WO2019076018A1 - 一种用于检测目的基因低频突变的扩增子文库的构建方法 - Google Patents

一种用于检测目的基因低频突变的扩增子文库的构建方法 Download PDF

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WO2019076018A1
WO2019076018A1 PCT/CN2018/083822 CN2018083822W WO2019076018A1 WO 2019076018 A1 WO2019076018 A1 WO 2019076018A1 CN 2018083822 W CN2018083822 W CN 2018083822W WO 2019076018 A1 WO2019076018 A1 WO 2019076018A1
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sequence
primer
tested
mutation
downstream
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French (fr)
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郑乔松
师晓
陈敏
张凯华
国晓玲
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北京泛生子基因科技有限公司
北京泛生子医学检验实验室有限公司
重庆今创泛生医学检验实验室有限公司
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Priority to US16/757,222 priority Critical patent/US20210095393A1/en
Publication of WO2019076018A1 publication Critical patent/WO2019076018A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
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    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/143—Modifications characterised by incorporating a promoter sequence
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for constructing an amplicon library for detecting low frequency mutation of a target gene.
  • Tumors are highly heterogeneous, and the pathogenic mutations may be present in extremely low proportions.
  • the mutation frequency of the mutation site or mutation region of the target gene in the blood, urine and cerebrospinal fluid of the tumor patient may affect the future tumor. Judgment of medication or the direction of tumor development. Therefore, detecting the mutation frequency of a mutation site or a mutation region of a target gene in cfDNA in blood, urine, and cerebrospinal fluid of a tumor patient has become a research focus, and it is necessary to sequence a mutation site or a mutation region to detect a mutation frequency.
  • the error rate of the most accurate Hiseq sequencing in the second generation sequencing is about 0.2%.
  • the current DNA polymerase amplification error rate is also between 10 -7 -10 -5 , so how can Eliminating amplification errors and sequencing errors in the sequencing results directly reflects the low-frequency variation of the original template molecules in the sample, which becomes the key to the problem.
  • NGS next-generation sequencing
  • ARMS amplification refractory mutation system
  • the method provided by the present invention is applicable to all second generation sequencing platforms, including the following steps:
  • the Barcode primer F1 is sequentially composed of a sequencing adaptor 1, a barcode sequence for distinguishing different samples, and a universal sequence 1;
  • the upstream primer F2 is sequentially composed of a universal sequence 1, a molecular tag, a specific base sequence, and an upstream specific primer sequence;
  • the downstream external primer R1 is composed of a sequencing linker 2 and a universal sequence 2 in sequence;
  • the downstream internal primer R2 is composed of a universal sequence 2 and a downstream specific primer sequence in sequence;
  • the sequencing linker 1 and the sequencing linker 2 are selected corresponding sequencing blocks according to different sequencing platforms;
  • the barcode sequences are all nucleotides having a length of 8-12 nt, no contiguous base, and a GC content of 40-60%;
  • the universal sequence 1 and the universal sequence 2 are both 16-25 nt in length, and have no contiguous bases, and have a GC content of 35-65%, and have no obvious secondary structure;
  • the specific base sequence is GAT;
  • the upstream specific primer sequence and the downstream specific primer sequence are primers for amplifying the region of the target gene to be detected
  • the molecular tag is a 10-12 random base
  • the sequencing platform is Illumina platform, the sequencing linker 1 is I5, and the sequencing linker 2 is I7;
  • the sequencing platform is an Ion Torrent platform
  • the sequencing linker 1 is A
  • the sequencing linker 2 is P.
  • the molar ratio of the Barcode primer F1, the upstream primer F2, the downstream external primer R1, and the downstream internal primer R2 is 6: (10-6): (1-3): (1-3).
  • the mutation is a low frequency mutation, specifically a mutation frequency as low as 0.1%.
  • the sample to be tested is cfDNA isolated from ex vivo blood of a tumor patient, cfDNA isolated from ex vivo urine of a tumor patient, cfDNA isolated from ex vivo cerebrospinal fluid of a tumor patient, or genomic DNA extracted from ex vivo tumor tissue of a tumor patient.
  • the mutation of the target gene to be detected in the sample to be tested is detected by detecting a mutated base or a mutant amino acid of the target gene to be tested, or detecting a mutation frequency of the target gene to be tested.
  • the calculation method of the mutation frequency is as follows:
  • the DNA molecule carrying the same molecular tag is an amplification product of an initial DNA template, and is named as one family;
  • the mutation rate in the family if the mutation rate of the family is ⁇ 80%, the family is recorded as a DNA family with a molecular tag mutation
  • Mutation rate (number of DNA molecules with mutations in codons encoding amino acid residues in the same family / total number of DNA molecules in the same family) * 100%;
  • Mutation frequency number of DNA families with molecular tags in the sequencing results / number of all molecularly tagged DNA families in the sequencing results *100%.
  • Another object of the present invention is to provide a method for detecting a mutation in a region to be examined of a target gene in a cfDNA of a sample to be tested.
  • the method provided by the invention comprises the following steps:
  • the nucleotide sequence of the universal sequence 1 is sequence 1;
  • the nucleotide sequence of the universal sequence 2 is sequence 2;
  • the nucleotide sequence of the sequencing linker 1 is sequence 3;
  • the nucleotide sequence of the sequencing linker 2 is sequence 4;
  • the barcode sequences for distinguishing different samples are respectively sequence 5-sequence 14;
  • the gene to be tested is NRAS, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 15 and sequence 16 or sequence 17 and sequence 18, respectively;
  • the gene to be tested is ALK, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 19 and sequence 20 or sequence 21 and sequence 22 or sequence 23 and sequence 24 or sequence 25 and sequence 26 or sequence 27, respectively. Sequence 28 or sequence 29 and sequence 30 or sequence 31 and sequence 32;
  • the gene to be tested is PIK3CA, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 33 and sequence 34 or sequence 35 or sequence 36, respectively;
  • the gene to be tested is ROS, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 37 and sequence 38, respectively;
  • the gene to be tested is EGFR, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 39 and sequence 40 or sequence 41 and sequence 42 or sequence 43 and sequence 44 or sequence 45 and sequence 46 or sequence 47, respectively.
