Low-frequency mutations enrichment sequencing method for free target DNA in plasma

Abstract

The present invention provides a low-frequency mutation enrichment sequencing method for free target DNA in plasma, comprising plasma DNA extraction and library construction, general library TT COLD PCR amplification enrichment, probe enrichment capture, PCR and sequencing of captured products, and positive and negatice double-strand error-correction low-frequency information analysis.

Claims

1. A method for identifying low-frequency mutations in a target DNA present in plasma obtained from human peripheral blood, comprising the following steps: (1) extracting the target DNA from the plasma and constructing a DNA library containing fragments from the extracted target DNA; (2) amplifying and enriching such DNA fragments in the library containing predetermined mutations by first determining the Tm value of the library followed by universal library TT-COLD PCR; (3) enriching for fragments containing low-frequency mutations using hybridization capture probes and then amplifying and sequencing the resulting hybridization capture products which include fragments containing low-frequency mutations; and (4) analyzing the sequences of the fragments containing the low-frequency mutations using a forward and reverse double-strand error correction procedure so as to identify low-frequency mutations in the target DNA.

2. The method according to claim 1, wherein the DNA library in step (1) is constructed according to a three-step enzymatic reaction comprising terminal repair, addition of “A”, and library linker ligation.

3. The method according to claim 1, wherein step (2) comprises bypassing the specific Tc values for each DNA fragment in the library, enriching all the DNA fragments in the library using a pair of universal primers under one serial cycling condition, setting Tc min≈TM-2.5, followed by a gradual increase in Tc at a rate of 0.5° C., and performing full cold PCR under each Tc condition, respectively.

4. The method according to claim 1, wherein the Tm value of the library in step (2) is determined by a method comprising the following: a) amplifying by fluorescence quantitative PCR free target DNA from normal human plasma in a ligation library using a pair of primers to obtain a melting curve, and b) obtaining the Tm value of the library from analysis of the melting curve; wherein the nucleotide sequence of the pair of primers is the following: TABLE-US-00031 upstream primer: (SEQ ID NO: 3) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT TCCGATCT, and downstream primer: (SEQ ID NO: 4) CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTGGAGTTCAGACG TGTGCTCTTCCGATCT, where xxxxxxxx is an index tag.

5. The method according to claim 3, wherein the pair of universal primers in step (2) is a pair of universal library TT-COLD PCR primers, the nucleotide sequence of which is the following: TABLE-US-00032 upstream primer: (SEQ ID NO: 3) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT TCCGATCT, and downstream primer: (SEQ ID NO: 4) CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTGGAGTTCAGACG TGTGCTCTTCCGATCT, where xxxxxxxx is an index tag.

6. The method according to claim 3, wherein the one serial cycling condition is in the following: TABLE-US-00033 98° C. 30 sec — 98° C. 10 sec 3 cycles 55-65° C. 15 sec 72° C. 30 sec 98° C. 10 sec 4 cycles 70° C. 2 min Tc1 = TM-2° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec . . . Tc2; Tc3; Tc4 4 cycles/Tc (ΔT = 0.5° C.) 98° C. 10 sec 4 cycles 70° C. 2 min Tc5 = TM-0° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec 72° C. 2 min — 4° C. Storage  —.

7. The method according to claim 1, wherein the enriching in step (3) comprises using an enrichment probe chip for hybridization capture after the amplified library is qualified in quality control and the hybridization capture products are amplified and sequenced; wherein the enrichment probe chip is designed according to the following: the capture range of the chip is determined based on the purpose of the target gene, at least one hotspot mutation site is determined within a certain base range with reference to the database to which the target DNA belongs, with several primary types of mutations among multiple mutation types present with respect to this site taken for reference, and the corresponding frequency of occurrence is used as the proportion occupied by the mutation type in the total probe coverage level at the site, wherein a probe designed based on the human genome reference sequence hg19 is replaced with a probe designed based on a mutant base of the hotspot mutation, with the probes for other sites remaining unchanged, and the difference ratio between the total coverage of the probe for the hotspot mutation and the coverage of normal probe for the other sites is not less than 3:1, so as to achieve enrichment of the hotspot mutation during capture.

8. The method according to claim 1, wherein the forward and reverse double-strand error correction procedure in step (4) is the following: 1) based on the sequencing results, the first 12 bp bases of a tested sequence 1 and the first 12 bp bases of a tested sequence 2 of paired tested sequences are cut as tags, arranged according to alphabetical order, and connected having to form an index of 24 bp, while forward and reverse strands are selected according to the arrangement and combination of the tags of the tested sequences; 2) external sorting is carried out on the index to achieve the purpose of gathering together all the tested sequences of the same DNA template; 3) center clustering is carried out on the gathered tested sequences having the same index, each large cluster with the same index is gathered into small clusters according to the Hamming distance between the sequences, with the Hamming distance between any two pairs of paired tested sequences in each small cluster not exceeding 10, so as to achieve the purpose of distinguishing tested sequences having the same index but coming from different DNA templates; 4) repeated clusters of the same DNA template obtained in step 3) are screened, and if the numbers of tested sequences of the forward strand and the reverse strand both reach two pairs or more, subsequent analysis is performed; 5) the clusters that satisfy the conditions in 4) are corrected to generate a pair of error-free new tested sequences, and for each sequenced base in the DNA template, if a certain base type of the sequenced base in the tested sequence of the forward strand reaches a consistence rate of 80% and in the tested sequence of the reverse strand also reaches a consistence rate of 80%, the base type for this base in the new tested sequence is recorded as this base type, thereby obtaining a new tested sequence which represents the original DNA template sequence; 6) the new tested sequence is aligned again with the genome by bwa mem algorithm, and a tested sequence with an alignment quality of less than 30 is screened out; 7) statistics is carried out based on the tested sequences obtained in step 6) to obtain the base type distribution for each site, the coverage of the statistical target region, the average sequencing depth, the forward and reverse strand matching ratio, and the low-frequency mutation rate in the capture region; 8) SNV/InDel/SV/CNV is called based on the alignment of information between the sample from a patient and a control, mutect process is used to call somatic SNV mutation, gatk process is used to call somatic InDel mutation, contra.py process is used to call CNV, and som Var process is used to call SV, wherein the screening parameters used are the following: control site mutation rate ≤2%, the number of varied tested sequences after error correction ≥2, and mutation prediction p value ≤0.05; and 9) the varied function, the support number of the varied tested sequence, the frequency of mutation, amino acid mutation, and the condition of such mutation in an existing mutation database are annotated.

