, and 25 μL of 50% glycerol diluted with TE buffer (10mM Tris-HCl, 1mMEDTA) were mixed on ice and placed at 30°C for 1 hour. The product after the reaction was transposon complex 1, and the concentration of transposon in transposon complex 1 was 4pmol / μL. The prepared transposon complex 1 can be stored at -20°C for 1 year.

[0142] 3) Preparation of transposon 2

[0143] ① Connect capture oligo to a carrier modified with streptavidin protein through biotin-streptavidin interaction. Here, the carrier is Dynabeads M-280streptation (112.06D, Invitrogen). Take 43μL M-280 beads, add 4 times the concentration of magnetic bead salt buffer (50mM Tirs-HCl, 150mMNaCl, 0.02% Tween-20) to wash twice, then add 125μL of binding buffer (200mM Tirs-HCl, 600mMNaCl, 0.4mM EDTA), and then add 5μL of 100μM capture oligo. Finally, add molecular grade water to make up the volume to 500μL, mix thoroughly, and place on a vertical mixer to bind at 25℃ for 1 hour.

[0144] ② After the reaction, wash once with low salt buffer, and then wash once with 2 times concentration of pfucx mix (2mM PfuCxBuffer (X), 5% DMSO, 1000mM Betaine, 6mM MgSO4, 0.6mM dNTP), add 50μL of 2 times concentration of pfucxmix on ice, 10μL of 100μM ex primer, hot start Pfu Turbo Cxenzyme (AGILENT) at 98℃ for 3 minutes, then add 1.33μL, and finally add 38.67μL of molecular grade water to make up the volume to 50μL. After thorough mixing, place in a PCR instrument and run the following program: 70℃ for 3 minutes, 54℃ for 1 minute, 72℃ for 2 minutes, and store at 4℃. The second step to the third step (54℃ for 1 minute, 72℃ for 2 minutes) were repeated for 5 cycles.

[0145] ③After the reaction is completed, wash once with low salt buffer, then wash once with TE buffer (10mM Tris-HCl, 1mMEDTA), add 8μL of TE buffer, place on ice, add 4μL of USER (10U / μL), the total volume is 12μL, mix thoroughly, place on a vertical mixer, react at 37℃ for 1 hour. After the reaction is completed, place on a magnetic stand, aspirate the supernatant into a new PCR tube, and store at -20℃ for later use. This supernatant is transposon 2.

[0146] 4) Preparation of transposition complex 2

[0147] Use 11.8 μL of 1U / μL Tn5 transposase within the shelf life, 11 μL of transposon 2 obtained in step 3), and 5.2 μL of 50% glycerol diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA). Mix them on ice and react at 30°C for 1 hour. The product after the reaction is transposition complex 2. The prepared transposition complex 2 can be stored at -20°C for 1 year.

[0148] 5) Transposition complex 1 fragments genomic DNA

[0149] Prepare 2 tubes of a mixed solution of the transposition complex 1 obtained in step 2) and the long-fragment DNA molecule on ice.

[0150] Take 12μL of 5-fold transposase shearing buffer (HEPES-KOH 50mM, DMF 50%, MgCl225mM) per tube, 40ng of long-fragment DNA molecules, 0.675μL of transposition complex 1, and use molecular reaction-grade water to make up the system to 60μL. Place it in a PCR instrument and run the following reaction at 55℃ for 10 minutes and store at 4℃. After the reaction is completed, take it out and place it at room temperature, add 6.6μL of 1% SDS, shake it thoroughly and mix it, and incubate it at room temperature for 10 minutes. After the reaction is completed, the above product is purified by two-step method using XP magnetic beads (Agencourt AMPureXP-Medium, A63882, AGENCOURT), add 20μL of magnetic beads, remove the magnetic beads after binding at room temperature, absorb the supernatant, add 14μL of magnetic beads to the supernatant, discard the supernatant after binding at room temperature, wash twice with 80% ethanol, dry it at room temperature, and elute the DNA with TE buffer. The purified product is stored at -20℃ for later use. This product is the product of genomic DNA fragmentation to 700bp-5000bp and ligation to a known adapter sequence after insertion of transposition complex 1. After combining the two tubes, take 1 μL of the product and use a double-stranded concentration detection kit (lifetech) for concentration detection. Take 10 ng for electrophoresis detection. The results are as follows: Figure 1 shown.

