CN115992243A

CN115992243A - Primer combination, kit and library construction method for detecting ovarian cancer

Info

Publication number: CN115992243A
Application number: CN202211415748.9A
Authority: CN
Inventors: 姚小明; 张雅妮; 徐世美
Original assignee: Shenzhen Kairuisi Medical Technology Co ltd
Current assignee: Zhejiang Kairuisi Medical Technology Co ltd
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-04-21
Anticipated expiration: 2042-11-11
Also published as: CN115992243B

Abstract

Primer combination, kit and library construction method for detecting ovarian cancer, wherein the primer combination is used for targeted amplification of a locus in at least one of the following genes: PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, tele, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1. The invention can detect the gene mutation sites in a plurality of areas of 18 ovarian cancer oncogenes at the same time and directly reflect the specific mutation sites of related genes.

Description

Primer combination, kit and library construction method for detecting ovarian cancer

Technical Field

The invention relates to the field of gene detection, in particular to a primer combination, a kit and a library construction method for detecting ovarian cancer.

Background

Ovarian malignancy is a common malignancy in gynaecology. The ovarian tissue has complex composition, is the organ with the largest primary tumor types of all organs of the whole body, has great difference in tissue structure, biological behaviors and good development age of different types of ovarian tumors, and has various tissue types. Ovarian epithelial cancer is the most common histopathological type among them, accounting for 85% -90% of ovarian malignancies. Because of difficult early diagnosis and poor treatment effect, the death rate of the traditional Chinese medicine is the first place of gynecological malignant tumor, even exceeds the sum of cervical cancer and endometrial cancer, and seriously threatens the health of women. Ovarian cancer is the gynaecologic tumor with highest female death rate in China, and according to the statistics of Chinese cancer annual report, about 5.21 ten thousand new patients exist in 2015, and about 2.25 ten thousand patients die from the ovarian cancer in the same year.

Over the past few decades, mortality rates have increased due to ovarian cancer, which often requires surgical removal of accessories, even the uterus and fallopian tubes, which means that young patients lose fertility and, at the same time, loss of estrogen accelerates aging. Ovarian cancer has different clinical stage and obviously different prognosis. Ovarian cancer was found to be highly curable at an early stage. With the increasing progress of population aging in China, the incidence rate of ovarian cancer is increased year by year, and the incidence age tends to be younger. Few special symptoms are found in early stages of ovarian cancer, and early stage ovarian cancer may undergo intraperitoneal metastasis. Most ovarian cancer patients do not develop clinical symptoms until advanced stages, so 75% of patients have advanced at the time of diagnosis of ovarian cancer, and only 25% of patients are found in stage i. The survival rate of patients with advanced ovarian cancer (III-IV stage) in 5 years is only 20% -40%, but the survival rate of patients with early ovarian cancer (I-II stage) in 5 years is more than 70%. Therefore, the efficient, accurate and sensitive early screening method is the most important step for treating the ovarian cancer, and the effective early screening of the ovarian cancer can help to realize early diagnosis of the ovarian cancer, so that the survival rate of ovarian cancer patients is greatly improved. Thus, there is a greater need for timely prevention and early detection of ovarian cancer.

Currently, ovarian cancer is screened annually for high risk populations by examination of the pelvic cavity, transvaginal ultrasound, and CA125 biomarker detection. Clinical practice has shown, however, that there are significant limitations to the above approach. The accuracy of diagnosis of ovarian cancer by B ultrasonic is low, micro tumor is difficult to find, and the diagnosis is easily influenced by subjective experience of an examination operation doctor. The serum carcinoembryonic antigen CA125 has low detection specificity and sensitivity, can not be detected in early ovarian cancer usually, besides ovarian malignant tumor, many other diseases such as endometriosis, early pregnancy, inflammation, uterine fibroids, digestive tract malignant tumor, chronic hepatitis, liver cirrhosis and other serum CA125 are also highly expressed and are easily influenced by menstrual cycle and pregnancy, and the CA125 is rarely or hardly found in 20% of the serum of ovarian cancer patients. The combined application of CA125 and ultrasonic detection can only improve the positive detection rate by 20%.