9. The method according to claim 8, wherein in step 1), based on the sequence bases at the two ends of an inserted fragment, each fragment will form a pair of paired tested sequences by paired-end sequencing; the first 12 bp bases of a tested sequence 1 and the first 12 bp bases of a tested sequence 2 of paired tested sequences are taken as tags, arranged according to alphabetical order, and connected to form an index of 24 bp; and using the 24 bp as an index of the paired tested sequences, a strand is marked as a forward strand if the tag of the tested sequence 1 is in the front and a strand is marked as a reverse strand if the tag of the tested sequence 2 is in the front.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a process chart of the method of the present invention.

(2) FIG. 2 shows the Tm values of normal human plasma ligation libraries.

BEST MODE OF THE INVENTION

(3) The following examples further illustrate the present invention, but should not be construed as limitation to the present invention. Any modification or substitution of the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention.

(4) Unless otherwise specified, all the chemical reagents used in the examples are conventional commercially-available reagents. The technical means used in the examples are conventional ones known to those skilled in the art. The sequencing device used in the examples of the present invention is the Illumina HiSeq2500. In the sequencing step of the present invention, the sequencing device is not limited to the above sequencing device.

(5) In the examples of the present invention, all gene names adopt official symbols in NCBI-Gene. The synonymous mutation in the present invention means that the codon representing an amino acid is mutated to other codons due to a change of a certain base, but said other codons still encode the same amino acid. Missense mutation means that a codon encoding a certain amino acid becomes a codon encoding another amino acid after substitution of a base, so that the type of amino acids and the sequence of the polypeptide chain are changed. Some missense mutations can make the polypeptide chain lose its original function, and many protein abnormalities are caused by missense mutations. a mutation resulting in a termination codon, also referred to as nonsense mutation, means that a codon representing an amino acid is mutated to a termination codon due to a change of a certain base, so that the synthesis of a peptide chain is terminated in advance. A mutation resulting in the loss of a termination codon according to the present invention means that a termination codon is mutated to other codons due to a change of a certain base, so that the synthesis of a peptide chain cannot be terminated normally.

Example 1 Method for Enrichment and Sequencing of Low-Frequency Mutations of Target DNA in Plasma (ER-Seq Method)

(6) (1) Extraction of target DNA from plasma and construction of a library. The plasma was derived from human peripheral blood and the method for library construction was performed according to a three-step enzymatic reaction, i.e. terminal repair, addition of “A” and library linker ligation. The primers for the library linker were provided as follows:

(7) The first strand of the linker: TACACTCTTTCCCTACACGACGCTCTTCCGATCT,

(8) The second strand of the linker: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC.

(9) (2) Universal library TT-COLD PCR amplification and enrichment. It comprised the following steps:

(10) 1) determining the Tm value of the library; the Tm value of the library was determined by the following method: fluorescence quantitative PCR was performed on the library of the target DNA in plasma using one pair of primers, and analysis was carried out according to melting curve to obtain the Tm value of the library; the sequence of the primers were provided as follows:

(11) upstream primer:

(12) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGA TCT, and

(13) downstream primer:

(14) CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTGGAGTTCAGACGTGTGC TCTTCCGATCT, wherein, xxxxxxxx is an index tag;

(15) 2) bypassing specific Tc values present for each inserted fragment, enriching various types of mutations on all fragments in the library based on one pair of universal primers under one serial cycling condition; setting Tc min≈TM−2.5, followed by a gradual increase in Tc at a rate of 0.5° C., and performing FULL COLD PCR under each Tc condition, respectively;

(16) the one pair of universal primers was universal library TT-COLD PCR primer, and its nucleotide sequence was: upstream primer: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, and downstream primer: CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT, wherein, xxxxxxxx was an index tag;

(17) the one serial cycling condition was:

(18) TABLE-US-00010 98° C. 30 sec — 98° C. 10 sec 3 cycles 55-65° C. 15 sec 72° C. 30 sec 98° C. 10 sec 4 cycles 70° C. 2 min Tc1 = TM-2° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec . . . Tc2; Tc3; Tc4 4 cycles/Tc (ΔT = 0.5° C.) 98° C. 10 sec 4 cycles 70° C. 2 min Tc5 = TM-0° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec 72° C. 2 min — 4° C. Storage  —.

(19) (3) Enrichment and capture with probes, and amplification and sequencing of products captured by hybridization, The enrichment and capture with probes in step (3) referred to using an enrichment probe chip for capture via hybridization after the amplified library was qualified in quality control, and the products captured by hybridization were subjected to PCR amplification and then on-machine sequencing;

(20) the design method for the enrichment probe chip was set as follows: the capture range of the chip was determined based on the purpose of the target gene, at least one most important hotspot mutation site was determined within a certain base range with reference to the database to which the target DNA belongs, several primary types of mutations among multiple mutation types present at this site were taken for reference, corresponding frequency of occurrence was used as the proportion occupied by the mutation type in the total probe coverage level at the site; with respect to the hotspot mutation, a probe designed based on a human genome reference sequence hg19 was replaced with a probe designed based on a mutant base, the probes for other sites were maintained unchanged, and the difference ratio between the total coverage of the probe for hotspot mutation and the coverage of the normal probe for other regions was not less than 3:1, so as to achieve enrichment of hotspot mutation during capture.

(21) (4) The specific method for analysis on low-frequency information with forward and reverse double-strand error correction (RealSeq Pipeline) was provided as follows:

(22) 1) based on the sequence bases at two ends of an inserted fragment, (which was a DNA fragment linked with the linker primer in the library) as tags, each fragment formed a pair of paired tested sequences by paired-end sequencing; the first 12 bp bases of tested sequence 1 and the first 12 bp bases of tested sequence 2 of the paired tested sequences were taken as tags, arranged according to alphabetical order, and connected having smaller tags in the front to form an index of 24 bp; using the 24 bp as an index of the paired tested sequences, the strand was marked as a forward strand if the tag of the tested sequence 1 is in the front, and strand was marked as a reverse strand if the tag of the tested sequence 2 is in the front;

(23) 2) external sorting was carried out on the index to achieve the purpose of gathering together all the tested sequences of the same DNA template;

(24) 3) center clustering was carried out on the gathered tested sequences having the same index, each large cluster with the same index was gathered into several small clusters according to the Hamming distance between the sequences, with the Hamming distance between any two pairs of paired tested sequences in each small cluster not exceeding 10, so as to achieve the purpose of distinguishing tested sequences having the same index but coming from different DNA templates;

(25) 4) repeated clusters of the same DNA template obtained in step 3) was screened; if the numbers of tested sequences of the forward strand and the reverse strand both reached two pairs or more, subsequent analysis was performed;

