METHOD FOR CONSTRUCTING LIBRARY OF CELL-FREE DNAS IN BODY FLUIDS AND APPLICATION THEREOF

Abstract

A method for constructing a library of cell-free DNAs in body fluids, comprising directly acting a transposase or an endonuclease on a body fluid sample, fragmenting the cell-free DNAs within, and performing amplification to obtain a library. Also provided is a test kit using the present method for prenatal diagnosis or early detection of cancer.

Claims

1. A method for constructing a library of cell-free DNAs in a body fluid sample, comprising steps of 1) directly treating the body fluid sample by an enzyme, such that the cell-free DNAs in the body fluid sample are fragmented under the enzyme, and 2) amplifying the fragmented DNAs obtained in step 1) to obtain the library of cell-free DNAs in the body fluid sample.

2. The method according to claim 1, wherein the enzyme in step 1) is a transposase or an endonuclease.

3. The method according to claim 2, wherein the transposase is Tn5 transposase and the endonuclease is MNase or DNase.

4. The method according to claim 1, wherein the step 1) comprises utilizing a transposase to treat the body fluid sample containing the cell-free DNAs, such that the cell-free DNAs are fragmented and added with an adapter through a transposition reaction mediated by the transposase, thereby obtaining DNA fragments containing adapter sequences.

5. The method according to claim 1, wherein the step 1) comprises utilizing an endonuclease to treat the body fluid sample containing the cell-free DNAs, such that the cell-free DNAs are fragmented by the endonuclease, and adding an adapter at both ends of the fragmented DNAs, thereby obtaining DNA fragments containing adapter sequences.

6. The method according to claim 4, wherein the adapter for the transposition reaction is an adapter mixture prepared by steps of: a) annealing a primer A of 5′-CTGTCTCTTATACACATCT-3′ (SEQ ID NO: 1) and a primer B of 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 2) to obtain a first adapter, b) annealing a primer A of 5′-CTGTCTCTTATACACATCT-3′ (SEQ ID NO: 1) and a primer C of 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 3) to obtain a second adapter. c) mixing the first adapter and the second adapter to obtain the adapter mixture.

7. The method according to claim 6, wherein the transposition reaction is conducted by incubating the transposase-embedding complex and the body fluid sample at a condition suitable for transposition reaction.

8. The method according to claim 1, wherein the amplification in step 2) comprises two amplification processes; the cycle number N to be used in a second amplification process is determined by the qPCR reaction after a first amplification process.

9. The method according to claim 1, further comprising a step of 3) subjecting the library of cell-free DNAs in the body fluid sample obtained in step 2) to cyclization and enzyme digestion.

10. A method for obtaining epigenetic information of an individual, comprising steps of 1) obtaining a library of cell-free DNAs in a body fluid sample of an individual according to the method of claim 1, 2) sequencing and analyzing the library of cell-free DNAs in the body fluid sample obtained in step 1) to obtain epigenetic information of the individual.

11. (canceled)

12. A method for prenatal diagnosis or early detection of cancer, comprising performing the method for constructing a library of cell-free DNAs in a body fluid sample of claim 1.

13. (canceled)

14. (canceled)

15. The method according to claim 4, wherein the step 1) further comprises extracting the DNA fragments containing adapter sequences in the body fluid sample.

16. The method according to claim 5, wherein the step 1) further comprises extracting the fragmented DNAs in the body fluid sample after the fragmentation reaction.

17. The method according to claim 6, wherein the adapter mixture is embedded with the transposase to obtain a transposase-embedding complex for the transposition reaction.

18. The method according to claim 6, wherein the adapter mixture is embedded with the Tagment Enzyme Advanced V5S containing transposase.

19. The method according to claim 6, wherein a volume ratio of the adapter mixture to the Tagment Enzyme Advanced V5S is 1:20 to 1:25.

20. The method according to claim 7, wherein the volume ratio of the transposase-embedding complex to the body fluid sample for transposition reaction is 1:50 to 1:80.

21. The method according to claim 7, wherein the temperature for transposition reaction is 35 to 40° C.

22. The method according to claim 9, wherein the cyclization comprises denaturing the double-stranded DNAs in the library of cell-free DNAs in the body fluid sample into single-stranded DNAs, and ligating the single-stranded DNAs with an oligonucleotide fragment complementary to a partial region of the single-stranded DNA through base-complementary pairing, wherein the enzyme digestion is performed by using exonuclease I and exonuclease III to remove non-cyclized DNAs.

