Sequencing library, preparation method and use thereof

Abstract

The present invention provides a sequencing library, and the sequencing library has an inserted fragment which is an equidirectional alternating concatemer of a sequence to be tested and a tag sequence. The present invention further provides a method for preparing the sequencing library. The present invention also provides a sequencing method. The sequencing library and sequencing method as provided in the present invention are capable of removing DNA amplification errors and sequencing errors under any sequencing depths, so that mutations of DNA molecules could be ultra-accurately determined. The sequencing library of the present invention is suitable for construction of a sequencing library of trace short DNA fragments and even of single-strand DNAs.

Claims

1. A method for preparing a sequencing library, comprising the following steps: (1) a sequence to be tested is linked to a tag sequence to obtain a double-strand or single-strand linked sequence; (2) when the linked sequence obtained in step (1) is a double-strand sequence, the linked sequence is converted into single-strand sequences, then cyclized; when the linked sequence obtained in step (1) is a single-strand sequence, the linked sequence is directly cyclized; (3) the cyclized linked sequence obtained in step (2) is subjected to DNA amplification based on strand displacement reaction to obtain an equidirectional alternating concatemer consisting of the sequence to be tested and the tag sequence; (4) the equidirectional alternating concatemer is fragmented, and sequencing adaptors are linked to both terminals of each resulted fragment to obtain a sequencing library; wherein, the sum of the length of the sequence to be tested and the length of the tag sequence is less than half of the sequencing length of a sequencer; and the resulted fragment in step (4) has a length greater than the sequencing length of a sequencer; the tag sequence is linked to the 5-terminal of the sequence to be tested; and the equidirectional alternating concatemer comprises at least two repetitive units, and each repetitive unit comprises one sequence to be tested and one tag sequence.

2. The method according to claim 1, wherein the tag sequence comprises 4-20 consecutive determined bases and 0-18 consecutive random bases.

3. The method according to claim 2, wherein the determined bases and the random bases are arranged in a mode of sequential arrangement or mosaic arrangement.

4. The method according to claim 1, wherein the sequencing library is used for a second-generation sequencing or a third-generation sequencing.

5. A sequencing method, comprising a process of preparing a sequencing library, wherein the process for preparing the sequencing library comprises the following steps: (1) a sequence to be tested is linked to a tag sequence to obtain a double-strand or single-strand linked sequence; (2) when the linked sequence obtained in step (1) is a double-strand sequence, the linked sequence is converted into single-strand sequences, then cyclized; when the linked sequence obtained in step (1) is a single-strand sequence, the linked sequence is directly cyclized; (3) the cyclized linked sequence obtained in step (2) is subjected to DNA amplification based on strand displacement reaction to obtain an equidirectional alternating concatemer consisting of the sequence to be tested and the tag sequence, that is, a sequencing library is prepared and obtained; (4) the equidirectional alternating concatemer is fragmented, and sequencing adaptors are linked to both terminals of each resulted fragment to obtain a sequencing library; wherein, the sum of the length of the sequence to be tested and the length of the tag sequence is less than half of the sequencing length of a sequencer; and the resulted fragment in step (4) has a length greater than the sequencing length of a sequencer; the tag sequence is linked to the 5-terminal of the sequence to be tested; and the equidirectional alternating concatemer comprises at least two repetitive units, and each repetitive unit comprises one sequence to be tested and one tag sequence.

6. The method according to claim 5, wherein the tag sequence comprises 4-20 consecutive determined bases and 0-18 consecutive random bases.

7. The method according to claim 6, wherein the determined bases and the random bases are arranged in a mode of sequential or mosaic arrangement.

8. The method according to claim 5, wherein the sequencing method is a second-generation sequencing method or a third-generation sequencing method.

9. The method according to claim 5, wherein the sequencing method is used for genomic DNA sequencing, target fragment trapping sequencing, single-strand DNA fragment sequencing, fossil DNA sequencing and sequencing of free DNA in body fluid.

10. The method according to claim 2, wherein the tag sequence comprises 6-13 consecutive determined bases.

11. The method according to claim 2, wherein the tag sequence comprises 0-13 consecutive random bases.

12. The method according to claim 3, wherein the determined bases are arranged in front of or behind the random bases.

13. The method according to claim 6, wherein the tag sequence comprises 6-13 consecutive determined bases.

14. The method according to claim 6, wherein the tag sequence comprises 0-13 consecutive random bases.

15. The method according to claim 7, wherein the determined bases are arranged in front of or behind the random bases.

16. The method according to claim 9, wherein said target fragment trapping sequencing is exon trapping sequencing.

17. The method according to claim 9, wherein said body fluid is blood, urine, or saliva.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: shows ring sizes and distribution thereof after single-strand cyclization of Example 5 of the present invention.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

(2) The embodiments of the present invention are further illustrated as follows in conjunction with the examples, but those skilled in the art would understand that these examples are merely used for illustrating the present invention, rather than limiting the scope of the present invention. For those which specific conditions are not given in the examples, they would be carried out according to conventional conditions or conditions recommended by manufacturers. When reagents or instruments are not given manufacturers, they would be commercially available conventional products.

(3) One of innovation points of the present invention lies in linking a tag sequence to a short fragment DNA molecule (total length of them is less than half of sequencing length of sequencer), performing single-strand cyclization, rolling circle replication, to obtain an equidirectional alternating concatemer of the sequence to be tested and the tag sequence, constructing sequencing library and sequencing. Specifically, it can be implemented by the following two schemes.

(4) Scheme I:

(5) Firstly, DNA is randomly broken into fragments with a length less than half of the sequencing read length of second-generation sequencer (the sum of the length after breaking and the length of a tag sequence should be less than half of the read length), then a tag sequence is linked, in which the first strand (positive strand) of the tag sequence is modified by phosphorylation at 5-terminal, while a T base is protruded at 3-terminal; the second strand (negative strand) is not subjected to modification of phosphorylation at 5-terminal, while a G base is protruded at 3-terminal. After high-temperature denaturation, the tag sequence at incision is removed, thus a DNA sequence containing single-strand tag sequence is formed, which is then subjected to high-temperature denaturation and immediate cooling so as to convert DNA into single strand. After being converted into single strand, the DNA containing tag sequence is cyclized by using single-strand cyclase. The cyclized DNA is amplified by rolling circle chain displacement based on random primers, so that the cyclized DNA molecule is amplified in a large amount. The resultant amplification product is an equidirectional alternating concatemer comprising target DNA molecule and tag sequence. Nucleotide sequence of the equidirectional alternating concatemer can be used for constructing a standard second-generation sequencing library (fragments inserted during construction of library should have a size greater than the sequencing length of a sequencer, so as to ensure that the resulted repetitive units are independent from each other).