(26) 5) the clusters that satisfy the conditions in 4) were corrected to generate a pair of error-free new tested sequences; for each sequenced base in the DNA template, if a certain base type of the sequenced base in the tested sequence of the forward strand reached a consistence rate of 80%, and in the tested sequence of the reverse strand also reached a consistence rate of 80%, the base type for this base in a new tested sequence was recorded as this base type, otherwise recorded as N, thereby obtaining the new tested sequence which represents the original DNA template sequence;

(27) 6) the new tested sequence was aligned again with the genome by bwa mem algorithm, and the tested sequence with an alignment quality of less than 30 was screened out;

(28) 7) statistics was carried out based on the tested sequences obtained in step 6) to obtain the base type distribution for each site, the coverage of the statistical target region, the average sequencing depth, the forward and reverse strand matching ratio, and the low-frequency mutation rate in the capture region;

(29) 8) Call SNV/InDel/SV/CNV: based on the alignment of information between the sample from a patient and a control, the mutect process was used to call somatic SNVmutation; the gatk process was used to call somatic InDel mutation; the contra.py process was used to call CNV with; and the som Var process was used to call SV;

(30) the screening parameters used were: control site mutation rate ≤2%; the number of varied tested sequences after error correction ≥2; mutation prediction p value ≤0.05; and

(31) 9) Mutation Annotation: the varied function, the support of the varied tested sequence, the frequency of mutation, amino acid mutation, and the condition of such mutation in an existing mutation database were annotated.

Example 2 Establishment of Enrichment and Sequencing Method for Low Frequency Mutation of ctDNA in Plasma

(32) 1. Extraction of ctDNA from Plasma and Library Construction

(33) (1) 1-2 tubes (5 mL/tube) of the peripheral blood was drawn from the subject into an EDTA anticoagulant tube, gently shaken upside down (to prevent cell rupture) 6-8 times to mix thoroughly, and subjected to the following treatment within 4-6 hours on the day of blood sampling: the sample was centrifuged at 4° C. and 1600 g for 10 minutes; after centrifugation, the supernatant (plasma) was dispensed into a plurality of 1.5 mL/2 mL centrifuge tubes, and the middle layer of leukocytes could not be sucked during the sucking; centrifugation was carried out at 4° C. and 1600 g for 10 minutes, the remaining cells were removed, and the supernatant (plasma) was transferred to a new 1.5 mL/2 mL centrifuge tube, during which process the leukocytes at the bottom of the tube could not be sucked, to obtain the desired separated plasma; after treatment of plasma samples was finished, the resulting plasma and remaining blood cells were stored in a refrigerator at −80° C. to avoid repeated freezing and thawing.

(34) (2) Extraction and quantitation of plasma cfDNA/ctDNA: approximately 2-3 ml of the separated plasma was taken, and plasma cfDNA was extracted therefrom according to the extracting reagent instruction of QIAamp Circulating Nucleic Acid Kit (Qiagen). The extracted DNA was quantified by Qubit (Invitrogen, the Quant-iT™ dsDNA HS Assay Kit), and the total amount was about 30-50 ng.

(35) (3) Preparation of a library of the sample: the cfDNA extracted from plasma was subjected to 3-step enzymatic reaction according to the instruction for library construction of KAPA LTP Library Preparation Kit.

(36) 3.1 Terminal Repair

(37) TABLE-US-00011 DNA sample 50 μL Terminal repair pre-mixed solution 20 μL water 8 μL 10 × KAPA terminal repair buffer 7 μL KAPA terminal repair enzyme mixture 5 μL total volume 70 μL

(38) The materials were mixed well and incubated at 20° C. for 30 min.

(39) After that, 120 μL of Agencourt AMPure XP reagent was added, and the mixture was purified with beads, and finally dissolved in 42 μL of ddH.sub.2O, and subjected to the next step of reaction with the beads.

(40) 3.2 Addition of A

(41) TABLE-US-00012 A-tailing reaction pre-mixed solution: 50 μL water + DNA 42 μL 10 × KAPA A-Tailing Buffer(Blue) 5 μL (A-tailing reaction buffer) KAPA A-Tailing Enzyme(Blue) 3 μL (enzyme for A-tailing reaction) Total volume 50 μL

(42) The materials were mixed well and incubated at 30° C. for 30 min

(43) After that, 90 μL of PEG/NaCl SPRI solution was added, and mixed thoroughly; and the mixture was purified with beads, and finally dissolved in (35-linker)μL of ddH.sub.2O, and subjected to the next step of reaction with the beads.

(44) 3.3 Linker Ligation

(45) TABLE-US-00013 Ligation pre-mixed solution 45 μL water (35-linker) μL 5 × KAPA ligation buffer 10 μL KAPA T4 DNA ligase 5 μL 15 μM linker(50:1) final concentration 10 nM/ng Total volume 50 μL

(46) The materials were mixed well and incubated at 16° C. for 16 hours.

(47) With respect to the linker primer, please refer to Table 1 for the first and second strands of the linker. After that, 50 μL of PEG/NaCl SPRI solution was added twice, and the mixture was purified with beads twice, and finally dissolved in 25 μL of ddH.sub.2O.

(48) 2. Universal Library TT-COLD PCR:

(49) 1) Fluorescent quantitative PCR was performed using universal library primers for normal human plasma ligation libraries based on the same instruments and reagents, and the reaction reagents included KAPA HiFi HotStart ReadyMix and SYBR dye. By analysis of the melting curve, the Tm value (DNA melting temperature) of the library was obtained, as shown in FIG. 2. The universal library primer is shown in Table 1.

(50) TABLE-US-00014 TABLE 1 Primer sequence information primer Sequence information (5′-3′) First TACACTCTTTCCCTACACGACGCTCTTCCGATCT strand of the linker Second GATCGGAAGAGCACACGTCTGAACTCCAGTCAC strand of the linker Universal AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT library ACACGACGCTCTTCCGATCT upstream primer Universal CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTG library GAGTTCAGACGTGTGCTCTTCCGATCT downstream primer Note: xxxxxxxx: index tag

(51) 2) Universal Library TT COLD PCR: the reaction system was:

(52) TABLE-US-00015 Plasma ctDNA ligation library: 20 μL 2 × KAPA HiFi HotStart Ready Mix 25 μL 10 μM universal library upstream primer 2.5 μL 10 μM universal library downstream primer 2.5 μL Total volume 50 μL

(53) The above materials were mixed well.