It is currently accepted by the scientific community that all cancers are thought to be caused by somatic mutations (mutations in any cell in the body other than germ cells), although so far little is known to scientists about the mutation process involved. Cancer is essentially a genetic disease, all cancers are derived from genetic mutation, but not all genetic mutation is derived from the inheritance of parents, and many environmental factors such as virus infection, chemicals, rays and the like can cause genetic mutation, thereby causing cancer to occur. Studies have shown that there are a variety of genetic mutations in ovarian cancer, driving mutations have been found in many different genes, including ERBB2, ERBB3, FGFR2, HRA, KRAS, BRAF, PIK3CA, PIK3R1, ARHGAP35, RRAS2, NF1, PPP2R1A, PTEN, ZFHX3, FOXA2, KMT2D, ARID B, and FBXW7, etc., and that many studies have been performed to report mutation detection mutations for screening and diagnosis of ovarian cancer, and that single pathological typing of ovarian cancer has failed to meet clinical needs, requiring the identification of the mutation status of cancer tissue from the genetic level, providing a molecular basis for clinical use and efficacy prediction of targeted drugs. In view of this, there is an increasing clinical demand for accurate detection of ovarian cancer genetic mutations, and development of a method for detecting ovarian cancer-related polygenic mutations is necessary.

At present, common methods for detecting gene mutation include a fluorescent quantitative PCR method, a gene chip method, a Sanger method, a high throughput sequencing method and the like. The fluorescent quantitative PCR method and the gene chip method can only detect the site of one or a plurality of known mutations, and the Sanger sequencing method can only sequence a certain section of region of one sample at a time, and the common defects of detection are that the fluorescent quantitative PCR method and the gene chip method can not be used for detecting the site of the unknown mutation, the detection efficiency is low, the result is difficult to interpret, the repeatability is poor, the false positive and the false negative are more, and the like.

Disclosure of Invention

According to a first aspect, in one embodiment there is provided a primer combination for targeted amplification of a site in at least one of the following genes: PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, tele, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1.

According to a second aspect, in one embodiment there is provided a kit comprising the primer combination of the first aspect.

According to a third aspect, there is provided in one embodiment a library construction method comprising:

a linker ligation step comprising ligating a linker with a molecular tag to a nucleic acid sample to obtain a linker-ligated sample;

a first round of nested PCR amplification step comprising amplifying the adaptor-ligated sample using an outer primer to obtain a first round of amplification product;

and a second round of amplification step of nested PCR, wherein the second round of amplification step comprises the step of amplifying the sample connected with the connector by using an inner primer to obtain a second round of amplification product.

According to the primer combination, the kit and the library construction method for detecting ovarian cancer, which are disclosed by the embodiment, the mutation sites of genes in a plurality of areas of 18 ovarian cancer oncogenes can be detected at the same time, and the specific mutation sites of related genes can be directly reflected.

In one embodiment, the molecular tag nested multiplex PCR adopted by the invention can effectively eliminate false positive caused in the PCR process, improve the detection limit of a low-frequency mutation detection sample, and can detect 0.1% of low-frequency variation. Can efficiently and sensitively detect the mutation condition of the ovarian cancer oncogene, and simultaneously reduces the cost and simplifies the operation steps. Can be used for screening ovarian cancer, early diagnosis, auxiliary diagnosis, monitoring after cancer healing, targeted drug screening and the like.

Drawings

FIG. 1 is a flow chart of library construction in one embodiment.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.

The high-flux sequencing technology NGS is an international advanced novel sequencing technology, can perform sequence determination on hundreds of thousands to millions of DNA molecules at a time in parallel, and greatly improves the sequencing flux. Meanwhile, along with the rapid development of bioinformatics, through multi-gene and multi-site integrated detection and analysis, gene sites closely related to tumors can be detected and analyzed at one time, and the gene mutation information of patients can be detected.