23. The method according to claim 22, wherein the single-stranded DNAs are cyclized by using an oligonucleotide fragment of 5′-GCCATGTCGTTCTGTGAGCCAAGG-3′ (SEQ ID NO: 4).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a graph showing data of cycle number at plateau phase determined by qPCR reaction in Example 1.

[0056] FIG. 2 is a graph showing sample amplification curves in Example 1.

[0057] FIG. 3 is a graph showing Agilent 2100 detection results of part samples after double size selection by magnetic beads in Example 1.

[0058] FIG. 4 is a graph showing the correlation between the data obtained by sample sequencing in Example 1.

[0059] FIG. 5 is graphs showing the enrichment of transcription initiation regions of housekeeping gene and silent gene respectively among the fragments obtained after sequencing in Example 1.

[0060] FIG. 6 is a graph showing clustering results of sample sequencing data and tissue sequencing data in Example 1.

DETAILED DESCRIPTION

[0061] In order to facilitate understanding the present disclosure, the present examples herein are specified as below. Those skilled in the art should understand that the described examples are merely to promote understanding of the present disclosure, which should not be considered as a specific limitation to the present disclosure.

EXAMPLE 1

Library Construction of Cell-Free DNAs in Peripheral Blood and Analysis of Epigenetic Information

[0062] This example includes a series of steps including plasma sample preparation, direct transposition of the plasma sample by Tn5 transposase, DNA amplification after transposition, Tn5 library construction, PE50+10 sequencing, screening nucleosome fragments and the like. Tn5 transposase was prepared according to the instructions of TruePrep Mini DNA Sample Prep Kit from Vazyme Company.

[0063] 1.1 Plasma Sample Preparation

[0064] 1.1.1 Plasma Sample Collection

[0065] 10 mL of whole blood samples from healthy human were collected, centrifuged at 1600 g and 4° C. for 10 minutes, and the supernatant (i.e. plasma) was collected into a 15 mL new centrifuge tube.

[0066] 1.1.2 Plasma Purification

[0067] Plasma purification was conducted according to either of the following methods (Methods I and II).

[0068] 1.1.2.1 Method I—Filtration by filter head

[0069] 3 mL plasma was filtered by a 10 mL syringe equipped with 0.2 μm filter head and transferred into a new eppendorf (EP) tube, with 0.5 mL plasma lost.

[0070] 1.1.2.2 Method II—Twice centrifugation method

[0071] The plasma was centrifuged again at 16000 g and 4° C. for 10 minutes, and the supernatant was collected into a 15 mL new centrifuge tube.

[0072] 1.2 Transposition of Partial Plasma

[0073] Partial plasma was subjected to the transposition reaction according to the present method, in which the plasma was directly used for transposition reaction.

[0074] 1.2.1 Preparation of Adapter Mix

[0075] 1.2.1.1 Reference primers and sequences thereof are:

TABLE-US-00001 a primer A of (SEQ ID NO: 1) 5′-CTGTCTCTTATACACATCT-3′, a primer B of (SEQ ID NO: 2) 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′, and a primer C of (SEQ ID NO: 3) 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′.

[0076] 1.2.1.2 The primer A, primer B and primer C were dissolved to 100 μM by an annealing buffer.

[0077] 1.2.1.3 The following reaction systems were prepared, referring to Table 1.

TABLE-US-00002 TABLE 1 Preparation of Adapter Mix reaction systems Annealing reaction 1 Annealing reaction 2 primer A (100 μM) 10 μL primer A (100 μM) 10 μL primer B (100 μM) 10 μL primer C (100 μM) 10 μL Total volume 20 μL Total volume 20 μL

[0078] 1.2.1.4 A first annealing product (i.e. a first adapter) and a second annealing product (i.e. a second adapter) were respectively vortexed to thoroughly mix and centrifuged briefly to make the solution back the tube bottom, after that the tubes were placed in a PCR instrument and subjected to the following reaction procedures: 75° C. for 15 minutes; 60° C. for 10 minutes; 50° C. for 10 minutes; 40° C. for 10 minutes; and 25° C. for 30 minutes.

[0079] 1.2.1.5 After the completion of reaction, the first annealing product and the second annealing product in a same volume were mixed uniformly. The obtained mixture is named as Adapter Mix, stored at −20° C.