(54) By bypassing the specific Tc values present for each inserted fragment, various mutations on all the fragments in the library were enriched based on the 1 pair of universal library primers shown in Table 1 under 1 serial cycling condition. Specifically, the method was obtaining Tc min≈TM−2.5 by an empirical formula, followed by a gradual increase in Tc at a rate of 0.5° C., and FULL COLD PCR was performed under each Tc condition. PCR reaction program settings are shown in Table 2.

(55) TABLE-US-00016 TABLE 2 98° C. 30 sec — 98° C. 10 sec 3 cycles 55-65° C. 15 sec 72° C. 30 sec 98° C. 10 sec 4 cycles 70° C. 2 min Tc1 = TM-2° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec . . . Tc2; Tc3; Tc4 4 cycles/Tc (ΔT = 0.5° C.) 98° C. 10 sec 4 cycles 70° C. 2 min Tc5 = TM-0° C. 20 sec 55-65° C. 15 sec 72° C. 30 sec 72° C. 2 min — 4° C. Storage  —.

(56) 3. Enrichment and Capture with Probes and On-Machine Sequencing

(57) 1) Design of Enrichment Probe Chip for Tumor:

(58) The capture range of the chip was determined based on TCGA, ICGC, COSMIC and like databases and relevant reference documents, with reference to the design principle for conventional capture probes for chips;

(59) In the capture range, at least one most important hotspot mutation site (SNV>3) was determined for each 200 bps with reference to TCGA, COSMIC and other relevant databases; several primary mutation types among multiple mutation types present at this site were taken for reference, and corresponding frequency of occurrence was used as the proportion occupied by the mutation type in the total probe coverage level at the site;

(60) When the chip was designed, with respect to relevant hotspot mutation, a probe designed based on REF was replaced with a probe designed based on a mutant base, other probes were maintained unchanged, and the difference ratio between the total coverage of the probe for hotspot mutation and the coverage of normal probe for other regions was at least 3:1, so as to achieve enrichment of hotspot mutation during capture.

(61) 2) After amplification, library was subjected to quality control and enrichment-probe capture, followed by amplification and on-machine sequencing of products captured via hybridization.

(62) After the amplified library was qualified in quality control, the above enrichment probe chips for tumor were used for capture via hybridization according to the instructions provided by the chip manufacturer (Roche). Finally, the resulting material was eluted and dissolved in 21 μL ddH.sub.2O, with beads subjected to hybridization and elution.

(63) Amplification System for Products Captured Via Hybridization:

(64) TABLE-US-00017 Products captured via hybridization 20 μL 2 × KAPA HiFi HotStart Ready Mix 25 μL FellowCell Primer 1 2.5 μL FellowCell Primer 2 2.5 μL Total volume 50 μL

(65) PCR reaction conditions: initial denaturation at 98° C. for 45 sec; denaturation at 98° C. for 15 sec, annealing at 65° C. for 30 sec, extension at 72° C. for 30 sec, totaling 10 cycles; extension at 72° C. for 60 sec, storage at 4° C.

(66) FellowCell Primer 1 and Primer 2 were primers contained in the Hiseq on-machine test platform, which were used for amplifying the captured DNA template to obtain enough output to meet the requirements of on-machine sequencing.

(67) The beads from the previous step were removed first, and then 50 μL of Agencourt AMPure XP reagent was added again. The mixture was purified with beads, and finally dissolved in 25 μL ddH.sub.2O and subjected to QC and on-machine sequencing. Illumina HiSeq 2500 PE101+8+101 program was used for on-machine sequencing. In sequencing experimental operation, operations for on-machine sequencing were carried out according to the manufacturer's instructions (see cBot officially published by Illumina/Solexa).

(68) 4. Analysis on Low-Frequency Information with Forward and Reverse Double-Strand Error Correction (RealSeq Pipeline Method):

(69) 1) based on the sequence bases at two ends of an inserted fragment (which was a DNA fragment linked with the linker primer in the library) as tags, each fragment formed a pair of paired tested sequences by paired-end sequencing; the first 12 bp bases of tested sequence 1 and the first 12 bp bases of tested sequence 2 of paired tested sequences were taken as tags, arranged according to alphabetical order, and connected having smaller tags in the front to form an index of 24 bp; using the 24 bp as an index of the paired tested sequences, a strand was marked as a forward strand if the tag of the tested sequence 1 is in the front, and a strand was marked as a reverse strand if the tag of the tested sequence 2 is in the front;

(70) 2) external sorting was carried out on the index to achieve the purpose of gathering together all the tested sequences amplified from the same DNA template;

(71) 3) center clustering was carried out on the gathered tested sequences having the same index, each large cluster with the same index was gathered into several small clusters according to the Hamming distance between the sequences, with the Hamming distance between any two pairs of paired tested sequences in each small cluster not exceeding 10, so as to achieve the purpose of distinguishing tested sequences having the same index but coming from different DNA templates;

(72) 4) repeated clusters of the same DNA template obtained in step 3) was screened; if the numbers of tested sequences of the forward strand and the reverse strand both reached two pairs or more, subsequent analysis was performed;

(73) 5) the clusters that satisfy the conditions in 4) were corrected to generate a pair of error-free new tested sequences; for each sequenced base in the DNA template, if a certain base type for the base in the tested sequence of the forward strand reached a consistence rate of 80%, and in the tested sequence of the reverse strand also reached a consistence rate of 80%, the base type for this base in a new tested sequence was recorded as this base type, otherwise recorded as N, thereby obtaining the new tested sequence which represents the original DNA template sequence;

(74) 6) the new tested sequence was aligned again with the genome by bwa mem algorithm, and the tested sequence with an alignment quality of less than 30 was screened out;

(75) 7) statistics was carried out based on the tested sequences obtained in step 6) to obtain the base type distribution for each site, the coverage of the statistical target region, the average sequencing depth, the forward and reverse strand matching ratio, and the low-frequency mutation rate in the capture region;

(76) 8) Call SNV/InDel/SV/CNV: based on the alignment of information between the sample from a patient and a control sample, the mutect process was used to call somatic SNV mutation; the gatk flow was used to call somatic InDel mutation; the contra.py flow was used to call CNV with; and the som Var flow was used to call SV;

(77) the screening parameters used were: mutation rate for a control site ≤2%; the number of varied tested sequences after error correction ≥2; mutation prediction p value ≤0.05; and

(78) 9) Mutation Annotation: the varied function, the support number of the varied tested sequence, the frequency of mutation, amino acid mutation, and the condition of such mutation in an existing mutation database were annotated.