[0151] 6) Transposition complex 2 inserts into the DNA product fragmented by transposition complex 1

[0152] Prepare a mixture of the transposase complex 2 obtained in step 4) and the product of step 5) on ice. Take 4 μL of 5-fold transposase disruption buffer (HEPES-KOH 50mM, DMF 50%, MgCl2 25mM), 20ng of fragmented DNA molecules, 4 μL of transposase complex 2, and use molecular reaction grade water to make up the system to 20 μL. Place it in a PCR instrument and run the following reaction at 55°C for 10 minutes and store at 4°C. After the reaction is completed, take it out and place it at room temperature, add 2.2 μL of 1% SDS, shake it thoroughly to mix, and incubate it at room temperature for 10 minutes. Proceed to the next step of the reaction.

[0153] 7) Polymerase chain reaction

[0154] Prepare the PCR reaction mixture on ice as follows:

[0155] 2X Pfu Turbo Cx Mix 3 50 μL PCR primer 1 (100 μM) 0.5 μL PCR primer 2 (100 μM) 0.5 μL Pfu Turbo Cx enzyme 1.33 μL 10% Tween 20 5.5 μL 20% Triton x-100 5.5 μL <![CDATA[H2O]]> 14.47 μL Total 77.8 μL

[0156] After sufficient oscillation and mixing, place in a PCR instrument, incubate at 98°C for 3 minutes, and store at 4°C. After the reaction is complete, take out and place on ice, add 22.2 μL of all the products in step 6), oscillate and mix thoroughly, place on a PCR machine, and run the following program:

[0157]

[0158] After the reaction, 80 μL XP magnetic beads were used to purify the product. The purification method was as described in the official standard operating procedures. 1 μL of the purified product was tested for concentration using a double-stranded concentration detection kit (Lifetech), and 10 ng was tested for agarose gel electrophoresis. The results were as follows: Figure 2 shown.

[0159] 8) Use BGIseq-500 for sequencing. The library obtained in the previous step needs to be subjected to single-stranded circularization reaction. For details of the operation, please refer to the circularization step of the BGIseq-500 standard DNA small fragment library construction process.

[0160] 9) Through data analysis, we output the 19bp transposon sequence inserted into the DNA, count the number of reads supported by each transposon, analyze the base distribution under each transposon, and finally we screened 3 sequences. They are recognition sequence 1: 5-CTGGCTCTTATACATAGGT-3, recognition sequence 2: 5-CTGGCTCTTATACAGAGTT-3, and recognition sequence 3: 5-CTGGCTCTTATACAAATGT-3. We then constructed the library and sequenced the 3 sequences. The insertion efficiency of the transposon was evaluated through data analysis, and the inserted fragment of the transposon, the CG content of the first 15bp of the insertion position, the CG separation, and the AT separation were calculated. The results showed that the results of the 3 sequences in terms of alignment rate, coverage, insertion efficiency, inserted fragment, and overall GC content were basically consistent with the existing recognition sequence results, and the repetition rate was slightly higher than that of the control library, which may be caused by differences in sequencing data (as shown in Table 1). However, further careful analysis of the CG content, CG separation, and AT separation of the first 15bp of the insertion position revealed that the results of the three recognition sequences we screened were better than the existing recognition sequences. The specific data showed that in the first 15bp of the transposon insertion position, the GC content of the three sequence libraries was closer to 40% for 10bp-11bp, the GC separation was smaller for 8bp, and the AT separation was smaller for 10bp (as shown in Table 2, where the data of the first 9bp was quoted from TransGen statistical data, and the last 6bp was from the self-test library.)