[0080] 1.2.2 The Adapter Mix was embedded with the Tn5 transposase to obtain a transposase-embedding complex.

[0081] 1.2.2.1 The following reaction components in Table 2 were respectively added into a sterilized PCR tube. In Table 2, the Tagment Enzyme Advanced V5S is BGI V5S reagent containing 1000U Tn5 transposase (Supplier: BGI, Catalog Number: BGE005S).

TABLE-US-00003 TABLE 2 Embedding system of Adapter Mix and Tn5 transposase Reagent Volume (μL) Tagment Enzyme Advanced V5S 98 Adapter Mix 4 Coupling Buffer V5S 98 Total volume 200

[0082] 1.2.2.2 The mixture in the tube was gently pipetted by a pipettor to thoroughly mix.

[0083] 1.2.2.3 The mixture in the tube was reacted at 25° C. for 60 minutes. The reaction product is named as the Tagment Enzyme Advanced Mix V5S, stored at −20° C.

[0084] 1.2.3 Fragmentation of Blood Cell-Free DNAs

[0085] A plasma transposition reaction system was prepared, referring to Table 3. The mixture was uniformly mixed after prepared on ice, and then subjected to the transposition reaction at 37° C. on a constant temperature metal mixer for 60 minutes.

TABLE-US-00004 TABLE 3 Plasma transposition reaction system Reagent Volume (μL) plasma 500 5×TAG buffer 140 Tagment Enzyme Advanced Mix V5S 10 PBS 50 Total volume 700

[0086] 1.3 Extraction of Plasma Cell-Free DNAs After Transposition Reaction

[0087] 1.3.1 The plasma cell-free DNAs after transposition reaction were extracted by using Magen blood cell-free DNA extraction kit (MAGEN MD5432-01).

[0088] 1.3.2 After the transposition reaction, 25 μL proteinase K and 35 μL MagBind magnetic beads were added to a 1.5 mL new centrifuge tube.

[0089] 1.3.3 The plasma transposition reaction system after transposition reaction obtained

[0090] 1.2.3were transferred to the tube containing proteinase K in 1.3.2 and then shaken for 5 seconds.

[0091] 1.3.4 700 μL of buffer MLE was added to the mixture in 1.3.3 and vortexed to mix, followed by incubated with shaking at 55° C. for 15 minutes.

[0092] 1.3.5 The tube in 1.3.4 was transferred to a magnetic separator, and stilled for 5 to 10 minutes for adsorption of magnetic beads.

[0093] 1.3.6 The solution in tube was carefully aspirated and discarded.

[0094] 1.3.7 320 μL of buffer MW1 was added into the tube, and vortexed to mix for 15 seconds.

[0095] 1.3.8 The tube in 1.3.7 was transferred to the magnetic separator, stilled for 3 to 5 minutes to adsorb magnetic beads, followed by aspirating the solution and discarded.

[0096] 1.3.9 320 μL of buffer MW2 was added into the tube, and vortexed to mix for 15 seconds.

[0097] 1.3.10 The tube in 1.3.9 was transferred to the magnetic separator, stilled for 3 to 5 minutes to adsorb magnetic beads, followed by aspirating the solution and discarded.

[0098] 1.3.11 Steps 1.3.9 and 1.3.10 were repeated.

[0099] 1.3.12 The tube was centrifuged briefly to concentrate the droplets on the tube wall, and then transferred to the magnetic separator, aspirating the solution and discarded.

[0100] 1.3.13 The tube was dried in air for 5 to 10 minutes.

[0101] 1.3.14 20 μL of buffer AE was added and pipetted to mix.

[0102] 1.3.15 The tube was stilled at room temperature for 3 minutes.

[0103] 1.3.16 The tube was transferred to the magnetic separator, stilled for 3 minutes to dissolve DNAs.

[0104] 1.3.17 The DNA solution was transferred to a 1.5 mL new centrifuge tube.

[0105] 1.3.18 The DNA concentration was detected by using the Qubit device.

[0106] 1.4 Amplification of fragmented DNAs

[0107] 1.4.1 A PCR reaction system was prepared in a 0.2 mL PCR tube according to Table 4.