Example 3 Early Screening of Tumor

(79) 1. Chip Design

(80) A chip, ONCOcare-ZS, for early screening of tumors (lung cancer, colorectal cancer, gastric cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer and liver cancer, etc.) was completed based on the design principle of enrichment probe chips. The chip includes Driver Genes related with common cancers with high incidence, high-frequency mutation genes, important genes in 12 cancer-related signaling pathways, totaling 227 genes, 680 Kb, 5220 hotspot mutation. The gene list is shown in Table 3.

(81) TABLE-US-00018 TABLE 3 Gene list of the early-screening chip ONCOcare-ZS ABL1 BMPR1A CREBBP FAT1 IDH2 MLH1 PDGFRA SETD2 VHL ABL2 BRAF CRKL FBXW7 IGF1R MLH3 PDGFRB SF3B1 WT1 ACVR1B BRCA1 CRLF2 FGFR1 IL7R MLL2 PHF6 SMAD2 XPO1 AKT1 BRCA2 CSF1R FGFR2 INSRR MLL3 PIK3CA SMAD4 AKT2 BRD4 CTNNB1 FGFR3 IRS2 MPL PIK3CB SMARCA4 AKT3 BRIP1 CYLD FGFR4 JAK1 MRE11A PIK3R1 SMARCB1 ALK C11orf30 DAXX FH JAK2 MSH2 PMS1 SMO APC CARD11 DDR2 FLCN JAK3 MSH6 PMS2 SOCS1 AR CASP8 DNMT1 FLT1 KDM5C MSR1 PPP2R1A SOX9 ARAF CBL DNMT3A FLT3 KDM6A MTOR PRDM1 SPOP ARID1A CCDC6 EGFR FLT4 KDR MUTYH PTCH1 SRC ARID1B CCND1 ELAC2 FOXL2 KIF1B MYD88 PTEN SRSF2 ARID2 CCND2 EP300 FUBP1 KIT NBN PTPN11 STAG2 ASXL1 CCND3 EPCAM GAB2 KLF4 NCOR1 QC21 STAT3 ATM CCNE1 EPHA2 GATA1 KMT2A NF1 RAD50 STK11 ATR CDC73 EPHA3 GATA2 KRAS NF2 RAD51C SYK ATRX CDH1 EPHA5 GATA3 MAP2K1 NFE2L2 RAF1 TERT AURKA CDK4 ERBB2 GNA11 MAP2K2 NOTCH1 RARA TET2 AURKB CDK6 ERBB3 GNAQ MAP3K1 NOTCH2 RB1 TMEM127 AXIN1 CDK8 ERBB4 GNAS MAPK1 NOTCH3 RET TNFAIP3 AXIN2 CDKN1A ERCC3 H3F3A MAX NPM1 RNASEL TOP1 AXL CDKN1B ERG HGF MCL1 NRAS RNF43 TP53 B2M CDKN2A ESR1 HIST1H3B MDM2 NTRK1 ROS1 TRAF7 BAP1 CDKN2B EWSR1 HNF1A MDM4 NTRK3 RUNX1 TSC1 BARD1 CEBPA EXT1 HRAS MED12 PALB2 SDHAF2 TSC2 BCL2 CHEK1 EXT2 HSD17B3 MEN1 PAX5 SDHB TSHR BCOR CHEK2 EZH2 HSD3B2 MET PBRM1 SDHC U2AF1 BCR CIC FAM123B IDH1 MITF PCM1 SETBP1 VEGFA

(82) 2. Analysis of Sequencing Result

(83) One patient with small pulmonary nodules was subjected to sequencing and analysis according to the method described in Example 1, wherein the chip ONCOcare-ZS of the present example was used in the step of enrichment and capture with probes. The statistical results of the sequencing data are shown in Table 4 below:

(84) TABLE-US-00019 TABLE 4 sequencing results Matching rate of Low-frequency Sample Total data the forward and error-correction Effective data name Size of nodules output(G) reverse strands depth utilization ratio CD148 0.7 cm × 0.5 13.2 76.62% 1153.6X 3.78% cm × 0.3 cm Note: Matching rate of the forward and reverse strands: the ratio of the clusters present on both forward and reverse strands of 3 tested sequences to overall clusters on the 3 tested sequences, to evaluate the matching circumstance of forward and reverse strands in the available data; Effective data utilization ratio: the ratio of the number of tested sequences at least satisfying 2+/2− cluster after error correction to the total number of tested sequences; Low-frequency error-correction depth: average coverage for the bases in the target region after effective error correction of data.

(85) Analysis of the results: Two driver mutations, TP53 p. [Val272Leu] and EGFR p. [Leu861Arg] were detected in the plasma of the patient, indicating that the patient had a higher risk of cancer. It was confirmed by subsequent clinical pathology that the patient had invasive adenocarcinoma T1aN0M0, IA. In addition, conventional high-throughput sequencing analysis of corresponding tissue and plasma and plasma digital PCR validation results were shown as follows:

(86) TABLE-US-00020 TABLE 5 Mutation Mutation frequency frequency by by Conventional Conventional tissue plasma Mutation Mutation NGS NGS frequency by frequency Gene cHGVS pHGVS analysis analysis Digital PCR by ER-seq TP53 c.[814G > T] p.[Val272Leu] 13% — 0.13%  0.4% EGFR c.[2582T > G] p.[Leu861Arg] 3% — — 0.15%

Example 4 Instructing Individualized Medication Against Tumor

(87) 1. Chip Design

(88) A probe, ONCOcare-drug, for instructing individualized medication against tumor was completed based on the design principle of enrichment probe chips. The chip includes high-frequency genes of 12 kinds of common cancers, important genes in 12 signaling pathways of cancer, common target drug and chemotherapeutic drug genes, totaling 559 genes, 850 KB, 2,400 hotspot target mutations. The gene list is shown in Table 6.