[0161] Table 1 Comparison of sequencing results between the screened recognition sequence library and the existing recognition sequence library

[0162]

[0163] Table 2 Comparison of GC content, GC separation and AT separation results of the selected recognition sequences and existing recognition sequences

[0164]

[0165]

[0166] Example 2 Tn5 transposition complex tag sequence screening

[0167] 1) Design and synthesize the following sequence

[0168] Transposon 3-F: 5′-CGATCCTTGGTGATCCGTCAGTGCNNNNNNNNNNTTGTCTTCCTAGATGTGTATAAGAGACAG-3′; (as shown in SEQ ID NO: 12) Transposon 1-R: 5′-pho-CTGTCTCTTATACACATCT-3′;

[0169] Transposon 4-F: 5'-GAGACGTTCTCGACTCAGCAGAAGATGTGTATAAGAGACAG-3'; (as shown in SEQ ID NO: 13)

[0170] PCR primer 1: 5'-CGATCCTTGGTGATCCGTCAGTGC-3'; (as shown in SEQ ID NO: 14)

[0171] PCR primer 2: 5′pho-GAGACGTTCTCGACTCAGCAGA-3′ (as shown in SEQ ID NO: 15).

[0172] 2) Preparation of transposition complex 3

[0173] 20 μL of transposon 3-F and transposon 1-R at a concentration of 100 μM were mixed, placed on a PCR instrument, incubated at 70°C for 1 minute, slowly cooled to 20°C at a rate of 0.1°C / s, and reacted at 20°C for 30 minutes to finally obtain transposon 3 at a concentration of 50 uM. 20 μL of transposon 4-F and transposon 1-R at a concentration of 100 μM were mixed, placed on a PCR instrument, incubated at 70°C for 1 minute, and 20 μL of transposon 1-R were mixed, placed on a PCR instrument, incubated at 70°C for 1 minute, slowly cooled to 20°C at a rate of 0.1°C / s, and reacted at 20°C for 30 minutes to finally obtain transposon 4 at a concentration of 50 uM. Use 11.8 μL of 1U / μL Tn5 transposase within the shelf life, mix 1.6 μL transposon 3 and 1.6 μL transposon 4, and 25 μL of 50% glycerol diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA) on ice, and then react at 30°C for 1 hour. The product after the reaction is transposition complex 3. The concentration of transposons in transposition complex 3 is 4 pmol / μL. The prepared transposition complex 3 can be stored at -20°C for 1 year.

[0174] 3) Transposition complex 3 fragments genomic DNA

[0175] A mixed solution of the transposition complex 3 obtained in step 2) and the long-fragment DNA molecule was prepared on ice.

[0176] Take 20μL of 5-fold transposase shearing buffer (HEPES-KOH 50mM, DMF 50%, MgCl2 25mM), 20ng of long DNA molecules, 4μL diluted to 0.25pmol / μL transposition complex 3, use molecular reaction grade water to make up the system to 100μL, place in PCR instrument to run the following reaction, 55℃ for 10 minutes, and store at 4℃. After the reaction is completed, take out and place at room temperature, add 11μL of 1% SDS, shake and mix thoroughly, and incubate at room temperature for 10 minutes. After the reaction is completed, the above product is purified with 166.5μL XP magnetic beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT), and 20μL TE buffer is used to dissolve. The purification method is shown in the official standard operating procedures.

[0177] 1 μL of the purified product was used for concentration detection using a double-stranded concentration detection kit (LifeTech), and 8 ng was used for electrophoresis detection. The results were as follows: Figure 3 The remaining product was made up to 18 μL and then all was used for the next step reaction.