The main flow high throughput sequencing technologies are mainly two kinds: one is large and whole genome-wide/transcriptome-wide pool sequencing, the other is small and refined targeted pool sequencing. Although the whole genome and whole exon sequencing methods can meet clinical needs in terms of throughput, they cannot be popularized in clinical applications due to high price. Therefore, the targeted library-building sequencing plays a positive role in clinical auxiliary diagnosis and treatment of diseases and tumor gene detection by virtue of the advantages of low cost, high data utilization rate, flexible combination and the like.

The library construction method in the targeted library construction sequencing technology is further divided into: the hybridization capturing method is used for establishing a library and the multiplex PCR method is used for establishing a library. The targeted multiplex PCR library establishment does not need to interrupt sample DNA, and target sequences are amplified simultaneously only by adding various primers, so that the library establishment flow is reduced, and the library establishment time is greatly shortened. Multiplex PCR is a PCR technique in which multiple pairs of primers are added to one reaction system, and different gene fragments of the same DNA sample are amplified simultaneously. By analyzing the sequences of the different gene fragments, whether the gene has deletion, insertion or point mutation is judged.

According to a first aspect, in an embodiment, a primer combination is provided for targeted amplification of a site in at least one of the following genes: PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, tele, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1.

In one embodiment, the primer set comprises an outer primer, an inner primer.

In one embodiment, the outer primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, the sequence number is 2n, and n is an integer not less than 0.

In one embodiment, the inner primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, wherein the sequence number is 2n+1, and n is an integer not less than 0.

In one embodiment, the primer combination is used to detect ovarian cancer.

According to a second aspect, in an embodiment, a kit is provided comprising the primer combination of the first aspect.

In one embodiment, at least one of a molecular tagged linker, library pretreatment reagent, targeted amplification reagent, library amplification reagent, I5 tag, I7 tag, purification reagent is also included.

In one embodiment, the library pretreatment reagent comprises at least one of a terminal addition "A" reagent, a linker ligation reagent.

In one embodiment, the purification reagent comprises magnetic beads, including but not limited to IGT ^TM Pure Beads or Agencou rt AMPure XP magnetic Beads.

According to a third aspect, in an embodiment, there is provided a library construction method comprising:

and a linker ligation step comprising ligating a molecular tagged linker to the nucleic acid sample to obtain a linker-ligated sample.

And a first round of amplification step of nested PCR, wherein the first round of amplification step comprises the step of amplifying the sample connected with the connector by using an outer primer to obtain a first round of amplification product.

In one embodiment, the first amplification step of the nested PCR comprises a primer that is complementary to at least a portion of the sequence-specific reverse complement of the molecular tagged linker.

In one embodiment, in the first amplification step of the nested PCR, the reaction system further comprises a targeted amplification reagent.

In one embodiment, the second amplification step of the nested PCR comprises a primer that is complementary to at least a portion of the sequence-specific reverse complement of the molecular tagged linker.

In one embodiment, the method further comprises a purification step comprising subjecting the second round amplification product to magnetic bead purification to obtain a purified product.

In one embodiment, the method further comprises a library amplification step comprising amplifying the purified product to obtain a library amplified product.

In one embodiment, in the library amplification step, the reaction system contains at least one of library amplification reagent, I5 tag, I7 tag.

In one embodiment, a further purification step is included, comprising magnetic bead purification of the library amplification products to obtain a library useful for on-press sequencing.

In one embodiment, in the adaptor-ligation step, the nucleic acid sample is a sample after the end repair, addition of "A" reaction.

According to a fourth aspect, in an embodiment, there is provided a library prepared by the library construction method of the third aspect.

In one embodiment, the molecular tag UMI (Unique molecular identifer) is added in the multiplex PCR process to help correct errors generated in the amplification and sequencing processes, for UMI marked reads, sequencing reads with the same UMI can be formed into a group in the data analysis process, and a final single-stranded consensus sequence (SSCS) can be obtained through data correction of the same group, the repeated sequence is removed according to the UMI sequence and the position on the genome, and the single-stranded consensus sequence is formed), so that a real mutation result is obtained, and background noise is reduced.