(89) TABLE-US-00021 TABLE 6 Gene list of the chip ONCOcare-drug for instructing individualized medication against tumor ABL1 C1R DIS3 FGF19 HSPA4 MIR142 PAX5 RB1 SRSF2 ABL2 C1S DNMT1 FGF23 IDH1 MITF PBRM1 REL SSTR2 ACVR1B CARD11 DNMT3A FGF3 IDH2 MLH1 PCBP1 RET STAG2 ACVR2A CASP8 DOT1L FGF4 IFNAR1 MLH3 PCM1 RHEB STAT4 AJUBA CBFB DUSP6 FGF6 IFNAR2 MLL PDGFRA RICTOR STAT5B AKT1 CBL EDNRA FGF7 IGF1 MLL2 PDGFRB RNASEL STK11 AKT2 CBLB EGFR FGFR1 IGF1R MLL3 PDK1 RNF43 SUFU AKT3 CBR1 EGR3 FGFR2 IGF2 MLL4 PHF6 ROBO1 SUZ12 ALK CCND1 EIF4A2 FGFR3 IKBKB MPL PIGF ROBO2 SYK ALOX12B CCND2 ELAC2 FGFR4 IKBKE MRE11A PIK3C2A ROS1 TAF1 ANGPT1 CCND3 ELF3 FH IKZF1 MS4A1 PIK3C2B RPA1 TBL1XR1 ANGPT2 CCNE1 EML4 FLCN IL7R MSH2 PIK3C2G RPL22 TBX3 APC CD79A EP300 FLT1 INHBA MSH3 PIK3C3 RPL5 TEK APCDD1 CD79B EPCAM FLT3 IRF4 MSH4 PIK3CA RPS14 TERT AR CDC25C EPHA2 FLT4 IRS2 MSH5 PIK3CB RPS6KB1 TET2 ARAF CDC42 EPHA3 FNTA ITGB2 MSH6 PIK3CG RPTOR TFG ARFRP1 CDC73 EPHA5 FOXA1 JAK1 MSR1 PIK3R1 RUNX1 TGFBR2 ARHGAP35 CDH1 EPHB1 FOXA2 JAK2 MTOR PIK3R2 RUNX1T1 TIPARP ARID1A CDK12 EPHB2 FOXL2 JAK3 MUC1 PLK1 RXRA TLR4 ARID1B CDK2 EPHB6 FPGS JUN MUTYH PML RXRB TMEM127 ARID2 CDK4 EPPK1 FUBP1 KAT6A MYC PMS1 RXRG TNFAIP3 ARID5B CDK6 ERBB2 FYN KDM5A MYCL1 PMS2 SDHAF2 TNFRSF14 ASXL1 CDK8 ERBB3 GAB2 KDM5C MYCN PNRC1 SDHB TNFRSF8 ATM CDKN1A ERBB4 GATA1 KDM6A MYD88 POLQ SDHC TNFSF11 ATR CDKN1B ERCC2 GATA2 KDR NAV3 PPP2R1A SDHD TNFSF13B ATRX CDKN2A ERCC3 GATA3 KEAP1 NBN PRDM1 SEMA3A TOP1 AURKA CDKN2B ERG GID4 KIF1B NCOA1 PRKAA1 SEMA3E TOP2A AURKB CDKN2C ESR1 GNA11 KIF5B NCOA2 PRKAR1A SETBP1 TOP2B AXIN1 CDX2 ETV1 GNA13 KIT NCOR1 PRKCA SETD2 TP53 AXIN2 CEBPA ETV6 GNAQ KLF4 NEK11 PRKCB SF1 TRAF7 AXL CFLAR EWSR1 GNAS KLHL6 NF1 PRKCG SF3B1 TSC1 B2M CHD1 EXT1 GNRHR KRAS NF2 PRKDC SH2B3 TSC2 B4GALT3 CHD2 EXT2 GPR124 LCK NFE2L2 PRSS8 SIN3A TSHR BACH1 CHD4 EZH2 GRIN2A LIMK1 NFE2L3 PSMB1 SLAMF7 TSHZ2 BAK1 CHEK1 FAM123B GRM3 LRRK2 NFKBIA PSMB2 SLC4A1 TSHZ3 BAP1 CHEK2 FAM46C GSK3B LYN NKX2-1 PSMB5 SLIT2 TUBA1A BARD1 CHUK FANCA H3F3A MALAT1 NKX3-1 PTCH1 SMAD2 TUBB BCL2 CIC FANCC H3F3C MAP2K1 NOTCH1 PTCH2 SMAD3 TUBD1 BCL2A1 CRBN FANCD2 HCK MAP2K2 NOTCH2 PTEN SMAD4 TUBE1 BCL2L1 CREBBP FANCE HDAC1 MAP2K4 NOTCH3 PTP4A3 SMARCA1 TUBG1 BCL2L11 CRIPAK FANCF HDAC2 MAP3K1 NOTCH4 PTPN11 SMARCA4 TYR BCL2L2 CRKL FANCG HDAC3 MAP3K13 NPM1 PTPRD SMARCB1 U2AF1 BCL6 CRLF2 FANCI HDAC4 MAPK1 NR3C1 RAC1 SMARCD1 USP9X BCOR CROT FANCL HDAC6 MAPK3 NRAS RAC2 SMC1A VEGFA BCORL1 CSF1R FANCM HDAC8 MAPK8 NSD1 RAD21 SMC3 VEGFB BCR CTCF FAT3 HGF MAPK8IP1 NTRK1 RAD50 SMO VEZF1 BLM CTLA4 FBXW7 HIF1A MAX NTRK2 RAD51 SOCS1 VHL BMPR1A CTNNA1 FCGR1A HIST1H1C MC1R NTRK3 RAD51B SOX10 WHSC1L1 BRAF CTNNB1 FCGR2A HIST1H2BD MCL1 NUP93 RAD51C SOX17 WISP3 BRCA1 CUL4A FCGR2B HIST1H3B MDM2 PAK3 RAD51D SOX2 WWP1 BRCA2 CUL4B FCGR2C HNF1A MDM4 PAK7 RAD52 SOX9 XIAP BRIP1 CYLD FCGR3A HRAS MECOM PALB2 RAD54L SPEN XPA BTG1 CYP17A1 FCGR3B HRH2 MED12 PARP1 RAF1 SPOP XPC BTK DAXX FGF10 HSD17B3 MEF2B PARP2 RARA SPRY4 XPO1 C11orf30 DDR1 FGF12 HSD3B2 MEN1 PARP3 RARB SRC XRCC3 C1QA DDR2 FGF14 HSP90AA1 MET PARP4 RARG SRD5A2 YES1 ZNF217 ZNF703 ZRSR2 WT1 XRCC1 GSTP1 ERCC1 MTHFR SOD2 UMPS UGT1A1 CBR3 ATIC MTRR DPYD TPMT

(90) 2. Analysis of Sequencing Result

(91) One patient with advanced colorectal disease was analyzed according to the method described in Example 1, wherein the chip ONCOcare-Drug of the present example was used in the step of enrichment and capture with probes. The statistical results of the sequencing data are shown in Table 7 below:

(92) TABLE-US-00022 TABLE 7 Matching rate of Low-frequency Sample Total data the forward and error-correction Effective data name Clinical stages output(G) reverse strands depth utilization ratio CD160 IV stage 5.5 78.2% 520X 4.1% metastasis Note: Matching rate of the forward and reverse strands: the ratio of the clusters present on both forward and reverse strands of 3 tested sequences to overall clusters on the 3 tested sequences, to evaluate matching circumstance of forward and reverse strands in the available data; Effective data utilization ratio: the ratio of the number of tested sequences at least satisfying 2+/2− cluster after error correction to the total number of tested sequences; Low-frequency error-correction depth: average coverage for the bases in the target region after effective error correction of data.