[0178] 4) Polymerase chain reaction

[0179] Prepare the PCR reaction mixture on ice as follows:

[0180] 2X Pfu Turbo Cx Mix3 25 μL PCR primer 1 (100 μM) 0.25 μL PCR primer 2 (100 μM) 0.25 μL Pfu Turbo Cx enzyme (2.5U / μL) 0.75 μL <![CDATA[H2O]]> 5.75 μL Total 32 μL

[0181] After sufficient oscillation and mixing, place in a PCR instrument, incubate at 98°C for 3 minutes, and store at 4°C. After the reaction is complete, take out and place on ice, add 18 μL of all the products in step 3), oscillate and mix thoroughly, place on a PCR machine, and run the following program:

[0182]

[0183] After the reaction, 40 μL XP magnetic beads were used to purify the product. The purification method was as described in the official standard operating procedures. 1 μL of the purified product was tested for concentration using a double-stranded concentration detection kit (Lifetech), and 10 ng was tested for agarose gel electrophoresis. The results were as follows: Figure 4 shown.

[0184] 5) Use BGIseq-500 for sequencing to measure the random N tag sequence. The library obtained in the previous step needs to be subjected to single-stranded circularization reaction. For details of the operation, please refer to the circularization step of the BGIseq-500 standard DNA small fragment library construction process.

[0185] 6) Output 10 bp transposon tag sequences through data analysis, and select tag sequences with uniform insertion efficiency based on the number of reads supported by each transposon tag sequence.

[0186] Example 3 Method for screening tag sequences using T4 DNA ligase

[0187] 1) Design and synthesize the following sequence

[0188] Linker-top: 5-CGATCCTTGGTGATCCGTCAGTGCNNNNNNNNNNTTGTC TTCCT-3; (as shown in SEQ ID NO: 17)

[0189] Linker-bottom: 5pho-GGAAGACAATCTGCTGAGTCGAGAACGTCTC-3 (as shown in SEQ ID NO: 18).

[0190] 2) Preparation of adapter

[0191] Mix 20 μL of 100 μM adapter-top and 20 μL of adapter-bottom, place on a PCR instrument, incubate at 70°C for 1 minute, slowly cool to 20°C at a rate of 0.1°C / s, react at 20°C for 30 minutes, and finally obtain a 50 μM adapter. Take part of the adapter product and dilute it with TE buffer to a 20 μM adapter for later use.

[0192] 3) Fragmentation of genomic DNA

[0193] Genomic DNA can be fragmented by ultrasound or enzyme cleavage. Here, the enzyme cleavage method is used to fragment genomic DNA. Prepare the genomic DNA and the fragmentation reaction mixture on ice, take 200ng of genomic DNA dissolved in TE buffer, add 5μL of 5-fold concentrated fragmentation reaction buffer (enzymatics), 10μL of fragmentation reaction enzyme mixture (enzymatics), add TE buffer to 50μL, shake and mix thoroughly, and place it on the PCR instrument to run the following program:

[0194] Heating cover 70℃ on 4℃ hold 32℃ 16min 65℃ 15min 4℃ hold

[0195] After the reaction, add 30 μL TE, shake and mix, take 5 μL for agarose gel electrophoresis, and the electrophoresis detection results are as follows Figure 5The remaining product was purified with 60 μL XP magnetic beads. The purification method is shown in the official standard operating procedures. The purified product was re-dissolved with 25 μL TE, and 5 μL was taken for agarose gel electrophoresis. The electrophoresis detection results are shown in Figure 5 As shown in Figure 2, the remaining products were all put into the next step reaction.

[0196] 4) End repair

[0197] Prepare the fragmented DNA product obtained in step 3) and the end repair reaction mixture on ice according to the following system:

[0198] Fragmented DNA 18.1 μL 10X PNK buffer (enzymatics) 2.5 μL dNTP(25mM) 1 μL T4PNK Kinase(enzymatics) 1.5 μL T4 DNA polymerase (3U / μL) 1.5 μL Klenow Fragment(BGI) 0.4 μL Total volume 25 μL

[0199] After thorough oscillation and mixing, place in a PCR instrument at 20°C for 30 minutes and store at 4°C. After the reaction is completed, add 25 μL XP magnetic beads for purification. The purification method is described in the official standard operating procedures. The purified product is re-dissolved in 20 μL TE and immediately proceeded to the next reaction.