(6) Scheme II:

(7) Firstly, DNA is randomly broken into fragments with a length less than half of the sequencing read length of a second-generation sequencer (the sum of the length after breaking and length of a tag sequence to be linked subsequently should also be less than half of the read length), then a specific tag sequence is linked thereto (same as Scheme I). After being converted into single strand, the DNA containing the tag sequence is cyclized by using a single-strand cyclase. The cyclized DNA is subjected to rolling circle amplification by using DNA polymerase (such as Phi29 DNA polymerase) with chain displacement function, in which the primer is the second strand (i.e., negative strand) in the tag sequence. After amplification, the first strand (i.e., positive strand) in the tag sequence is used as a primer to synthesize a duplex from the single-strand linear DNA after rolling. The double-strand DNA is composed of repetitive units comprising the tag sequence and the target DNA. After the double-strand DNA is purified, it can be used for constructing a standard second-generation sequencing library, in which fragments inserted during construction of library should have a size greater than the sequencing length of sequencer, so as to ensure the resultant repetitive units are independent from each other.

Example 1: Construction of Equidirectional Alternating Concatemer Library for Whole-Genome DNA Sequence to be Tested and Tag Sequence According to Scheme I (Illumina Platform)

(8) 1) DNA Fragmentation

(9) Instruments and reagents:

(10) Ultrasonic breaker: Covaris: S2 Focused-ultrasonicator

(11) Breaking tube: Covaris Microtube 616 mm, catalog #: 520045

(12) Agarose: Promega, Agarose, LE, Analytical Grade, catalog #: V3121

(13) Power of electrophoresis apparatus: Beijing Liuyi Instrument Plant, DYY-7C type

(14) Electrophoresis tank: Beijing Liuyi Instrument Plant, DYCP-31DN type electrophoresis tank

(15) QIAGEN MinElute Gel Extraction Kit (250), Catalog #: 28606

(16) Takara 20 bp DNA Ladder (Dye Plus), Takara Code, 3420A

(17) Ultrasonic breaker (Covaris S2 Focused-ultrasonicator) was used to break 1 g of purified PhiX 174 genome DNA into 150-200 bp (Intensity: 5, Duty Cycle: 10%, Cycles per Burst: 200, Temperature: 4 C., time: 60 s, number of cycles: 5), breaking system was in an amount of 50 l.

(18) 4% agarose gel electrophoresis (80V, 70 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 60-90 bp fragments (Takara 20 bp DNA Ladder), brief recovering steps: 6 times volume of buffer QG sol, adding with same volume of isopropanol, mixing homogeneously and then being separated by chromatography, eluted with buffer QG, eluted with buffer PE, dried by airing, eluted with 56 l ddH.sub.2O. See details in specification of QIAGEN MinElute Gel Extraction Kit.

(19) 2) End-Filling

(20) Reagents: New England Biolabs: NEBNext Ultra DNA Library Prep Kit for Illumina, Catalog #: E7370S

(21) Fragmented DNA: 55.5 l

(22) End Prep Enzyme Mix: 3 l

(23) End Repair Reaction Buffer (10): 6.5 l

(24) In total: 65 l

(25) 20 C. 30 min, 65 C. 30 min

(26) 3) Adding A at Terminal and Linking to Tag Sequence

(27) Reagents: New England Biolabs: NEBNext Ultra DNA Library Prep Kit for Illumina, Catalog #: E7370S

(28) Filled DNA: 65 l

(29) Blunt/TA Ligase Master Mix: 15 l

(30) Ligation Enhancer: 1 l

(31) Tag sequence UO-A (50 pmol): 1 l

(32) ddH.sub.2O: 1.5 l

(33) in total: 83.5 l

(34) 20 C. 30 min, 65 C. 10 min, then immediately placed on ice for 3 min.

(35) The product was purified with MinElute Reaction Cleanup Kit, eluted with 15 l of double-distilled water.

(36) Tag sequence: UO-A was obtained by annealing with mixture of 100 pmol UO-adaptor 1 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and 100 pmol UO-adaptor 2 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) of same volume (94 C. 5 min, gradually cooling to 25 C. with a rate of 0.1 C. per second).

(37) TABLE-US-00001 (SEQIDNO:1) UO-adaptor1:5-pTATGGGCAGTCGT-3 (SEQIDNO:2) UO-adaptor2:5-CGACTGCCCATAG-3

(38) Note: tag sequence included but was not limited to those formed with UO-adaptor 1 and UO-adaptor 2 in the example. Similarly hereinafter.

(39) 4) Single-Strand Cyclization

(40) Instruments and reagents:

(41) PCR apparatus: Eppendorf: Mastercycler pros

(42) New England Biolabs: Exonuclease I (E. coli), Catalog #: M0293

(43) New England Biolabs: Exonuclease III (E. coli), Catalog #: M0206

(44) Epicentre: CircLigase II ssDNA Ligase, Catalog #: CL9025K

(45) The above fragmented DNA was dried by distillation at 37 C. to 4.2 l.

(46) 95 C. 3 min (note: PCR apparatus should be capable of performing reaction of 100 l system, otherwise, 4.2 l would be dried out by distillation after 95 C. distillation), and immediately placed on ice for 3 min.

(47) After completion, added with:

(48) 10 circligase buffer: 0.5 l

(49) 10 mmol MnCl.sub.2: 0.25 l

(50) Circligase (100 u/ul): 0.25 l

(51) Cyclization was performed at 65 C. for 2 h, at 80 C. for 10 min.

(52) After completion of cyclization, linear and dimer DNAs were digested:

(53) Exonuclease I (E. coli): 0.25 l

(54) Exonuclease III (E. coli): 0.25 l

(55) 37 C. 1 h, 80 C. 20 min.

(56) 5) Multiple Strand Displacement (MDA) Reaction

(57) Whole-genome amplification (WGA) kit based on MDA mechanism was used for rolling circle amplification of the cyclized product.

(58) Instruments and reagents:

(59) PCR apparatus: Eppendorf: Mastercycler pros

(60) GE healthcare: illustra GenomiPhi HY DNA Amplification Kits, Product code: 25-6600-20

(61) Beckman Coulter, Inc: Agencourt AMPure XP, Item No. A63880

(62) Took the above cyclized DNA: 2.5 l

(63) Sample buffer: 22.5 l

(64) 95 C. 3 min, immediately placed on ice for 3 min.

(65) After completion, added with:

(66) Reaction buffer: 22.5 l

(67) Enzyme mix: 2.5 l

(68) In total 20 l

(69) 30 C. 1 h, 65 C. 10 min.

(70) The product was purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc). Briefly: the product after amplification was added with 1.8 times volume of magnetic beads, stood at room temperature for 5 min, absorbed with magnetic shelf for 5 min, subjected to removal of supernatant, washed with 70% alcohol twice, dried by airing, eluted with 50 l buffer AE (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). See details in the specification of the kit.

(71) The purified product was the equidirectional alternating concatemer of the sequence to be tested and the tag sequence.

(72) 6) Constructing Illumina Library for Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence.