(93) Analysis of the results: A total of 6 non-synonymous mutations in the Exon region were detected, and they were consistent with tissue mutations. Details of the mutations are shown in Table 8:

(94) TABLE-US-00023 TABLE 8 Mutation Mutation of Mutation of frequency by Tissue mutation Gene bases amino acids Mutation type ER-seq frequency TP53 c.C241T p.R81X mutation resulting in 10.4%  32.8% a termination codon APC c.1254T > A p.N418K missense mutation 6.3% 25.6% KRAS c.35G > A p.G12D missense mutation 3.8% 20.3% ALMS1 c.T3971G p.V1324G missense mutation 1.2% 15.4% MLH1 c.A1427T p.E476V missense mutation 2.5% 13.8% ZNF721 c.C2061G p.H687Q missense mutation 0.83%  10.2%

(95) The details for chemotherapy sites are shown in

(96) TABLE-US-00024 TABLE 9 Gene name RS number Detected base Gene name RS number Detected base XPC rs2228001 GT MTHFR rs1801133 AA TP53 rs1042522 CC CBR3 rs1056892 GG XRCC1 rs25487 CC MTHFR rs1801133 AA GSTP1 rs1695 AG ATIC rs4673993 TT ERCC1 rs11615 GG MTRR rs1801394 AA ERCC1 rs3212986 CC TP53 rs1042522 CC MTHFR rs1801133 AA DPYD rs3918290 CC SOD2 rs4880 AA DPYD rs67376798 TT GSTP1 rs1695 AG TPMT rs1800460 CC MTHFR rs1801133 AA TPMT rs1800462 CC MTHFR rs1801131 TT TPMT rs1800584 CC GSTP1 rs1695 AG UGT1A1 rs8175347 7TA/7TA UMPS rs1801019 GG

(97) Drug prediction: the database was interpreted in combination with the above detection results based on the target drug chemotherapy. The following conclusions were only for clinician's reference during development of therapeutic schedule:

(98) TABLE-US-00025 TABLE 10 Medication prompts for targeted drugs Being recommended Being recommended for colorectal for other Clinical II/III cancer by FDA cancers by FDA stage medicine Gene Positive Negative Positive Negative Positive Negative mutation correlation correlation correlation correlation correlation correlation KRAS No Everolimus No No Selumetinib No p.G12D Antroquinonol

(99) TABLE-US-00026 TABLE 11 Medication prompts for chemotherapeutics Drugs recommended by FDA (colorectal Drugs recommended Efficacy prediction cancer) by FDA (other cancers) Low risk of toxic and Capecitabine, paclitaxel/docetaxel, side effects or high fluorouracil purine compounds/ drug sensitivity purine analogues High risk of toxic and No anthracycline, side effects or low cyclophosphamide drug sensitivity

Example 5 Postoperative Monitoring of 12 Kinds of Common Cancers

(100) 1. Chip Design

(101) A chip, ONCOcare-JK, for postoperative monitoring of tumors (lung cancer, colorectal cancer, gastric cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer and liver cancer, etc.) was completed based on the design principle of enrichment probe chips. The chip includes Driver Genes related with common cancers with high incidence, high-frequency mutant genes, important genes in 12 cancer-related signaling pathways, totaling 508 genes, 500 Kb, 4,800 hotspot mutations. The gene list is shown in table 12.

(102) TABLE-US-00027 TABLE 12 Gene list for the postoperative monitoring chip ONCOcare-JK ABL1 CBLB DOT1L FGF7 IGF2 MSH2 PIK3CB SDHB TRAF7 ABL2 CBR1 DUSP6 FGFR1 IKBKB MSH3 PIK3CG SDHC TSC1 ACVR1B CCND1 EDNRA FGFR2 IKBKE MSH4 PIK3R1 SDHD TSC2 ACVR2A CCND2 EGFR FGFR3 IKZF1 MSH5 PIK3R2 SEMA3A TSHR AJUBA CCND3 EGR3 FGFR4 IL7R MSH6 PLK1 SEMA3E TSHZ2 AKT1 CCNE1 EIF4A2 FLCN INHBA MSR1 PML SETBP1 TSHZ3 AKT2 CD79A ELAC2 FLT1 IRF4 MTOR PMS1 SETD2 TUBA1A AKT3 CD79B ELF3 FLT3 IRS2 MUC1 PMS2 SF1 TUBB ALK CDC25C EML4 FLT4 ITGB2 MUTYH PNRC1 SF3B1 TUBD1 ANGPT1 CDC42 EP300 FNTA JAK1 MYC POLQ SH2B3 TUBE1 ANGPT2 CDC73 EPHA2 FOXA1 JAK2 MYCL1 PPP2R1A SIN3A TUBG1 APC CDH1 EPHA3 FOXA2 JAK3 MYCN PRDM1 SLAMF7 TYR AR CDK12 EPHA5 FOXL2 JUN NAV3 PRKCA SLC4A1 VEGFA ARAF CDK2 EPHB1 FPGS KDR NBN PRKCB SLIT2 VEGFB ARFRP1 CDK4 EPHB2 FUBP1 KEAP1 NCOA1 PRKCG SMAD2 VEZF1 ARID1A CDK6 EPHB6 FYN KIF1B NCOA2 PRKDC SMAD3 VHL ARID1B CDK8 EPPK1 GAB2 KIF5B NCOR1 PRSS8 SMAD4 WISP3 ASXL1 CDKN1A ERBB2 GATA1 KIT NEK11 PSMB1 SMARCA1 WT1 ATM CDKN1B ERBB3 GATA2 KLF4 NF1 PSMB2 SMC1A WWP1 ATR CDKN2A ERBB4 GATA3 KLHL6 NF2 PSMB5 SMC3 XIAP ATRX CDKN2B ERCC2 GID4 KRAS NOTCH1 PTCH1 SMO XPA AURKA CDKN2C ERCC3 GNA11 LCK NOTCH2 PTCH2 SOCS1 XPC AURKB CDX2 ERG GNA13 LIMK1 NOTCH3 PTEN SOX2 XPO1 AXIN1 CEBPA ESR1 GNAQ LRRK2 NOTCH4 PTP4A3 SOX9 XRCC3 AXIN2 CFLAR ETV1 GNAS MALATI NPM1 PTPN11 SPEN YES1 AXL CHD1 ETV6 GNRHR MAP2K1 NR3C1 PTPRD SPRY4 ZNF217 BACH1 CHD2 EWSR1 GPR124 MAP2K2 NRAS RAC1 SRC ZRSR2 BAK1 CHD4 EXT1 GRIN2A MAP2K4 NSD1 RAC2 SRD5A2 BAP1 CHEK1 EXT2 GRM3 MAP3K1 NTRK1 RAD21 SRSF2 BARD1 CHEK2 EZH2 GSK3B MAP3K13 NTRK2 RAD50 SSTR2 BCL2 CHUK FAM46C H3F3A MAPK1 NTRK3 RAD51 STAG2 BCL2A1 CIC FANCA H3F3C MAPK3 NUP93 RAF1 STAT4 BCL2L1 CRBN FANCC HCK MAPK8 PAK3 RARA STAT5B BCL2L2 CREBBP FANCD2 HDAC1 MAX PAK7 RARB STK11 BCL6 CRIPAK FANCE HDAC2 MC1R PALB2 RARG SUFU BCOR CRKL FANCF HDAC3 MCL1 PARP1 RB1 SUZ12 BCORL1 CRLF2 FANCG HDAC4 MDM2 PARP2 REL SYK BCR CTCF FANCI HDAC6 MDM4 PARP3 RET TAF1 BLM CTLA4 FANCL HDAC8 MED12 PARP4 RHEB TBX3 BMPR1A CTNNA1 FANCM HGF MEF2B PCM1 RNF43 TEK BRAF CTNNB1 FAT3 HIF1A MEN1 PDGFRA ROBO1 TERT BRCA1 CUL4A FBXW7 HNF1A MET PDGFRB ROBO2 TET2 BRCA2 CUL4B FCGR2A HRAS MITF PDK1 ROS1 TFG BRIP1 CYLD FCGR2B HRH2 MLH1 PHF6 RPA1 TGFBR2 BTG1 DAXX FCGR2C IDH1 MLH3 PIGF RPL5 TIPARP BTK DDR1 FCGR3A IDH2 MLL PIK3C2A RPS14 TLR4 CARD11 DDR2 FCGR3B IFNAR1 MLL2 PIK3C2B RXRA TOP1 CASP8 DIS3 FGF3 IFNAR2 MLL3 PIK3C2G RXRB TOP2A CBFB DNMT1 FGF4 IGF1 MLL4 PIK3C3 RXRG TOP2B CBL DNMT3A FGF6 IGF1R MS4A1 PIK3CA SDHAF2 TP53