TABLE-US-00005 TABLE 4 First amplification reaction system of transposition product Reagent Volume (μL) DNA solution 18 NEBNext High-Fidelity 2xPCR 25 Master Mix N5 primer (25 μM)  2.5 N7 primer (25 μM)  0.7 Total volume 20 *Note: the DNA solution for Example 1 is the purified DNA solution obtained in step 1.3.17, and the DNA solution for the following Comparative Example 1 is the purified DNA solution obtained in step 2.4. N5 primer: Pho-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC (SEQ ID NO: 5). N7 primer: TGTGAGCCAAGGAGTTGTTGTCTTCNNNNNNNNNNGTCTCGTGGGCTCGG (SEQ ID NO: 6),
in which, NNNNNNNNNN represents a label sequence consisting of 10 random bases, and the label sequence for each sample is different.

[0108] 1.4.2 The first amplification process was conducted according to the following parameters:

TABLE-US-00006 1 cycle 5 minutes 72° C. 30 seconds 98° C. 8 cycles 10 seconds 98° C. 30 seconds 63° C. 1 cycle 1 minute 72° C. holding  4° C.

[0109] 1.5 Q-PCR Determination and Second Amplification

[0110] 1.5.1 Preparation of Q-PCR reaction system (as shown in Table 5)

TABLE-US-00007 TABLE 5 qPCR reaction system for determination of cycle number to be used in the second amplification Reagent Volume (μL) DNA solution 4 2×SYBR ® Premix Ex Taq ™ II 10 Primer B (20 μM) 0.5 Primer C (20 μM) 0.5 ROX 0.08 NF-H.sub.2O 4.92 Total volume 20

[0111] 1.5.2 qPCR reaction was conducted according to the following parameters:

TABLE-US-00008 1 cycle 30 seconds 98° C. 40 cycles 10 seconds 98° C. 30 seconds 63° C. 1 minute 72° C.

[0112] As shown in FIG. 1, the cycle number corresponding to ⅓ of fluorescence intensity at plateau phase in the Rn/Cycle curve of qPCR linear amplification is the cycle number N to be used in the second amplification.

[0113] 1.5.3 The second amplification process was conducted according to the following parameters:

TABLE-US-00009 1 cycle 5 minutes 72° C. 30 seconds 98° C. N cycles 10 seconds 98° C. 30 seconds 63° C. 1 minute 72° C. 1 cycle 5 minutes 72° C. holding  4° C.

[0114] In which, N is the cycle number determined in step 1.5.2. The sample amplification curves are shown in FIG. 2.

[0115] 1.6 Double Size Selection by XP Magnetic Beads

[0116] 1.6.1 The volume of PCR tube was checked and made up to 50 μL with NF—H.sub.2O.

[0117] 1.6.2 40 μL magnetic beads (0.8×) were added to the PCR tube, mixed by pipetting and stilled at room temperature for 5 minutes.

[0118] 1.6.3 The PCR tube was placed on the magnetic separator for 2 minutes, and the supernatant was transferred to a new PCR tube, in which DNA fragments in the supernatant have a length less than 350 bp.

[0119] 1.6.4 35 μL magnetic beads (0.7×) were added, mixed uniformly and stilled at room temperature for 5 minutes.

[0120] 1.6.5 The PCR tube was placed on the magnetic separator for 2 minutes, and the supernatant containing small DNA fragments and RNAs was removed.

[0121] 1.6.6 The PCR tube was kept on the magnetic separator, and 150 μL of pre-cooled 80% ethanol was added to wash twice (30 seconds).

[0122] 1.6.7 The PCR tube was kept on the magnetic separator for 5 minutes to allow water evaporate.

[0123] 1.6.8 20 μL TE Buffer (AMBION AM9858) was added to elute DNAs, gently mixed by pipetting and incubated at room temperature for 5 minutes.

[0124] 1.6.9 The PCR tube was kept on the magnetic separator for 2 minutes, and the supernatant was transferred to a new PCR tube carefully, avoiding aspirating any magnetic beads.

[0125] 1.6.10 Qubit dsDNA High sensitivity assay kit (INVITROGEN Q32854) was used for DNA quantification.

[0126] 1.6.11 The resulting DNA was tested by using Agilent 2100, and the results are shown in FIG. 3.

[0127] 1.7 Library Cyclization and Enzyme Digestion

[0128] 1.7.1 The single-stranded mediator of 5′-GCCATGTCGTTCTGTGAGCCAAGG-3′ (SEQ ID NO: 4) was used.