(73) Commercially available kits for constructing standard Illumina libraries could be used, for example, TruSeq DNA Sample Preparation Kits, Nextera DNA Sample Preparation Kits. Specific steps comprised:

(74) (1) DNA Fragmentation of Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence

(75) Instruments and reagents:

(76) 1) Ultrasonic breaker: Covaris: S2 Focused-ultrasonicator

(77) 2) Breaking tube: Covaris Microtube 616 mm, No.: 520045

(78) 3) Agarose: Promega, Agarose, LE, Analytical Grade, catalog 14: V3121

(79) Ultrasonic breaker (Covaris S2 Focused-ltrasonicator) was used to break 2 g of purified direct repeat concatemer of DNA fragments was broken into 500-700 bp (Intensity: 3, Duty Cycle: 5%, Cycles per Burst: 200, Temperature: 4 C., time: 15 s, number of cycles: 5), the breaking system was in an amount of 85 l.

(80) (2) End-Filling

(81) Reagents: New England Biolabs: NEBNext End Repair Module, Catalog #:E6050

(82) QIAGEN: MinElute Reaction Cleanup Kit, Catalog #: 28206

(83) Fragmented DNA: 85 l

(84) NEBNext End Repair Reaction Buffer: 10 l

(85) NEBNext End Repair Enzyme Mix: 5 l

(86) In total: 100 l

(87) 20 C. 30 min.

(88) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 43 l ddH.sub.2O.

(89) (3) Adding A at Terminal

(90) Reagents: New England Biolabs: NEBNext dA-Tailing Module, Catalog #:E6053

(91) QIAGEN: MinElute Reaction Cleanup Kit, Catalog #: 28206

(92) Filled DNA: 42 l

(93) NEBNext dA-Tailing Reaction Buffer: 5 l

(94) Klenow Fragment (3.fwdarw.5 exo-): 3 l

(95) In total: 50 l

(96) 37 C. 30 min.

(97) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 35.5 l ddH.sub.2O.

(98) (4) Linking Sequencing Adaptor Sequence

(99) Reagents: Invitrogen: T4 DNA Ligase, Catalog #: 15224-041

(100) DNA added with A at terminal: 34.5 l

(101) Adaptor sequence 1 (50 pmol): 3 l

(102) 5DNA ligase buffer: 10 l

(103) T4 DNA Ligase: 2.5 l

(104) In total: 50 l

(105) 16 C. overnight (16 h).

(106) 2% Agarose gel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500700 bp fragments, eluting with 22 l ddH.sub.2O.

(107) Adaptor Sequence 1:

(108) TABLE-US-00002 MultiplexingAdaptor1.0: (SEQIDNO:3) 5-pGATCGGAAGAGCACACGTCT-3 MultiplexingAdaptor2.0: (SEQIDNO:4) 5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3

(109) Annealing adaptor sequences: taking 100 pmol Multiplexing Adapter 1.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and Multiplexing Adapter 2.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) with same volume, 94 C. 5 min, then being gradually cooled to 25 C. at rate of 0.1 C. per second. After annealing, adaptor sequence with concentration of 50 pmol was obtained.

(110) (5) PCR Amplification

(111) Instruments:

(112) PCR apparatus: Eppendorf: Mastercycler pros

(113) Thermo scientific: Phusion High-Fidelity PCR Master Mix with HF Buffer, Catalog #: F531L

(114) The above recovered DNA (about 30 ng)+ddH.sub.2O: 23 l

(115) MP PCR primer 1.0 (10 pmol): 1 l

(116) MP index primer 1 (10 pmol): 1 l

(117) 2 Phusion High-Fidelity PCR Master Mix: 25 l

(118) In total: 50 l

(119) PCR Amplification Circulation Conditions:

(120) Pre-denaturation at 98 C. for 45 s, circulation amplification (98 C. 15 s, 65 C. 30 s, 72 C. 60 s) 10 times, 72 C. 5 min, 4 C. cooling.

(121) 2% Agarosegel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500-700 bp fragments, eluting with 22 l ddH.sub.2O.

(122) The eluted DNAs were a constructed library, and this library could be used for sequencing in second-generation sequencing platform.

(123) Primer sequences were as follows:

(124) TABLE-US-00003 MPPCRprimer1.0: (SEQIDNO:5) 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC GCTCTTCCGATCT-3 MPindexprimer1: (SEQIDNO:6) 5-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGAC GTGTGCTCTTCCGATCT-3

Example 2: Construction of Equidirectional Alternating Concatemer Library for Human Exon Sequence to be Tested and Tag Sequence According to Scheme I (Illumina Sequencing Platform)

(125) 1) DNA Fragmentation

(126) The used instruments and reagents were as those of Example 1. Ultrasonic breaker was used to break 1 g of purified human peripheral blood genome DNA into 300 bp (Intensity: 4, Duty Cycle: 10%, Cycles per Burst: 200, Temperature: 4 C., time: 60 s, number of cycles: 2), the breaking system was in an amount of 50 l.

(127) 4% Agarose gel electrophoresis (80V, 70 min; 1TAE), cutting gel and recovering 80130 bp fragments, brief recovering steps: 6 times volume of buffer QG sol, adding with same volume of isopropanol, mixing and being separated by chromatography, eluted with buffer QG, eluted with buffer PE, dried by airing, eluted with 56 l ddH.sub.2O. See details in specification of QIAGEN MinElute Gel Extraction Kit.

(128) 2) End-Filling

(129) Reagents: see also those in Example 1.

(130) Fragmented DNA of step 1): 55.5 l

(131) End Prep Enzyme Mix: 3 l

(132) End Repair Reaction Buffer (10): 6.5 l

(133) In total: 65 l

(134) 20 C. 30 min, 65 C. 30 min.

(135) 3) Adding A at Terminal and Linking to Tag Sequence

(136) Reagents: see also those in Example 1.

(137) Filled DNA of step 2): 65 l

(138) Blunt/TA Ligase Master Mix: 15 l

(139) Ligation Enhancer: 1 l

(140) tag sequence UO-A (50 pmol): 1 l

(141) ddH.sub.2O: 1.5 l

(142) In total: 83.5 l

(143) 20 C. 30 min, 65 C. 10 min, then immediately placed on ice for 3 min.

(144) The product was purified with MinElute Reaction Cleanup Kit, eluted with 15 l ddH.sub.2O.

(145) Tag sequence: UO-A was obtained by annealing with mixture of 100 pmol UO-adaptor 1 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and 100 pmol UO-adaptor 2 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) of same volume (94 C. 5 min, gradually cooling to 25 C. with a rate of 0.1 C. per second).

(146) TABLE-US-00004 UO-adaptor1:5 -pTATGGGCAGTCGT-3 UO-adaptor2:5 -CGACTGCCCATAG-3

(147) 4) Single-Strand Cyclization of DNA

(148) Instruments and reagents: see those in Example 1.

(149) The fragmented DNA of step 3) was dried by distillation at 37 C. to reach 4.2 l.

(150) 95 C. 3 min (note: PCR apparatus should be capable of performing reaction of 100 l system, otherwise, 4.2 l would be dried out after 95 C. distillation), and immediately placed on ice for 3 min;

(151) After completion, added with:

(152) 10 circligase buffer: 0.5 l

(153) 10 mmol Mncl 2: 0.25 l

(154) Circligase (100 u/l): 0.25 l

(155) 65 C. 2 h, 80 C. 10 min;

(156) After completion of cyclization, linear and dimer DNAs were digested:

(157) Exonuclease I (E. coli): 0.25 l

(158) Exonuclease III (E. coli): 0.25 l

(159) 37 C. 1 h, 80 C. 20 min.