[0200] 5) Add "A"

[0201] Prepare the fragmented end repair product obtained in step 4) and the reaction mixture with "A" on ice according to the following system:

[0202] End repair products 18 μL water 7.05 μL 10X PNK buffer (enzymatics) 3 μL 10mM dATP 0.75 μL klenow exo-(5U / μL) 1.2 μL Total volume 30 μL

[0203] After thorough oscillation and mixing, place in a PCR instrument, incubate at 37°C for 30 minutes, and store at 4°C. After the reaction is complete, add 30 μL XP magnetic beads for purification. The purification method is described in the official standard operating procedures. The purified product is re-dissolved in 15 μL TE, and 13 μL is taken for the next ligation reaction.

[0204] 6) Add connector

[0205] Prepare the "A"-added product obtained in step 5) and the linker-added reaction mixture on ice according to the following system:

[0206] water 1.4 μL 3X HB buffer 9 μL T4 DNA ligase (enzymatics) 1.6 μL Total volume 12 μL

[0207] 3X HB buffer is a self-made reagent and needs to be prepared in advance. The formula is as follows: 50% PEG 80004.8mL, pH7.82MTris-HCl 0.6mL, 0.1MATP 0.24mL, 10mg / mL BSA 0.12mL, 1M Magnesium Chloride0.24mL, 1M DTT 0.012mL, molecular grade water is added to 8mL, and stored at -20℃ after aliquoting for later use.

[0208] After the above mixture is prepared, shake it thoroughly and set aside. Add 2 μL of the 20 μM linker prepared in step 2) to the 13 μL product obtained in step 6) on ice, and then add 12 μL of the prepared connection reaction mixture, with a total volume of 27 μL. Shake it thoroughly and place it in a PCR instrument at 20°C for 30 minutes and store it at 4°C. After the reaction is completed, add 33 μL TE, then add 30 μL XP magnetic beads for purification, dissolve the purified product back with 20 μL TE, and take 1 μL for concentration detection using a double-stranded concentration detection kit (lifetech).

[0209] 8) Use BGIseq-500 for sequencing to measure the random N tag sequence. The library obtained in the previous step needs to be subjected to single-stranded circularization reaction. For details of the operation, please refer to the circularization step of the BGIseq-500 standard DNA small fragment library construction process.

[0210] 9) Output 10 bp transposon tag sequences through data analysis, and select tag sequences with uniform insertion efficiency based on the number of reads supported by each transposon tag sequence.

[0211] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sequencing element, characterized in that Include any of the following: (I), a transposition complex and a primer set; the transposition complex comprises a transposase and a transposon; and / or (II), ligase, primer set and nucleic acid adaptor molecule; The transposition complex includes one or more of transposition complex 1, transposition complex 2 or transposition complex 3; The transposon includes one or more of transposon 1, transposon 2, transposon 3 or transposon 4; The transposition complex 1 includes the transposase and the transposon 1; The transposon 1 includes transposon 1-F and transposon 1-R; The transposon 1-F comprises a primer binding fragment, a tag fragment, a connection fragment and a binding fragment of the transposon 1-R; The primer binding fragment is identical to the upstream primer sequence of the primer set; The transposition complex 2 includes the transposase and the transposon 2; The transposon 2 is prepared by reacting the vector connection fragment and the extended primer fragment; The vector connection fragment includes a transposase recognition fragment to be screened, a complementary fragment of the extended primer fragment, and a fragment containing A; The extended primer fragment is identical to the downstream primer sequence of the primer set; The transposition complex 3 includes the transposase, the transposon 3 and the transposon 4; The transposon 3 is prepared by reacting transposon 3-F and transposon 1-R; The transposon 4 is prepared by reacting transposon 4-F and transposon 1-R; The transposon 3-F comprises a primer binding fragment, a tag fragment to be screened, a connection fragment and a binding fragment of the transposon 1-R; the primer binding fragment is identical to the upstream primer sequence of the primer set; The transposon 4-F includes an extended primer segment and a binding segment of the transposon 1-R; The nucleic acid linker molecule includes a linker-top and a linker-bottom; The adapter-top comprises a primer binding fragment, a tag fragment to be screened and a connection fragment; the primer binding fragment is identical to the upstream primer sequence of the primer set; The adapter-bottom includes a complementary segment of the connection segment and a complementary segment of the extension primer segment.