  • the gene to be tested is MET, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 49 and sequence 50 or sequence 51 and sequence 52 or sequence 53 and sequence 54 or sequence 55 and sequence 56, respectively;
  • the gene to be tested is BRAF, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 57 and sequence 58 or sequence 59 and sequence 60, respectively;
  • the gene to be tested is KRAS, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 61 and sequence 62 or sequence 63 and sequence 64, respectively;
  • the gene to be tested is TP53, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 65 and sequence 66 or sequence 67 and sequence 68 or sequence 69 and sequence 70 or sequence 71 and sequence 72 or sequence 73, respectively. Sequence 74 or sequence 75 and sequence 76;
  • the gene to be tested is ERBB2, and the corresponding upstream specific primer sequence and downstream specific primer sequence are sequence 77 and sequence 78, respectively.
  • test sample is cfDNA isolated from ex vivo blood of a tumor patient, cfDNA isolated from ex vivo urine of a tumor patient, cfDNA isolated from ex vivo cerebrospinal fluid of a tumor patient, or genomic DNA extracted from ex vivo tumor tissue of a tumor patient.
  • a third object of the present invention is to provide a method for detecting the mutation frequency of a mutation site or a mutation region of a gene of interest in a sample to be tested.
  • the method provided by the present invention comprises the steps of the above method, wherein the sample to be tested is cfDNA isolated from ex vivo blood of a tumor patient, cfDNA isolated from tumor urine of a tumor patient, and cfDNA isolated from ex vivo cerebrospinal fluid of a tumor patient. Or genomic DNA extracted from ex vivo tumor tissue of a tumor patient.
  • the above method or the above DNA library is useful for guiding tumor administration or for judging the direction of tumor development.
  • a fourth object of the present invention is to provide a method for guiding the administration of a tumor to a patient to be tested or for judging the direction of tumor development.
  • the method provided by the invention firstly detects the mutation frequency of the mutation site or the mutation region of the target gene in the cfDNA of the patient to be tested by using the steps described in the above method, and then guides the tumor of the patient to be tested according to the mutation frequency or determines the direction of tumor development.
  • a fifth object of the present invention is to provide a kit for detecting an amplicon library of a mutation of a gene to be detected in a sample to be tested.
  • the kit provided by the present invention comprises the Barcode primer F1, the upstream primer F2, the downstream external primer R1, and the downstream internal primer R2 in the above method.
  • Figure 1 shows the function of the molecular tag.
  • A, B and C are different mutation sites, respectively.
  • Figure 2 shows the distribution of amplification products detected by Agilent 2200 TapeStation Systems after completion of construction of the cfDNA library extracted from the blood sample of Subject 1.
  • Fig. 3 shows the results of sequencing of the amplicon library obtained by the one-step method of the cfDNA extracted from the blood sample of the subject 1 on the Ion Torrent platform.
  • Example 1 Construction of an amplicon library for detecting low frequency mutations of a gene of interest
  • the mutation frequency of the mutation site or mutation region of the target gene in the blood, urine and cerebrospinal fluid of the tumor patient may affect the judgment of the future tumor drug or the direction of tumor development.
  • the blood and urine of the tumor patient are detected.
  • the mutation frequency of the mutation site or mutation region of the target gene in the cfDNA in the cerebrospinal fluid construct an amplicon library for detecting the low frequency mutation of the target gene, as follows:
  • a region of the known gene of interest is selected as the region to be tested to synthesize the following primers:
  • Barcode primer F1 sequencing linker 1 + barcode sequence + universal sequence 1;
  • Upstream primer F2 universal sequence 1 + molecular tag + specific base sequence + upstream specific primer sequence
  • Downstream external primer R1 sequencing linker 2+ universal sequence 2;
  • Downstream primer R2 universal sequence 2+ downstream specific primer sequence
  • the barcode sequence is used to distinguish sequences of different samples, and one sample to be tested corresponds to a barcode sequence.
  • the length of the Barcode sequence is 8-12 nt, requiring no continuous base, the GC content is 40-60%, and the primers introduced into the Barcode sequence are introduced. No obvious secondary structure, etc.
  • F1 is used to distinguish different samples, as long as it is the same sample, F1 is the same, regardless of the detection site.
  • the universal sequences 1 and 2 are 16-25 nt in length and require no contiguous bases, the GC content is 35-65%, the primers introduced into the sequences have no obvious secondary structure, and the like, the sequence can be changed as needed, and the embodiment adopts
  • Sequencing linker 1 and sequencing linker 2 were determined according to the sequencing platform:
  • sequencing linkers 1 and 2 are I5 and I7, respectively, and the linker sequence and the primer sequences on the chip are complementary, and the linker is added to attach the nucleic acid fragment to the vector.
  • sequencing linkers 1 and 2 are A and P (sequences 3 and 4, respectively), the A linker is used for sequencing, complementary to specific primers, and the P linker is complementary to the sequence on the vector to serve Carrier connection.
  • the specific base sequence is GAT, which is not part of the gene-specific amplified fragment. Its function is to facilitate the bioinformatics analysis of the sequencing results, and to improve the efficiency of data screening by identifying the GAT sequence.