(160) 5) Multiple Strand Displacement (MDA) Reaction

(161) Whole-genome amplification (WGA) kit based on MDA mechanism was used for rolling circle amplification of the cyclized product.

(162) Instruments and reagents: see those in Example 1.

(163) The above cyclized DNA: 2.5 l

(164) Sample buffer: 22.5 l

(165) 95 C. 3 min, immediately placed on ice for 3 min;

(166) After completion, added with:

(167) Reaction buffer: 22.5 l

(168) Enzyme mix: 2.5 l

(169) In total 20 l

(170) 30 C. 1 h, 65 C. 10 min;

(171) The product was purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc). Briefly: the product after amplification was added with 1.8 times volume of magnetic beads, stood at room temperature for 5 min, absorbed with magnetic shelf for 5 min, subjected to removal of supernatant, washed with 70% alcohol twice, dried by airing, eluted with 50 l buffer AE (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). See details in the specification of the kit.

(172) The purified product was the equidirectional alternating concatemer of the sequence to be tested and the tag sequence.

(173) 6) Constructing Exon Trapping Library (Illumina Sequencing Platform) for the Above Prepared Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence.

(174) Commercially available kits for constructing exon trapping libraries could be used, for example, Agilent: SureSelect Human All Exon Kits.

(175) (1) DNA Fragmentation of Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence

(176) Instruments and reagents: see those in Example 1.

(177) Ultrasonic breaker was used to break 2 g of the purified equidirectional alternating concatemer of sequence to be tested and tag sequence into 500-700 bp (Intensity: 3, Duty Cycle: 5%, Cycles per Burst: 200, Temperature: 4 C., time: 15 s, number of cycles: 5), the breaking system was in an amount of 85 l.

(178) (2) End-Filling

(179) Reagents: see those in Example 1.

(180) Fragmented DNA of step (1): 85 l

(181) NEBNext End Repair Reaction Buffer: 10 l

(182) NEBNext End Repair Enzyme Mix: 5 l

(183) In total: 100 l

(184) 20 C. 30 min;

(185) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 43 l ddH.sub.2O.

(186) (3) Adding A at Terminal

(187) Reagents: see those in Example 1.

(188) Filled DNA of step (2): 42 l

(189) NEBNext dA-Tailing Reaction Buffer: 5 l

(190) Klenow Fragment (3.fwdarw.5 exo-): 3 l

(191) In total: 50 l

(192) 37 C. 30 min;

(193) The product was purified with MinElute Reaction Cleanup Kit, eluted with 35.5 l ddH.sub.2O.

(194) (4) Linking Sequencing Adaptor Sequence

(195) Reagents: see those in Example 1.

(196) DNA added with A at terminal: 34.5 l

(197) Adaptor sequence 1 (50 pmol): 3 l

(198) 5DNA ligase buffer: 10 l

(199) T4 DNA Ligase: 2.5 l

(200) In total: 50 l

(201) 16 C. overnight (16 h);

(202) 2% Agarose gel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500-700 bp fragments, eluting with 22 l ddH.sub.2O.

(203) Adaptor Sequence 1:

(204) TABLE-US-00005 MultiplexingAdaptor1.0: 5-pGATCGGAAGAGCACACGTCT-3 MultiplexingAdaptor2.0: 5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3

(205) Annealing adaptor sequences: taking 100 pmol Multiplexing Adapter 1.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and Multiplexing Adapter 2.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) with same volume, 94 C. 5 min, then being gradually cooled to 25 C. with a rate of 0.1 C. per second. After annealing, adaptor sequence 1 with concentration of 50 pmol was obtained.

(206) (5) PCR Amplification

(207) Instruments and reagents:

(208) PCR apparatus: Eppendorf: Mastecycler pro s

(209) Agilent: Herculase II Fusion DNA Polymerases, Catalog #: 600677

(210) QIAGEN: MinElute Reaction Cleanup Kit, Catalog #: 28206

(211) Four reactions were performed in parallel model, and each of the reactions had following formula:

(212) The above recovered DNA (about 90 ng)+ddH.sub.2O: 36.5 l

(213) MP PCR primer 1.0 (10 pmol): 1 l

(214) MP index primer 1 (10 pmol): 1 l

(215) 5 Herculase II Reaction Buffer: 10 l

(216) dNTPs (100 mM; 25 mM each dNTP): 0.5 l

(217) Herculase H Fusion DNA Polymerase: 1 l

(218) In total: 50 l

(219) PCR amplification circulation conditions:

(220) Pre-denaturation at 98 C. for 2 min, circulation amplification (98 C. 30 s, 65 C. 30 s, 72 C. 30 s) 8 times, 72 C. 10 min, 4 C. cooling.

(221) After completion of PCR, the PCR products in 4 reaction tubes were concentrated (MinElute Reaction Cleanup Kit), eluted with 46 l ddH2O.

(222) 2% Agarose gel electrophoresis (80V, 90 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500700 bp fragments, eluted with 26 l ddH2O.

(223) Primer sequences were as follows:

(224) TABLE-US-00006 MPPCRprimer1.0: 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCGATCT-3 MPindexprimer1: 5-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGAC GTGTGCTCTTCCGATCT-3

(225) (6) Exon Probe Hybridization

(226) In the present test, Agilent: SureSelect Human All Exon Kits was used to perform exon probe hybridization for the above PCR reaction product. Briefly:

(227) Preparing hybridization buffer solution:

(228) SureSelect Hyb #1 (orange cap, or bottle): 25 l

(229) SureSelect Hyb #2 (red cap): 1 l

(230) SureSelect Hyb #3 (yellow cap): 10 l

(231) SureSelect Hyb #4 (black cap, or bottle): 13 l

(232) In total: 49 l

(233) 65 C. 5 min.

(234) Preparing trapping library mixture:

(235) SureSelect Library: 5 l

(236) SureSelect RNase Block (purple cap): 0.5 l

(237) ddH.sub.2O: 1.5 l

(238) In total: 7 l

(239) 65 C. 2 min.

(240) Preparing sample mixture:

(241) Purified DNA (about 700 ng): 3.4 l

(242) SureSelect Indexing Block #1 (green cap): 2.5 l

(243) SureSelect Block #2 (blue cap): 2.5 l

(244) SureSelect Indexing Block #3 (brown cap): 0.6 l

(245) In total: 9 l

(246) 95 C. 5 min, 65 C. hold.

(247) 13 l of the prepared hybridization buffer solution was added with the trapping library mixture (7 l), then added with the sample mixture (9 l), in total 29 l, hybridized at 65 C. for 24 h.

(248) Magnetic beads (Invitrogen: Dynabeads M-280 Streptavidin, Catalog #: 11205D) were used to trap the hybridized fragments (50 l of magnetic beads, washed with 200 l SureSelect Binding Buffer for 3 times, the magnetic beads were resuspended in 200 l SureSelect Binding Buffer, added with the hybridization product, stood at room temperature for 30 min, absorbed with magnetic beads, washed with SureSelect Wash 1 once, washed with SureSelect Wash 2 for 3 times, the magnetic beads were resuspended in 36.5 l ddH.sub.2O), see details in operation manual of Agilent: SureSelect Human All Exon Kits.