[0129] 1.7.2 324.5 ng of the purified DNAs obtained in step 1.6 was mixed with 5 μL single-stranded mediator (20 μM), and was made up to 70 μL volume with sterile ultrapure water.

[0130] 1.7.3 The tube containing the DNA mixture obtained in step 1.7.2 was transferred to a thermal cycler, reacted at 95° C. for 3 minutes, and then quickly placed on ice for 10 minutes.

[0131] 1.7.4 The single-stranded DNA ligation reaction system was prepared according to Table 6, mixed uniformly and then quickly centrifuged for 3 seconds.

TABLE-US-00010 TABLE 6 Single-stranded DNA ligation reaction system Reagent Volume (μL) DNA mixture 70 10×TA buffer 12 100 mM ATP 1.2 T4 DNA ligase (600 U/μL) 0.42 NF-H.sub.2O 36.38 Total volume 120 * Note: DNA mixture in Table 6 is the solution obtained in step 1.7.3.

[0132] 1.7.5 The single-stranded DNA ligation reaction system obtained in step 1.7.4 was transferred to a thermal cycler, reacted at 37° C. for 60 minutes, and temporarily stored at 4° C.

[0133] 1.7.6 The enzyme digestion reaction system was prepared according to Table 7, mixed uniformly, and quickly centrifuged for 3 seconds.

TABLE-US-00011 TABLE 7 Single-stranded DNA enzyme digestion reaction system Reagent Volume (μL) DNA ligation mixture 120 10×TA buffer 0.8 EXO I (20 U/μL) 3.9 EXO III (100 U/μL) 1.3 F-H.sub.2O 2 Total volume 128 * Note: EXO I is exonuclease I and EXO III is exonuclease III in Table 7.

[0134] 1.7.7 The enzyme digestion reaction system obtained in step 1.7.6 was transferred to a thermal cycler, reacted at 37° C. for 30 minutes, and temporarily stored at 4° C.

[0135] 1.8 Library Collection

[0136] 1.8.1 After vortexed to mix, 170 μL of PEG32 magnetic beads were added into 128 μL of PCR product obtained in step 1.7, gently pipetted for 10 times by a pipettor to thoroughly mix, and then incubated at room temperature for 10 minutes.

[0137] 1.8.2 The EP tube was briefly centrifuged and placed on the magnetic separator to separate magnetic beads and solution, stilled for 5 minutes to clear the solution which was carefully removed.

[0138] 1.8.3 200 μL of freshly prepared 80% ethanol was added to the EP tube kept staying on the magnetic separator for rinsing the magnetic beads, and the supernatant was carefully removed after incubating at room temperature for 30 seconds.

[0139] 1.8.4 The step 1.8.3 was repeated, in total of twice rinse by 80% ethanol.

[0140] 1.8.5 The EP tube was kept staying on the magnetic separator, and the lip was opened to dry in air for 10 minutes.

[0141] 1.8.6 The EP tube was taken out from the magnetic separator, and 25 μL of sterile ultrapure water was added to elute DNAs, followed by gently pipetting by a pipettor to thoroughly mix. After stilled at room temperature for 5 minutes, the EP tube was briefly centrifuged and placed on the magnetic separator to separate the magnetic beads and solution. After cleared (about 5 minutes), the supernatant was carefully drawn into a new EP tube and store at −20° C.

[0142] 1.8.7 1 μL of purified product was detected for ssDNA concentration.

[0143] 1.9 Sequencing by a Sequencer

[0144] 6 ng of constructed library obtained in step 1.8 was used to prepare DNA nanoballs (DNBs) through Rolling circle amplification (RCA) for 20 minutes according to the instructions of the BGI-SEQ500 sequencer, and subsequently sequenced on the sequencer via conventional PE50+10 strategy.

[0145] It should be noted that the blood cell-free DNA library obtained in the present disclosure can be sequenced through low-depth sequencing.

[0146] 1.10 Sequencing Data Analysis

[0147] 1.10.1 A Fastq file of sequencing data was subjected to quality filtering and alignment, after that a Bam file was obtained.

[0148] 1.10.2 The full-length and position information of each fragment was obtained through pairing the double-ended fragments of the sequencing data.

[0149] 1.10.3 Large fragments above 60 bp were screened out by filtering based on the fragment size. The correlation between the samples where the large fragments are derived, and the enrichment of these fragments in promoter and enhancer regions of different genes were calculated. The results are shown in FIGS. 4 and 5.