  • the upstream specific primer sequence and the downstream specific primer sequence are primers designed to amplify the gene according to the region of interest of the target gene, the upstream specific primer sequence size is 15-30 nt, and the downstream specific primer sequence size is 15-30 nt.
  • the molecular tag is a 10-12 random base that is used to label the starting cfDNA template.
  • Each random base has four base forms of ATCG, so there are 1048576 different molecular tags in 10 random bases.
  • the initial 20 ng DNA template has a copy number of 6000, while the cfDNA molecular fragment is shorter.
  • the effective template copy number that can be amplified is less than 6000, and 1048576 molecular tag forms can be used to add a specific "marker" to each initial template.
  • Figure 1 shows the five amplified products with the same molecular tag in the constructed library, in which the mutation at the A site is present on all 5 molecules, and the mutations at the B and C sites are shown. Only in one of the amplification products, the proportion is very low, it can be determined that the mutation of the A site is the mutation existing in the initial template molecule, and the mutation of the B and C sites is the PCR amplification of the library. A false positive mutation that occurs during or during sequencing. Therefore, the role of the molecular tag is to label the original template molecule, identify the mutations present in the original template, eliminate false positive mutations in the PCR and sequencing process, and improve the detection sensitivity.
  • Table 1 is the PCR amplification system
  • Table 2 shows the amplification procedure
  • the cycle conditions of the gradient annealing temperature of the first two cycles of the PCR process are the initial amplification of the original template, or it can be said that a specific molecular label is added to the different original template, and then the PCR conditions of the 19 cycles are original.
  • the template is amplified in the molecular tag.
  • the high concentration of F1, R1 and the low concentration of the primers F2, R2 also ensure that the intramolecular tag amplification is carried out in the next 19 cycles (that is, it is generally not expanding). Add other molecular labels during the addition process).
  • the PCR product was pipetted with 1.3 volumes of Agencourt AMPure XP Kit (BECKMAN COULTER, A63882) to purify the PCR product to obtain a DNA library for amplicon sequencing.
  • the specific purification steps are as follows:
  • the libraries amplified by the above different samples were mixed in equal proportions according to the measured concentrations, finally diluted to a specific concentration, and sequenced by a second-generation sequencer to obtain sequencing results.
  • the results of sequencing were analyzed by data processing and bioinformatics analysis to obtain mutations in the detected genes.
  • the data processing process includes sequencing data conversion, quality control, sequence alignment (reference genome is NCBI GRCh37/Hg19), mutation site analysis and other processes, and the mutation and detection frequency of the detected samples are obtained by data processing analysis.
  • the mutation frequency is calculated as follows:
  • the DNA molecule carrying the same molecular tag is an amplification product of an initial DNA template, and is named as one family;
  • the mutation rate in the family if the mutation rate of the family is ⁇ 80%, the family is recorded as a DNA family with a molecular tag mutation
  • Mutation rate (number of DNA molecules with mutations in codons encoding amino acid residues in the same family / total number of DNA molecules in the same family) * 100%;
  • Mutation frequency number of DNA families with molecular tags in the sequencing results / number of all molecularly tagged DNA families in the sequencing results *100%.
  • Example 3 constructing an amplicon library for detecting low frequency mutations of a target gene
  • the target gene is shown in Table 5.
  • the sample to be tested is derived from 49 subjects who have been identified as lung cancer patients.
  • the purpose of this example is to detect the gene mutation frequency of 49 patients shown in Table 5 by the method of the present invention.
  • Table 3 is the primer combination
  • primer design principles annealing temperature 55-65 ° C, as little as possible secondary structure, GC content 35%-65%, primer length 16-30nt, primers should not form a secondary structure, as shown in Table 4.
  • Table 4 shows the specific primer sequences corresponding to each gene as primer combinations.
  • the concentration of Barcode primer F1 was 1.67 ⁇ M
  • the concentration of the downstream external primer R1 was 2.78 ⁇ M
  • the concentration of the upstream primer F2 was 0.28 ⁇ M
  • the concentration of the primer R2 in the downstream was 0.28 ⁇ M.
  • FFPE samples formalized paraffin-embedded tissues after formalin fixation
  • blood samples of 49 subjects all confirmed cancer patients
  • Fig. 2 is the distribution map of the amplified product detected by Agilent 2200 TapeStation Systems after the completion of the specific sample library construction, and the abscissa is the fragment length.
  • the ordinate is the signal intensity (FU)
  • the lower peak is the 25 bp position marker
  • the upper peak is the 1500 bp position marker.
  • the PCR products obtained by PCR amplification are concentrated in the range of 160-230 bp.
  • Figure 3 shows the results of sequencing of the cfDNA extracted from the blood sample (sample 1) of patients diagnosed with lung cancer using the Ion Torrent platform.
  • the cfDNA test results of the FFPE samples and blood samples of the 49 subjects actually collected are shown in Table 5 below.
  • the comparison method of cfPE DNA and blood sample cfDNA detection was performed by Agilent's SureSelect customized service.
  • the detection method of sample cfDNA was constructed by this patent method. The results showed that the consistency between the FFPE sample and the patented method for the detection of cfDNA in blood samples was as high as 87.76%. This patented method was built with Agilent's SureSlect customized service.
  • the method of the library also has a consistency of 49.92% for 49 cases of cfDNA detection, and the two inconsistent site mutations have been verified by ddPCR and the variation results obtained by the patented method, which proves that the sensitivity of the method is superior to that of cfDNA. Detection of cfDNA by capture.
  • Table 5 shows the results of cfPE samples and blood samples of 49 subjects.
  • Table 6 shows the meaning of each mutation site.