In one embodiment, the invention provides a multiplex PCR specific primer, a kit and a detection method for detecting ovarian cancer polygenic mutation based on a targeting high-throughput sequencing technology, which can detect a plurality of genes and a plurality of site mutations simultaneously, effectively improve detection efficiency and accuracy, reduce cost, simplify operation steps and the like.

In one embodiment, the invention provides a PCR primer pair, a kit and a method for detecting ovarian cancer polygenic mutation based on targeted high-throughput sequencing of molecular tag nested multiplex PCR. The adopted molecular tag nested multiplex PCR technology can effectively eliminate false positive brought in the PCR process, and improves the detection sensitivity and accuracy.

In one embodiment, the invention provides a primer pair for detecting ovarian cancer polygene based on high-throughput targeted sequencing of molecular tag nested multiplex PCR, wherein the nucleotide sequences of 2 rounds of nested multiplex PCR primers are shown in SEQ ID No.1 to SEQ ID No.300, the initial position list of the amplified target fragment region on the chromosome is shown in table 1, the first round of nested PCR amplification uses an outer primer, and the second round of nested PCR amplification uses an inner primer: the oncogene is PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, POLE, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1. The mutation may be a substitution, insertion and/or deletion of one or more bases.

The outer primer is specifically a sequence with the sequence number of 2n in the nucleotide sequences shown in SEQ ID No. 1-300, and n is an integer more than or equal to 0. Such as the nucleotide sequence shown as SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, and further such as the nucleotide sequence shown as SEQ ID No.152, SEQ ID No.154, SEQ ID No.156, SEQ ID No.158, etc.

The upstream primer in the outer primer is the nucleotide sequence shown as SEQ ID No. 1-150, the sequence number is 2n, n is an integer which is more than or equal to 1, such as the nucleotide sequences shown as SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 and the like.

The downstream primer in the outer primer is the nucleotide sequence shown as SEQ ID No. 151-300, the sequence number is 2n, n is an integer which is more than or equal to 1, such as the nucleotide sequence shown as SEQ ID No.152, SEQ ID No.154, SEQ ID No.156SEQ ID No.158, and the like.

The inner primer is specifically a sequence with the sequence number of 2n+1 in the nucleotide sequences shown in SEQ ID No. 1-300, and n is an integer more than or equal to 0. Such as the nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, and such as the nucleotide sequence shown as SEQ ID No.151, SEQ ID No.153, SEQ ID No.155, SEQ ID No.157, etc.

The upstream primer in the inner primer is the nucleotide sequence shown as SEQ ID No. 1-150, the sequence number is 2n+1, n is an integer more than or equal to 0, such as the nucleotide sequences shown as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, and the like.

The downstream primer in the inner primer is the nucleotide sequence shown as SEQ ID No. 151-300, the sequence number is 2n+1, n is an integer more than or equal to 0, such as the nucleotide sequences shown as SEQ ID No.151, SEQ ID No.153, SEQ ID No.155, SEQ ID No.157, etc.

In one embodiment, the specific primers shown in SEQ ID No.1 to SEQ ID No.300 of the present invention are specifically designed sequences for 18 ovarian cancer oncogenes, specifically, 18 oncogene mutation site regions such as PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, POLE, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1 and the like can be amplified, and the amplified products are subjected to high-throughput sequencing to obtain sequence information, so that mutation status of the genes in a sample can be detected. Because all the detection is of the target region fragment of the gene, in order to ensure the detection specificity of each region and increase the stability, 2 rounds of nest type multiplex amplification are adopted on the basis of the principle of the multiplex PCR amplification library construction method. The primers shown in SEQ ID No.1 to SEQ ID No.300 can be used for amplifying target sequences effectively in 1 tube by using 2 rounds of nested multiplex PCR amplification technology, the target sequences are amplified effectively in 1 tube by PCR product purification by the first round of multiplex PCR reaction, the target sequences are amplified effectively in 1 tube by the second round of multiplex PCR reaction, the target sequences are purified by the PCR products, post-PCR reaction and re-purification are carried out, the obtained PCR products are used for preparing a sequencing library and carrying out high-throughput sequencing on an upper machine, and the constructed sequencing library is applicable to various high-throughput sequencing platforms such as Huada and Illumina after being detected to be qualified by Qubit 3.0.