(249) (7) PCR after Probe Hybridization

(250) Instruments and reagents:

(251) PCR apparatus: Eppendorf: Mastecycler pro s

(252) Agilent: Herculase II Fusion DNA Polymerases, Catalog #: 600677

(253) Beckman Coulter, Inc: Agencourt AMPure XP, Item No. A63880

(254) Four reactions were performed in parallel model, and each of the reactions had reaction formula as follows:

(255) Magnetic beads resuspended during exon probe hybridization: 36.5 l

(256) MP PCR primer 1.0 (10 pmol): 1 l

(257) MP PCR primer 2.0 (10 pmol): 1 l

(258) 5 Herculase II Reaction Buffer: 10 l

(259) dNTPs (100 mM; 25 mM each dNTP): 0.5 l

(260) Herculase II Fusion DNA Polymerase: 1 l

(261) In total: 50 l.

(262) PCR amplification circulation conditions:

(263) Pre-denaturation at 98 C. for 2 min, circulation amplification (98 C. 30 s, 65 C. 30 s, 72 C. 30 s) 12 times, 72 C. 10 min, 4 C. cooling.

(264) Primer sequences were as follows:

(265) TABLE-US-00007 MPPCRprimer1.0: 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCGATCT-3 MPPCRprimer2.0: (SEQIDNO:7) 5-CAAGCAGAAGACGGCATACGAGAT-3

(266) After completion of PCR, Agencourt AMPure XP magnetic beads were used for purification, briefly: the amplification product was added with 1.8 times volume of magnetic beads, stood at room temperature for 5 min, absorbed with magnetic shelf for 5 min, subjected to removal of supernatant, washed with 70% alcohol twice, dried by airing, eluted with 16 l ddH.sub.2O. See details in the specification of the kit.

(267) The eluted DNA was equidirectional alternating concatemer library of the constructed human exon sequence to be tested and the tag sequence, and the library could be used for sequencing in second-generation sequencing platform.

Example 3: Construction of Equidirectional Alternating Concatemer Library for Peripheral Blood Free DNA Sequence to be Tested and Tag Sequence According to Scheme I (Illumina Sequencing Platform)

(268) 1) Extracting Peripheral Blood Free DNA and Determining Fragment Size Thereof

(269) Instruments and reagents:

(270) QIAGEN: QIAamp Circulating Nucleic Acid Kit, catalog #: 55114

(271) Agilent: 2100 bioanalyzer

(272) 2 ml of blood plasma was collected, QIAamp Circulating Nucleic Acid Kit of QIAGEN was used to extract DNA (cell-free circulating DNA) in the plasma, and elution was performed by using 20 l ddH.sub.2O (the extraction method could be seen in the specification of the kit). 2100 bioanalyzer of Agilent was used to determine the size distribution of the extracted fragments. It could be seen in the results that the free DNA fragments of normal human subjects had a size centered around 172 bp, a distribution range of about (130 bp-230 bp), and a concentration of 0.354 ng/l, while the free DNA fragments of patients with liver cancer had a size centered around 164 bp, a distribution range of about (110 bp-210 bp), and a concentration of 4.78 ng/l.

(273) 2) End-Filling

(274) Reagents: see those in Example 1.

(275) Extracted-peripheral blood free DNA (50 ng)+ddH2O: 55.5 l

(276) End Prep Enzyme Mix: 3 l

(277) End Repair Reaction Buffer (10): 6.5 l

(278) In total: 65 l

(279) 20 C. 30 min, 65 C. 30 min.

(280) 3) Adding A at Terminal and Linking to Tag Sequence

(281) Reagents: see those in Example 1

(282) Filled DNA: 65 l

(283) Blunt/TA Ligase Master Mix: 15 l

(284) Ligation Enhancer: 1 l

(285) Tag sequence UO-A (50 pmol): 1 l

(286) ddH.sub.2O: 1.5 l

(287) In total: 83.5 l

(288) 20 C. 30 min, 65 C. 10 min, immediately placed on ice for 3 min.

(289) The product was purified with MinElute Reaction Cleanup Kit, eluted with 15 l ddH2O.

(290) Tag sequence: UO-A was obtained by annealing with mixture of 100 pmol UO-adaptor 1 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and 100 pmol UO-adaptor 2 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) of same volume (94 C. 5 min, gradually cooling to 25 C. with a rate of 0.1 C. per second).

(291) TABLE-US-00008 UO-adaptor1:5-pTATGGGCAGTCGT-3 UO-adaptor2:5-CGACTGCCCATAG-3

(292) 4) Single-Strand Cyclization of DNA

(293) Instruments and reagents: see those in Example 1.

(294) The extracted-peripheral blood free DNA was dried by distillation at 37 C. to reach 4.2 l.

(295) 95 C. 3 min (note: PCR apparatus should be capable of performing reaction of 100 l system, otherwise, 4.2 l would be dried out after 95 C. distillation), and immediately placed on ice for 3 min;

(296) After completion, added with:

(297) 10 circligase buffer: 0.5 l

(298) 10 mmol MnCl.sub.2: 0.25 l

(299) Circligase (100 u/l): 0.25 l

(300) 65 C. 2 h, 80 C. 10 min.

(301) After completion of cyclization, linear and dimer DNAs were digested:

(302) Exonuclease I (E. coli): 0.25 l

(303) Exonuclease III (E. coli): 0.25 l

(304) 37 C. 1 h, 80 C. 20 min.

(305) 5) Multiple Strand Displacement (MDA) Reaction

(306) Whole-genome amplification (WGA) kit based on MDA mechanism was used for rolling circle amplification of the cyclized product.

(307) Instruments and reagents: see those in Example 1.

(308) The above cyclized DNA: 2.5 l

(309) Sample buffer: 22.5 l

(310) 95 C. 3 min, immediately placed on ice for 3 min;

(311) After completion, added with:

(312) Reaction buffer: 22.5 l Enzyme mix: 2.5 l

(313) In total: 20 l

(314) 30 C. 1 h, 65 C. 10 min.

(315) The product was purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc). Briefly: the product after amplification was added with 1.8 times volume of magnetic beads, stood at room temperature for 5 min, absorbed with magnetic shelf for 5 min, subjected to removal of supernatant, washed with 70% alcohol twice, dried by airing, eluted with 50 l buffer AE (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0). See details in the specification of the kit.

(316) The purified product was the equidirectional alternating concatemer of the sequence to be tested and the tag sequence.

(317) 6) Constructing Illumina Sequencing Library for the Above Prepared Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence.

(318) Commercially available kits for constructing standard Illumian libraries could be used, for example, TruSeq DNA Sample Preparation Kits, Nextera DNA Sample Preparation Kits.

(319) (1) DNA Fragmentation of Direct Repeat Concatemer

(320) Instruments and reagents: see those in Example 1.