[0150] 1.10.4 These fragment data and the DNase-seq data of different human tissues from the ENCODE database were subjected to cluster analysis, and the results are shown in FIG. 6.

COMPARATIVE EXAMPLE 1

[0151] In this comparative example, the prior art method was conducted as follows, that is, the cell-free DNAs in blood were extracted firstly before the transposition reaction.

[0152] 2.1 Extraction of Blood Cell-Free DNAs Before Transposition Reaction

[0153] 2.1.1 The blood cell-free DNAs were directly extracted from the purified plasma obtained in step 1.2 of Example 1, specifically referring to step 1.3 of Example 1.

[0154] 2.1.2 Tn5 transposition reaction system was formulated on ice according to Table 8, mixed uniformly and subjected to transposition reaction at 37° C. on the constant temperature metal mixer for 30 minutes. During the transposition reaction, the Tn5 transposition reaction system was gently shaken for several times.

TABLE-US-00012 TABLE 8 Transposition reaction system for extracted blood cell-free DNAs Reagent Volume (μL) Extracted blood cell-free DNAs 17 5xTAG buffer 6 Tn5 V5S 1 PBS 6 Total volume 30

[0155] 2.1.3 Termination Reaction

[0156] 7.5 μL of 5×NT buffer was added, gently pipetted for 20 times and stayed at room temperature for 5 minutes.

[0157] 2.1.4 Collection of DNAs by 1.8× Magnetic Beads

[0158] 2.1.4.1 XP magnetic beads (AGENCOURT A63882) were taken out from the refrigerator at 4° C., mixed uniformly and placed at room temperature for 10 minutes.

[0159] 2.1.4.2 1.8 × XP magnetic beads were added into the tube in step 2.1.3, mixed uniformly by pipetting for 10 times, and stilled at room temperature for 5 minutes.

[0160] 2.1.4.3 The tube was placed on the magnetic separator, stilled for 2 minutes, such that the magnetic beads were absorbed on the magnetic separator and the solution becomes clear.

[0161] 2.1.4.4 The supernatant was removed while 5 μL of supernatant can be left to ensure no magnetic beads aspirated.

[0162] 2.1.4.5 150 μL of 80% ethanol was added and stilled for 30 seconds, after that the supernatant was discarded.

[0163] 2.1.4.6 The step 6.5 was repeated and the ethanol solution was maximally removed. The tube was dried in air until the magnetic beads are non-reflective.

[0164] 2.1.4.7 The EP tube was taken out from the magnetic separator, 24 μL of nanofiltration (NF) water was added to dissolve DNAs, pipetted for 10 times to mix uniformly, stilled at room temperature for 3 minutes.

[0165] 2.1.4.8 The EP tube was placed on the magnetic separator and stilled for 1 minute, after that the solution becomes clear.

[0166] 2.1.4.9 The supernatant was transferred to a new EP tube.

[0167] 2.1.4.10 Qubit dsDNA High sensitivity assay kit (INVITROGEN Q32854) was used for DNA quantification.

[0168] The DNA product obtained in step 2.1.4 of Comparative Example 1 was subsequently subjected to steps 1.4 to 1.10 of Example 1 to analyze the enrichment of large fragments above 60 bp from the library constructed in Comparative Example 1 in promoter and enhancer regions of different genes. The results are shown in FIG. 4.

[0169] As shown in FIG. 4, it can be clearly found that the correlation between samples obtained by the present method (Example 1) is significantly higher than the correlation between samples obtained by the prior art method (Comparative Example 1) after comparing and analyzing the results of Example 1 and Comparative Example 1.

[0170] As shown in FIG. 5, the present method (Example 1) exhibits a significantly increased region enrichment effect than the prior art method (Comparative Example 1).

[0171] As shown in FIG. 6, the fragment data obtained by the present method and the existing data of different human tissues can be clustered together according to cluster methods, indicating the present method is capable of capturing the cell-free DNA information from different human tissues, thus further for tissue traceability.

[0172] The applicant states that the detailed method of the present application is illustrated by the examples as described above, however the present application is not limited to this detailed method. That is, it does not mean that the present application has to rely on the detailed method to implement. Those skilled in the art should understand that any improvements to this application, the equivalent replacement of various raw materials of the product of this application, the addition of auxiliary components, the choice of specific means and the like all fall within the scope of protection and disclosure of this application.