2. The use of the sequencing element according to claim 1, including but not limited to any one of the following: (I), application in library construction; and / or (II), application in sequencing; and / or (III) Application in screening tag fragments; and / or (IV) Application in screening transposase recognition fragments.

3. The method for preparing the transposon in the sequencing element according to claim 1, characterized in that: The following steps are involved: S1: taking the transposon 1-F and the transposon 1-R, mixing them, and amplifying them to obtain the transposon 1; and / or S2: taking the vector connection fragment and the extension primer fragment, mixing them, and amplifying them to obtain the transposon 2; and / or S3: Mix the transposon 3-F and the transposon 1-R, and amplify them to obtain the transposon 3; and / or S4: taking the transposon 4-F and the transposon 1-R, mixing them, and amplifying them to obtain the transposon 4; S1 to S4 are in no particular order.

4. The method for preparing the transposition complex in the sequencing element according to claim 1, characterized in that: The following steps are involved: S1: embedding the transposase into the transposon 1 to obtain the transposition complex 1; and / or S2: embedding the transposase 2 into the transposon 2 to obtain the transposition complex 2; and / or S3: mixing the transposase, the transposon 3 and the transposon 4 to obtain the transposition complex 3; S1 to S3 are in no particular order.

5. A method for genome fragmentation, characterized in that The pre-treated samples were digested and fragmented to obtain fragmented genomes; The pretreatment includes one or more of a transposition reaction, an enzyme digestion reaction and / or an ultrasonic reaction; When the pretreatment is a transposition reaction, the transposition reaction uses the transposition complex 1 and / or transposition complex 3 in the sequencing element as claimed in claim 1; The enzyme cutting and interruption uses the transposition complex 2 in the sequencing element as claimed in claim 1; Preferably, when the pretreatment is an enzyme digestion reaction and / or an ultrasonic reaction, the enzyme digestion and interruption further include the steps of: end repair, A addition, and adapter addition; The adding of adapters uses the nucleic acid adapter molecules in the sequencing element as claimed in claim 1.

6. A method for screening tag fragments, characterized in that: The sample to be tested is fragmented, polymerized, chain-reacted, purified, and sequenced to obtain the labeled fragments; The fragmentation comprises one or more of a transposition reaction, an enzyme digestion reaction and / or an ultrasonic reaction; When the fragmentation is a transposition reaction, the transposition reaction uses the transposition complex 3 of the sequencing element as claimed in claim 1; Preferably, when the fragmentation is an enzyme cleavage reaction and / or an ultrasonic reaction, after the fragmentation and before the polymerization chain reaction, the steps of end repair, A addition, and adapter addition are further included; the adapter addition adopts the nucleic acid adapter molecule in the sequencing element as claimed in claim 1.

7. A method for screening transposase recognition fragments, characterized in that: Taking the first fragmented sample to be tested, performing a second fragmentation, polymerizing chain reaction, purifying, and circularizing, to obtain the transposase recognition fragment; The first fragmentation uses the transposition complex 1 in the sequencing element as claimed in claim 1; The second fragmentation uses the transposition complex 2 in the sequencing element as claimed in claim 1.

8. A tag identification fragment, characterized in that have: (1), the nucleotide sequence shown in SEQ ID NO: 8; or (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or (3) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (1) or (2).

9. A transposase recognition fragment, characterized in that It has: (4) the nucleotide sequence shown in SEQ ID NO: 9 to SEQ ID NO: 11; or (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (4) or (5).

10. Use of the tag recognition fragment according to claim 8 and / or the transposase recognition fragment according to claim 9, including but not limited to any one of the following: (I), application in library construction; and / or (II), application in sequencing; and / or (III) Application in screening tag fragments; and / or (IV) Application in screening transposase recognition fragments; and / or (V) Application in detecting the preference of nucleic acid binding proteins.

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