(321) Ultrasonic breaker was used to break 2 g of the purified equidirectional alternating concatemer of sequence to be tested and tag sequence into 500-700 bp (Intensity: 3, Duty Cycle: 5%, Cycles per Burst: 200, Temperature: 4 C., time: 15 s, number of cycles: 5), the breaking system was in an amount of 85 l.

(322) (2) End-Filling

(323) Reagents: see those in Example 1.

(324) Fragmented DNA: 85 l

(325) NEBNext End Repair Reaction Buffer: 10 l

(326) NEBNext End Repair Enzyme Mix: 5 l

(327) In total: 100 l

(328) 20 C. 30 min;

(329) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 43 l ddH2O.

(330) (3) Adding A at Terminal

(331) Reagents: see those in Example 1.

(332) Filled DNA: 42 l

(333) NEBNext dA-Tailing Reaction Buffer: 5 l

(334) Klenow Fragment (3.fwdarw.5 exo-): 3 l

(335) In total: 50 l

(336) 37 C. 30 min;

(337) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 35.5 l ddH2O.

(338) (4) Linking Sequencing Adaptor Sequence

(339) Reagents: see those in Example 1.

(340) DNA added with A at terminal: 34.5 l

(341) Adaptor sequence 1 (50 pmol): 3 l

(342) 5DNA ligase buffer: 10 l

(343) T4 DNA Ligase: 2.5 l

(344) In total: 50 l 16 C. overnight (16 h).

(345) 2% Agarose gel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500700 bp fragments, eluting with 22 l ddH2O.

(346) Adaptor Sequence 1:

(347) TABLE-US-00009 MultiplexingAdaptor1.0: 5-pGATCGGAAGAGCACACGTCT-3 MultiplexingAdaptor2.0: 5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3

(348) Annealing adaptor sequences: taking 100 pmol Multiplexing Adapter 1.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and Multiplexing Adapter 2.0 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) with same volume, 94 C. 5 min, then being gradually cooled to 25 C. with a rate of 0.1 C. per second. After annealing, adaptor sequence 1 with concentration of 50 pmol was obtained.

(349) (5) PCR Amplification

(350) Instruments and reagents: see those in Example 1.

(351) The above recovered DNA (about 30 ng)+ddH.sub.2O: 23 l

(352) MP PCR primer 1.0 (10 pmol): 1 l

(353) MP index primer 1 (10 pmol): 1 l

(354) 2 Phusion High-Fidelity PCR Master Mix: 25 l

(355) In total: 50 l.

(356) PCR amplification circulation conditions:

(357) Pre-denaturation at 98 C. for 45 s, circulation amplification (98 C. 15 s, 65 C. 30 s, 72 C. 60 s) 10 times, 72 C. 5 min, 4 C. cooling.

(358) 2% Agarose gel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500-700 bp fragments, eluting with 22 l ddH.sub.2O.

(359) The eluted DNAs were the constructed library, and this library could be used for sequencing in second-generation sequencing platform.

(360) Primer sequences were as follows:

(361) TABLE-US-00010 MPPCRprimer1.0: 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCGATCT-3 MPindexprimer1: 5-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGAC GTGTGCTCTTCCGATCT-3

Example 4: Construction of Equidirectional Alternating Concatemer Library for Sequence to be Tested and Tag Sequence According to Scheme II (Illumina Sequencing Platform)

(362) Steps:

(363) 1) DNA Fragmentation

(364) Instruments and reagents: see those in Example 1.

(365) Ultrasonic breaker was used to break 1 g of the purified Drosophila melanogaster genome DNA into 150-200 bp (Intensity: 5, Duty Cycle: 10%, Cycles per Burst: 200, Temperature: 4 C., time: 60 s, number of cycles: 5), the breaking system was in an amount of 50 l.

(366) 4% agarose gel electrophoresis (80V, 70 min; 1TAE), cutting gel and recovering 60-90 bp fragments, brief recovering steps: 6 times volume of buffer QG sol, adding with same volume of isopropanol, mixing homogeneously and then being separated by chromatography, eluted with buffer QG, eluted with buffer PE, dried by airing, eluted with 56 l ddH.sub.2O. See details in specification of QIAGEN MinElute Gel Extraction Kit.

(367) 2) End-Filling

(368) Reagents: see those in Example 1.

(369) Fragmented DNA: 55.5 l

(370) End Prep Enzyme Mix: 3 l

(371) End Repair Reaction Buffer (10): 6.5 l

(372) In total: 65 l

(373) 20 C. 30 min, 65 C. 30 min.

(374) 3) Adding A at Terminal and Linking to Tag Sequence

(375) Reagents: see those in Example 1.

(376) Filled DNA: 65 l

(377) Blunt/TA Ligase Master Mix: 15 l

(378) Ligation Enhancer: 1 l

(379) tag sequence UO-A (50 pmol): 1 l

(380) ddH2O: 1.5 l

(381) In total: 83.5 l

(382) 20 C. 30 min, 65 C. 10 min, immediately placed on ice for 3 min.

(383) The product was purified with MinElute Reaction Cleanup Kit, and eluted with 15 l ddH.sub.2O.

(384) Tag sequence: UO-A was obtained by annealing with mixture of 100 pmol UO-adaptor 1 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and 100 pmol UO-adaptor 2 (dissolved with annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) of same volume (94 C. 5 min, gradually cooling to 25 C. with a rate of 0.1 C. per second).

(385) TABLE-US-00011 UO-adaptor1:5 -pTATGGGCAGTCGT-3 UO-adaptor2:5 -CGACTGCCCATAG-3

(386) 4) Single-Strand Cyclization of DNA

(387) Instruments and reagents: see those in Example 1.

(388) The above fragmented DNA was dried by distillation at 37 C. to 4.2 l.

(389) 95 C. 3 min (note: PCR apparatus should be capable of performing reaction of 100 l system, otherwise, 4.2 l would be dried out by distillation after 95 C. distillation), and immediately placed on ice for 3 min.

(390) After completion, added with:

(391) 10 circligase buffer: 0.5 l

(392) 10 mmol MnCl.sub.2: 0.25 l

(393) Circligase (100 u/l): 0.25 l

(394) 65 C. 2 h, 80 C. 10 min;

(395) After completion of cyclization, linear and dimer DNAs were digested:

(396) Exonuclease I (E. coli): 0.25 l

(397) Exonuclease III (E. coli): 0.25 l

(398) 37 C. 1 h, 80 C. 20 min.

(399) 5) Rolling Circle Amplification

(400) Instruments and reagents:

(401) PCR apparatus: Eppendorf: Mastecycler pros

(402) New England Biolabs: phi29 DNA Polymerase, Catalog #: M0269L

(403) Single-strand cyclized DNA: 5.7 l

(404) Phi29 DNA Polymerase Reaction Buffer: 2 l

(405) Primer UO-a3 (10 pmol): 1 l

(406) ddH.sub.2O: 8.9 l

(407) In total: 17.6 l, 95 C. 3 min, immediately placed on ice for 3 min.

(408) After completion, added with:

(409) 10 mM dNTP: 1 l

(410) 100BSA: 0.4 l

(411) phi29 DNA Polymerase (10 U/l): 1 l

(412) In total: 20 l

(413) 30 C. 8 h, 65 C. 10 min.