  • the invention only relates to the one-step PCR reaction and the corresponding product purification step, which simplifies the operation process of the database construction and saves the construction time (the database can be completed within two hours, and the whole process from the library construction to the end of the machine and the completion of the bio-information analysis can be completed. Completed within 24 hours).
  • the database construction method can detect mutations as low as 0.1%.
  • the samples to be tested can be isolated free DNA such as blood, urine and cerebrospinal fluid, or genomic DNA extracted from traditional frozen tissues, paraffin sections and freshly puncture tissues.
  • the method can rapidly, easily, sensitively and specifically target different regions of free DNA in samples such as blood, urine and cerebrospinal fluid, and efficiently detect mutations as low as 0.1%. , greatly simplify the experimental operation, effectively avoid library loss and pollution, significantly reduce costs and improve efficiency.

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Abstract

提供了一种用于检测目的基因低频突变的扩增子文库的构建方法，包括如下步骤：1)设计合成Barcode引物、上游引物、下游外引物、下游内引物；2)用所述Barcode引物、所述上游引物、所述下游外引物和所述下游内引物对待测样本cfDNA进行一步PCR扩增，得到扩增产物，即为用于扩增子测序的DNA文库。该方法除能检测组织样本外，还能够对血液、尿液以及脑脊液等样本中游离DNA的不同区域进行靶向扩增。

Description

一种用于检测目的基因低频突变的扩增子文库的构建方法 技术领域
本发明属于生物技术领域，尤其涉及一种用于检测目的基因低频突变的扩增子文库的构建方法。
背景技术
肿瘤具有高度异质性，其中的致病突变可能以极低的比例存在，肿瘤患者的血液、尿液和脑脊液中的cfDNA中的目的基因的突变位点或突变区域的突变频率会影响将来肿瘤用药或者肿瘤发展方向的判断。因此，检测肿瘤患者的血液、尿液和脑脊液中的cfDNA中的目的基因的突变位点或突变区域的突变频率成为研究重点，这就需要对突变位点或者突变区域进行测序，检测突变频率。
目前二代测序最准的Hiseq测序本身的错误率就在0.2％上下，除此之外，当前的DNA聚合酶的扩增错误率也在10 -7-10 -5之间，因此，如何能够在测序结果中排除扩增错误和测序错误，直接反应出样本中原始模板分子的低频变异情况就成为了问题的关键。
肿瘤患者的血液、尿液和脑脊液中的cfDNA的含量很少，这对低频突变的检测来说是个难题。当前市场上主要有三种低频突变的检测方式：数字PCR、下一代测序(next-generation sequencing，NGS)和突变扩增系统(amplification refractory mutation system，ARMS)PCR。NGS具有高通量、低成本、快速、操作简便等优势，是目前国内最热门的低频突变检测技术。在NGS过程中，构建基因文库是整个测序进程中第一个步骤，也是最为关键步骤，基因文库的质量直接影响后续的测序工作。但是，市场上常规的建库方法均存在成本高、检测周期长、流程复杂、文库易被污染以及对检测人员要求高等缺陷，不适用于大量样本的测序建库。
发明公开
本发明的一个目的是提供用于检测待测样本目的基因待检区域突变情况的扩增子文库的构建方法。
本发明提供的方法适用于所有二代测序平台，包括如下步骤：
1)设计合成Barcode引物F1、上游引物F2、下游外引物R1、下游内 引物R2；
所述Barcode引物F1依次由测序接头1、用于区分不同样本的barcode序列和通用序列1组成；
所述上游引物F2依次由通用序列1、分子标签、特定碱基序列和上游特异性引物序列组成；
所述下游外引物R1依次由测序接头2和通用序列2组成；
所述下游内引物R2依次由通用序列2和下游特异性引物序列组成；
所述测序接头1和所述测序接头2为根据不同测序平台选择对应的测序接头；
所述barcode序列均为长度为8-12nt、无连续碱基，且GC含量为40-60％的核苷酸；
所述通用序列1和所述通用序列2的长度均为16-25nt，且无连续碱基，GC含量为35-65％，无明显二级结构；
所述特定碱基序列为GAT；
所述上游特异性引物序列和所述下游特异性引物序列是扩增所述目的基因待检区域的引物；
所述分子标签为10-12位随机碱基；
2)用所述Barcode引物F1、所述上游引物F2、所述下游外引物R1和所述下游内引物R2对待测样本cfDNA进行一步PCR扩增，得到扩增产物，即为用于扩增子测序的DNA文库。