(103) 2. Analysis of Sequencing Result

(104) One patient with lung adenocarcinoma who had an operation 3 months ago, was analyzed according to the method described in Example 1, wherein the chip ONCOcare-JK of the present example was used in the step of enrichment and capture with probes. The statistical results of the sequencing data are shown in Table 13 below:

(105) TABLE-US-00028 TABLE 13 Matching rate of Low-frequency Sample Total data the forward and error-correction Effective data name Clinical information output(G) reverse strands depth utilization ratio CD172 Right middle lung 10 73.4% 920X 3.92% adenocarcinoma T2aN0M0 Ib stage, 3 months after operation Note: Matching rate of the forward and reverse strands: the ratio of the clusters present on both forward and reverse strands of 3 tested sequences to overall clusters on the 3 tested sequences, to evaluate matching circumstance of forward and reverse strands in the available data; Effective data utilization ratio: the ratio of the number of tested sequences at least satisfying 2+/2− cluster after error correction to the total number of tested sequences; Low-frequency error-correction depth: average coverage for the bases in the target region after effective error correction of data.

(106) Analysis of the results: A total of 5 non-synonymous mutations in the Exon region were detected, and details of the mutations are shown in Table 14:

(107) TABLE-US-00029 TABLE 14 Mutation results of original Plasma monitoring results at carcinoma tissue sample 3 month after operation Mutation Mutation Mutation of of amino Mutation Mutation Mutation of of amino Mutation Mutation Gene bases acids type frequency Gene bases acids type frequency TP53 c.814G > T p.V272L missense  18% NOTCH1 c.2054A > C p.N685T missense 1.63% mutation mutation PDGFRA c.2235G > A p.M745I missense 11.5%  PDGFRA c.2235G > A p.M745I missense 1.02% mutation mutation ROS1 c.6316G > A p.A2106T missense  10% AR c.1369_1371 p.G457del missense 0.89% mutation delGGC mutation PTCH1 c.49_51del p.G17del Deletion   8% MEF2B c.844A > G p.R282G missense 0.76% GGC mutation mutation SETD2 c.22C > T p.P8S missense 7.6% PML c.851G > C p.R284P missense 0.47% mutation mutation NOTCH1 c.2054A > C p.N685T missense 6.2% mutation FUBP1 c.121A > T p.I41F missense 5.3% mutation

(108) A total of 19 mutations were detected, wherein 5 mutations were non-synonymous mutations in Exon. Relative to normal human baseline, the detected mutations were higher. In addition, NOTCH1 p.N685T and PDGFRA p.M745I present in the tissues still existed in the plasma after operation, indicating that there may be a higher risk of recurrence after operation. Clinical follow-up: there was a progress in disease of the patient. In addition, conventional high-throughput sequencing analysis of plasma and plasma digital PCR validation results were shown in table 15:

(109) TABLE-US-00030 TABLE 15 Mutation Mutation frequency by frequency Mutation Conventional plasma by Digital frequency by Gene cHGVS pHGVS NGS analysis PCR ER-seq NOTCH1 c.2054A > C p.N685T 0.78%  1.1% 1.63% PDGFRA c.2235G > A p.M745I — 0.42% 1.02%

(110) Industrial Practical Applicability

(111) The method for enrichment and sequencing of low-frequency mutations of target DNA in plasma, provided in the present invention, can accurately detect low-frequency of plasma DNA in 5-10 mL peripheral blood samples, with simple operation and strong practical applicability. In addition, the method has the following effects: high sensitivity, such that mutations at a low-frequency of 0.01% can be detected with high specificity; high specificity such that accurate detection of low-frequency mutations can be more effectively achieved with a specificity of 98% or greater; high-throughput, such that not only relevant genes of interest can be scanned at once to obtain more comprehensive information of the subject and more accurate relevant prediction, but also multiple samples can be detected simultaneously in a very short period of time, thereby reducing costs and facilitating clinical promotion; multidimensional applicability, such that this method can fully exploit the potential of plasma ctDNA, and lays a solid foundation for early screening, postoperative monitoring and accurate medical treatment of a variety of related tumors, thereby giving a big push to the development of clinical oncology.