(414) TABLE-US-00012 Primersequence:UO-a3: (SEQIDNO:8) 5 -ACGACTGCCCATAT-3

(415) 6) Converting Linear DNA into Double Strand

(416) Instruments and reagents:

(417) PCR apparatus: Eppendorf: Mastecycler pros

(418) New England Biolabs: phi29 DNA Polymerase, Catalog #: M0269L

(419) New England Biolabs: Exonuclease I (E. coli), Catalog #: M0293

(420) New England Biolabs: T4 DNA polymerase, Catalog #: m0203

(421) Epicentre: Ampligase Enzyme and Buffer, Catalog #:A3202K

(422) Beckman Coulter, Inc: Agencourt AMPure XP, Item No. A63880

(423) Rolling circle amplified DNA: 20 l

(424) Primer UO-a1 (10p): 1 l

(425) Ampligase 10 Reaction Buffer: 5 l

(426) 2.5 mM dNTP: 1 l

(427) ddH.sub.2O: 22.5 l

(428) 95 C. 3 min, immediately placed on ice for 3 min,

(429) After completion, added with:

(430) T4 DNA polymerase: 0.5 l

(431) 12 C. 2.5 h, 75 C. 20 min.

(432) After completion, added with:

(433) Ampligase DNA Ligase: 3 l

(434) 60 C. 1 h.

(435) After completion, added with:

(436) Exonuclease I: 1 l

(437) 37 C. 1 h, 80 C. 20 min.

(438) The product was purified with Agencourt AMPure XP magnetic beads. Briefly: the product after amplification was added with 1.8 times volume of magnetic beads, stood at room temperature for 5 min, absorbed with magnetic shelf for 5 min, subjected to removal of supernatant, washed with 70% alcohol twice, dried by airing, eluted with 20 l ddH.sub.2O. See details in the specification of the kit.

(439) The purified product was a direct repeat concatemer of the DNA fragment.

(440) TABLE-US-00013 PrimersequenceUO-a1:5-pTATGGGCAGTCGT-3

(441) 7) Constructing Illumina Sequencing Library for the Above Prepared Equidirectional Alternating Concatemer of Sequence to be Tested and Tag Sequence.

(442) After rolling circle amplification for 8 h, the obtained DNA had an amount ranging from dozens of nanograms to hundreds of nanograms, and the yield of DNA after rolling circle amplification could be elevated by increasing time of rolling circle amplification. According to the obtained DNA amount, a suitable commercially available kit could be chosen to construct standard Illumina library: if DNA in an amount of dozens of nanograms was obtained, Nextera DNA Sample Preparation Kits or other kits for constructing libraries based on small amount of DNA could be used; if DNA in an amount of hundreds of nanograms was obtained, TruSeq DNA Sample Preparation Kits or other kits for large initial amount of DNA could be used.

(443) This test used a method for constructing libraries based on transposase EZ-Tn5:

(444) (1) Assembling Transposons

(445) Epi_MA1 (10 pmol): 1 l

(446) Epi_MA2 (10 pmol): 1 l

(447) Glycerol: 0.5 l

(448) 1 U/l transposase EZ-Tn5 (epicentre): 2.5 l

(449) In total: 5 l

(450) 25 C. 20 min.

(451) (2) DNA Fragmentation

(452) The above transposons: 5 l

(453) 5LMW buffer: 2 l

(454) The above obtained direct repeat concatemer DNA (about 30 ng)+ddH2O: 3 l

(455) In total: 10 l

(456) 55 C. 10 min.

(457) The product was purified with MinElute Reaction Cleanup Kit, eluted with 24 l ddH2O.

(458) (3) PCR Amplification of the Recovered Product

(459) Instruments and reagents: see those in Example 1.

(460) The above recovered DNA (about 30 ng)+ddH2O: 23 l

(461) Epi_PCR primer 1.0 (10 pmol): 1 l

(462) Epi_index primer (10 pmol): 1 l

(463) 2 Phusion High-Fidelity PCR Master Mix: 25 l

(464) In total: 50 l

(465) PCR amplification circulation conditions:

(466) 72 C. 3 min (imperative), 98 C. 30 s, circulation amplification (98 C. 10 s, 65 C. 30 s, 72 C. 3 min) 10 times, 4 C. cooling.

(467) 2% Agarose gel electrophoresis (80V, 80 min; 1TAE), cutting gel and recovering (QIAGEN MinElute Gel Extraction Kit) 500800 bp fragments, eluted with 17 l ddH2O.

(468) The eluted DNA was the constructed library, and this library could be used for sequencing in second-generation sequencing platform.

(469) The above primer sequences were as follows:

(470) TABLE-US-00014 Epi_ME: (SEQIDNO:9) 5-CTGTCTCTTATACACATCT-3 Epi_Adaptor1: (SEQIDNO:10) 5-CTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG- 3 Epi_Adaptor2: (SEQIDNO:11) 5-CGGTCTGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG- 3 Epi_PCRprimer1.0: (SEQIDNO:12) 5-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATC AG-3 Epi_PCRindexprimer: (SEQIDNO:13) 5-CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTGCCAG CCCGCTCAG-3

(471) Epi_MA1: Obtained by annealing with 100 pmol Epi_ME (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and Epi_Adaptor 1 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 NaCl) with same volume. Conditions: 94 C. 5 min, following with gradually cooling to 25 C. with a rate of 0.1 C. per second.

(472) Epi_MA2: Obtained by annealing with 100 pmol Epi_ME (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) and Epi_Adaptor 2 (dissolved in annealing buffer solution: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl) with same volume. Conditions: 94 C. 5 min, following with gradually cooling to 25 C. with a rate of 0.1 C. per second.

(473) 5LMW buffer: 50 mM Tris-OAc, pH 8.0, 25 mM Mg(OAc).sub.2

(474) Constructing Oseq library according to Example 1.

Example 5: Library Construction and Data Analysis for Phage Phix174

(475) 1 g of phage Phix174 DNA was ultrasonically broken into 300 bp DNA fragments. 6080 bp fragments were recovered, linked to tag sequence, converted into single strand, and subjected to rolling circle amplification (see details in Example 1). The DNA after rolling circle amplification was subjected to second-generation sequencing library construction based on transposons (see details in Example 4). About 10G of bi-directional data (read length was 2100=200 bp) were obtained by using hiseq 2000. The data were treated and analyzed as follows:

(476) 1. Measured in total: 54391601 reads, in which the number of reads capable of forming rings (at least two repetitive units could be detected, similarly hereinafter) was: 33987941 reads.

(477) 2. Cyclization rate: OS2_in2: (135951764/4)/(217566404/4)=62.49%

(478) 3. For the formed rings, size range was: 30-162 bp, average size was: 72.5333 bp, standard deviation was: 14.06478, median was: 71 bp. Specific distribution was shown in FIG. 1.