上述方法中，
所述测序平台为Illumina平台，所述测序接头1为I5，所述测序接头2为I7；
或所述测序平台为Ion Torrent平台，所述测序接头1为A，所述测序接头2为P。
上述方法中，
所述PCR扩增中，所述Barcode引物F1、所述上游引物F2、所述下游外引物R1和所述下游内引物R2的摩尔比为6:(10-6):(1-3):(1-3)。
上述方法中，
所述突变为低频突变，具体为突变频率最低至0.1％。
上述方法中，
所述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
上述的方法制备DNA文库也是本发明保护的范围。
上述的方法或上述DNA文库在检测待测样本cfDNA中目的基因的突变位点或突变区域突变情况中的应用也是本发明保护的范围。
上述的方法或上述DNA文库在检测待测样本cfDNA中目的基因的突变位点或突变区域的突变频率中的应用也是本发明保护的范围。
上述检测待测样本目的基因待检区域的突变情况为检测待测样本目的基因待检区域的突变碱基或者突变氨基酸或者检测待测样本目的基因待检区域的突变频率。
突变频率的计算方法如下：
测序结果中，带有同一种分子标签的DNA分子为一种初始DNA模板的扩增产物，命名为1个家族；
检测该家族中的突变率，若该家族的突变率≥80％，则该家族记作带有分子标签的突变的DNA家族；
突变率＝(同一家族中编码氨基酸残基的密码子有突变的DNA分子的数量/同一家族中DNA分子总数)*100％；
突变频率＝测序结果中带有分子标签的突变的DNA家族的数量/测序结果中所有带有分子标签DNA家族的数量*100％。
备注：测序结果中带有同一分子标签的read(测序出的一条序列)数≥2才有统计意义
本发明另一个目的是提供一种检测待测样本cfDNA中目的基因待检区域的突变情况的方法。
本发明提供的方法，包括如下步骤：
1)按照上述第一个目的的方法制备DNA文库；
2)测序所述DNA文库，得到测序结果，根据测序结果分析待测样本cfDNA中目的基因待检区域的突变情况。
上述方法中，
所述通用序列1的核苷酸序列为序列1；
所述通用序列2的核苷酸序列为序列2；
所述测序接头1的核苷酸序列为序列3；
所述测序接头2的核苷酸序列为序列4；
所述用于区分不同样本的barcode序列分别为序列5-序列14；
所述待测基因为NRAS，对应的上游特异性引物序列和下游特异性引物序列分别为序列15和序列16或序列17和序列18；
所述待测基因为ALK，对应的上游特异性引物序列和下游特异性引物序列分别为序列19和序列20或序列21和序列22或序列23和序列24或序列25和序列26或序列27和序列28或序列29和序列30或序列31和序列32；
所述待测基因为PIK3CA，对应的上游特异性引物序列和下游特异性引物序列分别为序列33和序列34或序列35或序列36；
所述待测基因为ROS，对应的上游特异性引物序列和下游特异性引物序列分别为序列37和序列38；
所述待测基因为EGFR，对应的上游特异性引物序列和下游特异性引物序列分别为序列39和序列40或序列41和序列42或序列43和序列44或序列45和序列46或序列47和序列48；
所述待测基因为MET，对应的上游特异性引物序列和下游特异性引物序列分别为序列49和序列50或序列51和序列52或序列53和序列54或序列55和序列56；
所述待测基因为BRAF，对应的上游特异性引物序列和下游特异性引物序列分别为序列57和序列58或序列59和序列60；
所述待测基因为KRAS，对应的上游特异性引物序列和下游特异性引物序列分别为序列61和序列62或序列63和序列64；
所述待测基因为TP53，对应的上游特异性引物序列和下游特异性引物序列分别为序列65和序列66或序列67和序列68或序列69和序列70或序列71和序列72或序列73和序列74或序列75和序列76；
所述待测基因为ERBB2，对应的上游特异性引物序列和下游特异性引物序列分别为序列77和序列78。
上述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
本发明第3个目的是提供一种检测待测样本中目的基因的突变位点或突变区域的突变频率的方法。
本发明提供的方法，包括上述方法所述步骤，其中所述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
上述的方法或上述DNA文库在指导肿瘤用药或者判断肿瘤发展方向中的应用。
本发明第4个目的是提供一种指导待测患者肿瘤用药或者判断肿瘤发展方向的方法。
本发明提供的方法，先用包括上述方法所述步骤检测待测患者cfDNA中目的基因的突变位点或突变区域的突变频率，再根据突变频率指导待测患者肿瘤用药或者判断肿瘤发展方向。
本发明第5个目的是提供用于检测待测样本目的基因待检区域的突变情况的扩增子文库的试剂盒。
本发明提供的试剂盒，包括上述方法中的Barcode引物F1、上游引物F2、下游外引物R1、下游内引物R2。
附图说明
图1为分子标签的功能。A、B和C分别为不同的突变位点。
图2为受检者1血液样本提取的cfDNA文库构建完成后Agilent 2200 TapeStation Systems检测得到的扩增产物分布。
图3为受检者1的血液样本提取的cfDNA通过一步法获得的扩增子文库在Ion Torrent平台测序结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、构建用于检测目的基因低频突变的扩增子文库
肿瘤患者的血液、尿液和脑脊液中的cfDNA中的目的基因的突变位点或突变区域的突变频率会影响将来肿瘤用药或者肿瘤发展方向的判断，本实施例为了检测肿瘤患者的血液、尿液和脑脊液中的cfDNA中的目的基因的突变位点或突变区域的突变频率，构建用于检测目的基因低频突变的扩增子文库，具体如下：
一、用于检测目的基因低频突变的扩增子文库的引物组合设计合成
选择已知目的基因中一段区域作为待检区域设计合成如下引物：
该待检区域存在突变热点，只是待检样本中基因突变发生与否可以已知，也可未知