(479) 4. The constructed library of the equidirectional alternating concatemer of sequence to be tested and tag sequence was subjected to high-throughput Pair-End sequencing. Because the ring size was less than half of the sequencing length of sequencer, the concatemer of at least one unit must be covered by single-end sequencing once, the concatemer units must be tested twice by pair-end sequencing once, and inconsistent sequences could be removed by comparing the two concatemer sequences. By using this principle, the error rate of DNA in the measured data was calculated. If there is not infrequent mutation in sample, this method would have an error rate of 1e-5. Sequencing errors had different distributions on different bases (referring to bases of genome), in which sequencing error rates from C to T and G to A were relatively high, i.e., about 1e-4, and specific sequencing error rates were shown in Table 1. This kind of mutation pattern was also found in other researches for determining infrequent mutations, and these two kinds of mutations were likely caused by spontaneous deamination of cytosine or 5-methylated cytosine. After deamination, bases of one original single strand DNA changed, and only mutated bases could be observed in a plurality of independent determinations.

(480) TABLE-US-00015 TABLE 1 Sequencing error rates of different bases Type of sequencing error Sequencing error rate A => C 1.78E06 T => G 1.13E06 A => G 4.41E06 T => C 6.96E06 A => T 5.70E06 T => A 2.97E06 C => A 1.34E05 G => T 2.91E05 C => G 1.19E05 G => C 1.92E05 C => T 0.000153171 G => A 0.000443162

(481) It could be seen from the above calculation results that the method had a single-base error rate (10.sup.5) far lower than the error rate of second-generation sequencing (1%), and far lower than those of the improved methods in the prior art as well. Thus, the method could thoroughly solve the error rate problem of the second-generation sequencing, and could implement ultra-accurate sequencing of DNA molecules by using second-generation sequencing techniques. Another merit of the present method is that the sequencing precision is irrelative to sequencing depth, which could solve problem that accurate sequencing of DNA sequence by labeling methods could be achieved only under very high sequencing coverage multipliers, so that accurate sequencing of large genome (such as human genome) could be achieved.

Example 6: Library Construction and Data Analysis for E. coli

(482) DNAs of E. coli W3110 were collected, ultrasonically broken into DNA fragments with main band of 300 bp. 80150 bp fragments were recovered, linked to tag sequences, converted into single strand, subjected to rolling circle amplification. After the rolling circle amplification, the DNAs were subjected to conventional second-generation sequencing library construction (see details in Example 1). About 4G of bi-directional data (read length was 2150=300 bp) were obtained by using hiseq 2500. The data were treated and analyzed as follows:

(483) 1. Measured in total: 13787730 reads, in which the number of reads capable of forming rings was: 7578585 reads.

(484) 2. Cyclization rate: 54.96615468971325%

(485) 3. For the formed rings, size range was: 30-260 bp, average size was: 122.909 bp, standard deviation was: 17.74147 bp. median was: 122 bp.

(486) Sequencing error rates for bases were shown in Table 2.

(487) TABLE-US-00016 TABLE 2 Sequencing error rates for different bases Type of sequencing error Sequencing error rate A => C 2.66E07 T => G 4.10E07 A => G 2.79E06 T => C 2.47E06 A => T 1.58E06 T => A 1.29E06 C => A 5.68E06 G => T 3.85E06 C => G 3.20E06 G => C 1.14E06 C => T 0.000119 G => A 7.73E05

Example 7: Construction and Data Analysis for Sequencing Library with Random Tag Sequences

(488) PhiX174 DNA was collected, ultrasonically broken into DNA fragments with main band at 100200 bp. 60100 bp fragments were recovered, linked to tag sequences, converted into single strand, subjected to rolling circle amplification. After rolling circle amplification, the DNA was subjected to conventional second-generation sequencing library construction (see details in Example 1). Wherein the tag sequences linked to the DNA fragments to be tested were as follows:

(489) TABLE-US-00017 (SEQIDNO:14) UO-adaptor1N:5-pNNNNNNNNNTATGGGCAGTCGT-3 (SEQIDNO:15) UO-adaptor2:5-CGACTGCCCATAG-3.
About 4G of bi-directional data (read length was 2150=300 bp) were obtained by using hiseq 2000. The data were treated and analyzed as follows:

(490) 1. Measured in total: 19147560 reads, in which the number of reads capable of forming rings (at least two repetitive units could be detected, similarly hereinafter) was: 4580270 reads.

(491) 2. Cyclization rate: 23.92090689361987%.

(492) 3. For the formed rings, size range (after removal of tag sequences) was: 1-133 bp, average size was: 88.56275 bp, standard deviation was: 29.17562 bp. median was: 98 bp.

(493) Sequencing error rates for bases were shown in Table 3.

(494) TABLE-US-00018 TABLE 3 Sequencing error rates for different bases Type of sequencing error Sequencing error rate A => C 4.36E07 T => G 9.22E07 A => G 3.79E06 T => C 4.12E06 A => T 8.75E06 T => A 1.24E05 C => A 2.97E05 G => T 1.93E05 C => G 1.50E05 G => C 9.99E06 C => T 0.000103 G => A 0.000131

(495) The method of the present invention is capable of performing ultra-accurate determination of DNA molecule composition in cells, and can relatively truly present DNA composition in normal or diseased (e.g., cancer tissues) cell colonies. In aspect of cancer detection, this method can be used for detection whether a tissue or organ of a normal individual has potential carcinogenic mutations so as to achieve the goal of finding cancers in advance and prophylaxis of cancers. In aspect of cancer studying, this method can be used to determine distribution of DNA mutations in cancer colonies; can be used to find potential small clone colonies in cancer tissues so as to realize heterogeneous structure of tumors; can help to illustrate effects of mutations in occurrence and development of cancers; and can be used to find tumor stem cells. In aspect of cancer therapy, this method can be used to find tumor stem cell colonies, and then a specific drug target can be designed for the tumor stem cells, so that effect therapy of cancer can be achieved. For normal individuals, this method can be used to detect DNA mutations in normal cells, so as to retrieve growth pattern of normal tissues; an can be used to determine numbers of DNA mutations in certain tissue of individuals with different ages, so as to estimate DNA mutation rates; or can be used to detect whether diseases-associated mutations exist in a normal individual, so as to achieve prophylaxis of diseases.

(496) In the meantime, this method can be for effective library construction of free DNA in peripheral blood, and can effectively determine infrequent mutation sits in peripheral blood, and this kind of non-invasive detection method can be used for determination and evaluation of occurrence and development of cancers as well as harmful mutations in fetuses in antenatal diagnosis.

(497) Ancient human DNA sequence is a main means for studying human evolution, but there are lots of problems in sequencing ancient human DNAs, among which the most serious problems are that the extracted ancient human DNAs have very low contents, are seriously degraded, and heavily contaminated. This method can construct libraries by using very small amounts of DNA (either single or double strands), and the constructed libraries can be used for exon trapping (removal of microorganism genome contaminations), and thus these problems in ancient DNA library construction can be effectively solved.

(498) Although the specific embodiments of this invention have been described in details, those skilled in the art can understand that these details can be modified or changed according to the disclosed teachings, and all of these changes fall within the protection scope of the present invention. The protection scope of this invention is given by the appending claims and any equivalents thereof.