Barcode引物F1：测序接头1+barcode序列+通用序列1；
上游引物F2：通用序列1+分子标签+特定碱基序列+上游特异性引物序列；
下游外引物R1：测序接头2+通用序列2；
下游内引物R2：通用序列2+下游特异性引物序列；
其中，barcode序列是用来区分不同样本的序列，一个待测样本对应一个barcode序列，此Barcode序列长度为8-12nt，要求无连续碱基，GC含量为40-60％，引入Barcode序列的引物无明显二级结构等。F1是用来区分不同样本，只要是同一样本，F1均相同，与检测位点无关。
通用序列1和2长度为16-25nt，要求无连续碱基，GC含量为35-65％，引入序列的引物无明显二级结构等，该序列可根据需要变化，本实施例采用的是
通用序列1 GGCATACGTCCTCGTCTA(序列1)，大小18nt；
通用序列2 CGACATCGCCTCTGCTGT(序列2)，大小18nt。
测序接头1和测序接头2根据测序平台确定：
如果测序平台为Illumina平台，测序接头1和2分别为I5和I7，接头序列和芯片上的引物序列是互补的，加接头是为了把核酸片段连接到载体上。
如果测序平台为Ion Torrent平台，测序接头1和2分别为A和P(序列3和4)，A接头用来测序，与特异性引物互补，P接头与载体上序列互 补，用来将模板与载体连接。
特定碱基序列为GAT，不是基因特异扩增片段的部分，其作用是便于测序结果的生物信息分析，通过识别GAT序列来提高数据筛选的效率。
上游特异性引物序列和下游特异性引物序列为根据目的基因待检区域设计用于扩增其的引物，上游特异性引物序列大小为15-30nt，下游特异性引物序列大小为15-30nt。
分子标签为10-12位随机碱基，用来标记起始cfDNA模板。每位随机碱基有ATCG四种碱基形式，所以10位随机碱基总共存在1048576种不同的分子标签，以初始20ngDNA模板为例，其拷贝数为6000，而cfDNA的分子片段较短，所以能够扩增的有效模板拷贝数要小于6000，1048576种的分子标签形式完全可以为每一个初始模板加上特定的“标记”。通过分子标签来对测序结果的起始模板进行分类，就可以排除扩增错误及测序错误。
如图1所示，图1为所构建的文库中，带有同一个分子标签的5个扩增产物，其中A位点的突变在5个分子上都存在，而B和C位点的突变只在其中某一个扩增产物中存在，所占比例极低，就可以判定A位点的突变为初始模板分子中存在的突变，而B和C位点的突变则为建库的PCR扩增中或者测序过程中出现的假阳性的突变。所以，分子标签的作用是标记原始模板分子，识别原始模板中存在的突变，剔除PCR及测序过程中出现的假阳性突变，提高检测灵敏度。
二、检测方法的建立
1、将上述一的引物(F2，R1和R2)按特定比例混合，充分混匀后称引物mix，待用。
2、提取待检样本如肿瘤患者血液、尿液或脑脊液的cfDNA。
3、用不同样本对应的barcode引物和引物mix对cfDNA进行PCR扩增，向0.2ml的八连排管或96孔板中，依次加入如下表1所示的试剂，得到PCR扩增体系。
表1为PCR扩增体系
Figure PCTCN2018083822-appb-000001
Figure PCTCN2018083822-appb-000002
其中，引物mix中，R1、F2、R2引物加入初始浓度均为50μM，且R1:F2:R2(体积比)＝10:(1-5):(1-5)。
PCR扩增体系中，Barcode引物F1、上游引物F2、下游外引物R1和下游内引物R2的摩尔比如下：F1:R1:F2:R2的摩尔比＝6:(10-6):(1-3):(1-3)
4、在PCR仪(PCR仪使用Applied bio-system的2720 Thermal Cycler)上，运行如下表2所示扩增程序：
表2为扩增程序
Figure PCTCN2018083822-appb-000003
建库PCR过程中前两个循环的梯度退火温度的循环条件是对原始模板 进行初步扩增，也可以说是为不同原始模板加上特定的分子标签，而随后19循环的PCR条件是对原始模板进分子标签内扩增，同时，高浓度的F1，R1与低浓度的引物F2，R2，也保证了之后19个循环过程中所进行的为分子标签内扩增(即一般不会在扩增过程中添加其它分子标签)。
5、用移液枪吸取PCR反应液1.3倍体积的Agencourt AMPure XP Kit(BECKMAN COULTER,A63882)进行纯化回收PCR产物，得到用于扩增子测序的DNA文库。具体纯化步骤如下：
1)提前30分钟取出Agencourt AMPure XP Kit，充分涡旋后，室温静置。
2)PCR反应结束后，将磁珠再次充分涡旋，向体系中加入24μl磁珠，反复吹打5次以上或充分涡旋，室温静置5分钟。
3)将EP管转移至置于磁力架上，静置5分钟至溶液澄清后，用移液枪小心除去上清，注意不要触碰磁珠。
4)每管加入100μl新鲜配置的80％乙醇溶液，EP管置于磁力架上缓慢旋转2圈，静置5m，弃去上清。
5)重复4步一次。
6)将EP管打开，室温静置，使液体挥发干净，以磁珠表面无光泽为准，注意不要过分干燥磁珠。
7)从磁力架上取下EP管，加入30μl PCR级纯化水，涡旋混匀后，室温静置10分钟。
8)将上步的EP管置于磁力架上2分钟或直至溶液澄清后，用移液枪在远离磁石的一面小心吸取上清液，注意不要触碰磁珠。
至此，扩增子文库构建完成，采用QuBit定量来判断是否构建成功。
6、上机测序及结果分析
上述不同样本扩增的文库按照测定的浓度进行等比例混合，最后稀释到特定浓度，用二代测序仪测序，得到测序结果。
测序的结果经过数据处理、生物信息学分析之后得到检测基因的突变情况。数据处理过程包括测序数据的转换、质控、序列比对(参考基因组为NCBI GRCh37/Hg19)、突变位点分析等过程，通过数据处理分析后得到检测样本的突变情况和突变频率。
因文库扩增过程对原始模板进行分子标记，突变频率的计算方法如下：
测序结果中，带有同一种分子标签的DNA分子为一种初始DNA模板的扩增产物，命名为1个家族；
检测该家族中的突变率，若该家族的突变率≥80％，则该家族记作带有分子标签的突变的DNA家族；
突变率＝(同一家族中编码氨基酸残基的密码子有突变的DNA分子的数量/同一家族中DNA分子总数)*100％；
突变频率＝测序结果中带有分子标签的突变的DNA家族的数量/测序结果中所有带有分子标签DNA家族的数量*100％。
备注：测序结果中带有同一分子标签的read(测序出的一条序列)数≥2才有统计意义
实施例3、构建用于检测目的基因低频突变的扩增子文库
目的基因如表5所示，待测样本来源于49例已经鉴定为肺癌患者的受检者，本实施例的目的是用本发明的方法检测表5所示的49例患者的基因突变频率。
一、用于检测目的基因低频突变的扩增子文库的引物组合设计合成
根据实施例1的一的目的基因的突变位点或突变区域设计合成如下引物，具体见表3和表4：
表3为引物组合
Figure PCTCN2018083822-appb-000004
Figure PCTCN2018083822-appb-000005
特异性引物设计原则：退火温度55-65℃，尽可能少二级结构，GC含量35％-65％，引物长度16-30nt，引物之间不宜形成二级结构，具体如表4。
表4为各个基因对应的特异引物序列为引物组合
Figure PCTCN2018083822-appb-000006
Figure PCTCN2018083822-appb-000007
二、检测
1、与实施例1的二1相同，将三种引物(R1，F2和R2)按特定比例混合，充分混匀后称引物mix，F1:R1:F2:R2的摩尔比＝6:10:1:1，待用。
Barcode引物F1的浓度为1.67μM；
下游外引物R1的浓度为2.78μM；
上游引物F2的浓度为0.28μM；
下游内引物R2的浓度为0.28μM。
2、实际收集的49例受检者(均为已确诊的癌症患者)对应的FFPE样本(福尔马林固定后石蜡包埋的组织)与血液样本，提取FFPE样本的基因组DNA以及 血液样本的cfDNA。
3、与实施例1的二3相同；
4、与实施例1的二4相同；
5、与实施例1的二5相同；
受检者1文库的检测结果(包含了32个扩增子)如图2所示，图2为具体样本文库构建完成后Agilent 2200 TapeStation Systems检测得到的扩增产物分布图，横坐标为片段长度，纵坐标为信号强度(FU)，lower峰为25bp位置marker，upper峰为1500bp位置marker，如图2所示经PCR扩增后所得PCR产物集中在160-230bp范围内。
6、上机测序及结果分析，与实施例1的二5相同，结果如下：
图3为已诊断为肺癌患者的血液样本(样本1)提取的cfDNA采用本建库方法的Ion Torrent平台测序结果。
实际收集的49例受检者对应的FFPE样本与血液样本cfDNA检测结果如下表5所示，其中FFPE样本DNA和血液样本cfDNA检测的对比方法均采用安捷伦的SureSelect定制化服务进行捕获建库，血液样本cfDNA的检测方法二采用本专利方法进行建库，结果显示，FFPE样本与本专利方法对血液样本cfDNA所检测解决过的一致性高达87.76％，本专利方法与安捷伦的SureSlect定制服务进行捕获建库的方法同时对49例cfDNA检测的一致性高达95.92％，且该2例不一致的位点突变已经ddPCR验证与本专利方法所得变异检测结果一致，证明本专利方法对cfDNA的检测灵敏度要优于捕获法对cfDNA的检测。这些都充分说明了本发明的实际应用性和良好特异性。
表5为49例受检者对应的FFPE样本及血液样本cfDNA的检测结果
Figure PCTCN2018083822-appb-000008
Figure PCTCN2018083822-appb-000009
Figure PCTCN2018083822-appb-000010
注：无热点突变仅表示此本专利方法设计的此panel覆盖区域未检测到相关热点突变。
上述表5中各个突变位点代表的含义如表6所示：
表6为各个突变位点代表的含义
Figure PCTCN2018083822-appb-000011
Figure PCTCN2018083822-appb-000012
工业应用
本发明由于采取以上技术方案，具有以下优点：
1、操作简便，节省时间。传统的建库捕获技术操作繁琐，流程长，对操作人员要求高。本发明只涉及一步PCR反应及对应产物纯化步骤，简化了建库的操作流程，节省建库时间(两小时内可完成建库，从文库构建到上机结束及生信分析完成的整个流程可在24小时内完成)。
2、极高的检测灵敏度。该建库方法可检测低至0.1％的突变，待检样本可以是血液、尿液以及脑脊液等分离出的游离DNA，也可是传统的冰冻组织、石蜡切片及新鲜穿刺组织等提取的基因组DNA。
3、有效杜绝样本间交叉污染。在PCR起始便加入区分不同样本的barcode序列，且操作过程及步骤的简化有效的杜绝了建库过程中有可能造成的交叉污染。
4、降低建库成本。与传统的捕获技术相比，该文库制备所需成本大大降低。传统捕获建库时所使用的捕获探针成本高，其冗长的实验流程所涉及的试剂耗材也给捕获建库增加了很大的成本，相对而言，一步法建库过程将试剂耗材的使用量大大减少，建库成本也较传统捕获建库方法低得多。
5、节省空间。由于本方法只需一轮PCR，因此实验室要求分室只需3个房间(样本提取、PCR扩增间、文库纯化和测序)，与传统文库制备所需4房间(样本提取、PCR1、PCR2及文库纯化、测序)相比，节省空间需 求。
该方法除能检测组织样本外，还能够快速、简便、灵敏、特异的对血液、尿液以及脑脊液等样本中游离DNA的不同区域进行靶向扩增，并高效检测低至0.1％水平的突变，大大的简化实验操作，有效避免文库损失及污染，显著降低成本，提高效率。
灵活简单的建库方法以及极高的灵敏度是该专利的最大特点。

Claims (15)

  1. 用于检测待测样本目的基因待检区域的突变情况的扩增子文库构建方法，包括如下步骤：
    1)设计合成Barcode引物F1、上游引物F2、下游外引物R1、下游内引物R2；
    所述Barcode引物F1依次由测序接头1、用于区分不同样本的barcode序列和通用序列1组成；
    所述上游引物F2依次由通用序列1、分子标签、特定碱基序列和上游特异性引物序列组成；
    所述下游外引物R1依次由测序接头2和通用序列2组成；
    所述下游内引物R2依次由通用序列2和下游特异性引物序列组成；
    所述测序接头1和所述测序接头2为根据不同测序平台选择对应的测序接头；
    所述特定碱基序列为GAT；
    所述上游特异性引物序列和所述下游特异性引物序列是扩增所述目的基因待检区域的引物；
    2)用所述Barcode引物F1、所述上游引物F2、所述下游外引物R1和所述下游内引物R2对待测样本ctDNA进行一步PCR扩增，得到扩增产物，即为用于扩增子测序的DNA文库。
  2. 根据权利要求1所述的方法，其特征在于：
    所述barcode序列均为长度为8-12nt、无连续碱基，且GC含量为40-60％的核苷酸；
    或所述通用序列1和所述通用序列2的长度均为16-25nt，且无连续碱基，GC含量为35-65％，无明显二级结构；
    或所述分子标签为10-12位随机碱基；
    或所述测序平台为Illumina平台，所述测序接头1为I5，所述测序接头2为I7；
    或所述测序平台为Ion Torrent平台，所述测序接头1为A，所述测序接头2为P。
  3. 根据权利要求1或2所述的方法，其特征在于：
    所述PCR扩增中，所述Barcode引物F1、所述上游引物F2、所述下游外引物R1和所述下游内引物R2的摩尔比为6:(10-6):(1-3):(1-3)。
  4. 根据权利要求1-3中任一所述的方法，其特征在于：所述突变为低频突变。
  5. 根据权利要求1-4中任一所述的方法，其特征在于：所述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
  6. 权利要求1-5中任一所述的方法制备DNA文库。
  7. 权利要求1-5中任一所述的方法或权利要求6所述DNA文库在检测待测样本cfDNA中目的基因的突变位点或突变区域突变情况中的应用。
  8. 权利要求1-5中任一所述的方法或权利要求6所述DNA文库在检测待测样本cfDNA中目的基因的突变位点或突变区域的突变频率中的应用。
  9. 一种检测待测样本目的基因待检区域的突变情况的方法，包括如下步骤：
    1)权利要求1-5中任一所述的方法制备DNA文库；
    2)测序所述DNA文库，得到测序结果，根据测序结果分析待测样本目的基因待检区域的突变情况。
  10. 根据权要求9所述的方法，其特征在于：
    所述通用序列1的核苷酸序列为序列1；
    所述通用序列2的核苷酸序列为序列2；
    所述测序接头1的核苷酸序列为序列3；
    所述测序接头2的核苷酸序列为序列4；
    所述用于区分不同样本的barcode序列分别为序列5-序列14；
    所述待测基因为NRAS，对应的上游特异性引物序列和下游特异性引物序列分别为序列15和序列16或序列17和序列18；
    所述待测基因为ALK，对应的上游特异性引物序列和下游特异性引物序列分别为序列19和序列20或序列21和序列22或序列23和序列24或序列25和序列26或序列27和序列28或序列29和序列30或序列31和 序列32；
    所述待测基因为PIK3CA，对应的上游特异性引物序列和下游特异性引物序列分别为序列33和序列34或序列35或序列36；
    所述待测基因为ROS，对应的上游特异性引物序列和下游特异性引物序列分别为序列37和序列38；
    所述待测基因为EGFR，对应的上游特异性引物序列和下游特异性引物序列分别为序列39和序列40或序列41和序列42或序列43和序列44或序列45和序列46或序列47和序列48；
    所述待测基因为MET，对应的上游特异性引物序列和下游特异性引物序列分别为序列49和序列50或序列51和序列52或序列53和序列54或序列55和序列56；
    所述待测基因为BRAF，对应的上游特异性引物序列和下游特异性引物序列分别为序列57和序列58或序列59和序列60；
    所述待测基因为KRAS，对应的上游特异性引物序列和下游特异性引物序列分别为序列61和序列62或序列63和序列64；
    所述待测基因为TP53，对应的上游特异性引物序列和下游特异性引物序列分别为序列65和序列66或序列67和序列68或序列69和序列70或序列71和序列72或序列73和序列74或序列75和序列76；
    所述待测基因为ERBB2，对应的上游特异性引物序列和下游特异性引物序列分别为序列77和序列78。
  11. 根据权利要求7或8所述的应用或权利要求9或10所述的方法，其特征在于：所述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
  12. 一种检测待测样本中目的基因的突变位点或突变区域的突变频率的方法，包括权利要求1-4中任一所述方法所述步骤，其中所述待测样本为肿瘤患者的离体血液分离的cfDNA、肿瘤患者的离体尿液分离的cfDNA、肿瘤患者的离体脑脊液分离的cfDNA或肿瘤患者的离体肿瘤组织提取的基因组DNA。
  13. 权利要求1-5中任一所述的方法或权利要求6所述DNA文库或权 利要求9或10所述的方法在指导肿瘤用药或者判断肿瘤发展方向中的应用。
  14. 一种指导待测患者肿瘤用药或者判断肿瘤发展方向的方法，先用包括权利要求1-4中任一所述方法所述步骤检测待测患者cfDNA中目的基因的突变位点或突变区域的突变频率，再根据突变频率指导待测患者肿瘤用药或者判断肿瘤发展方向。
  15. 用于检测待测样本目的基因待检区域的突变情况的扩增子文库的试剂盒，包括权利要求1-4中任一所述方法中的Barcode引物F1、上游引物F2、下游外引物R1、下游内引物R2。
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