The primers are specifically as follows:

TABLE 1

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In one embodiment, high throughput targeted sequencing based on molecular tagged nested multiplex PCR to detect the primer pair sequences of ovarian cancer polygenes and the region of the amplified fragment of interest on the chromosome is also within the scope of the present invention.

In one embodiment, the invention provides a kit for detecting ovarian cancer polygene by high-throughput targeted sequencing based on molecular tag nested multiplex PCR, which comprises 2 rounds of multiplex PCR primers (an outer primer is used for nested PCR first round amplification, an inner primer is used for nested PCR second round amplification), library pretreatment reagents, targeted amplification reagents, library amplification reagents, I5 tags, I7 tags and purified magnetic beads.

In one embodiment, the end-added "A" reagent comprises end-added "A" buffer, end-added "A" enzyme.

In one embodiment, the adaptor ligation reagent comprises a adaptor with UMI, a DNA ligase, and a DNA ligation buffer.

In one embodiment, the targeted amplification reagent comprises a high fidelity DNA polymerase, PCR buffer, dNTP mix.

In one embodiment, the kit further comprises a primer AnchorSeq that is specifically complementary to the UMI-equipped linker ^TM An open Primer (for Illumina), as well as an outer Primer for nested PCR first round amplification and an inner Primer for nested PCR second round amplification.

In one embodiment, the library amplification reagents include high fidelity DNA polymerase, PCR buffer, dNTP mixture, anchorSeq ^TM UDI Primer。

In one embodiment, the purification beads are used to purify the size range DNA of interest from the targeted amplification products and library amplification products.

Example 1

The library building flow is shown in fig. 1, and the specific steps are as follows:

1. the genomic DNA standard for known mutations was tested 4 times, 25ng (2.5 ng/. Mu.L) each, and after disruption of genomic DNA, end repair was performed using a commercial kit, 3' end plus "A".

Preparing a reaction system:

TABLE 2

Reagent(s)	Volume of
		Fragmented DNA	10μL
End Repair&A-Tailing Buffer	7μL
		End Repair&A-Tailing Enzyme Mix	3μL
Nuclease-Free Water	40μL
		Total volume of	60μL

The PCR instrument parameters were set as follows:

TABLE 3 Table 3

2. Will be Anchor seq ^TM UMI Adapter (15. Mu.M, for Illumina, ai Jitai well) was diluted 10-fold to 1.5. Mu.M/. Mu.L in advance using Nuclear-Free Water. The previous step was completed with the sample with "A" at the 3' end for UMI-attached ligation.

Preparing a reaction system:

TABLE 4 Table 4

Reagent(s)	Volume of
		Sample after completion of the reaction in step 1	60μL
AnchorSeq ^TM UMI Adapter diluent	5μL
		Nuclease-Free Water	10μL
Ligation Buffer	30μL
		DNA Ligase	5μL
Total volume of	110μL

The PCR instrument parameters were set as follows:

TABLE 5

3. Using IGT ^TM The Pure Beads purified the ligated DNA samples of step 2 to give 13. Mu.L of eluate.

4. First round amplification by nested PCR using an outer primer pair, adding the outer mixed primer pair, a targeted amplification reagent, and a primer AnchorSeq complementary to the specificity of the UMI-equipped adaptor to a reaction system ^TM Anchor Primer (for Illumina), the target region was amplified in a first round of nested PCR.

The outer primer pair is specifically a sequence with the sequence number of 2n in the nucleotide sequences shown in SEQ ID No. 1-300, and n is an integer more than or equal to 0.

The following reaction system is prepared:

TABLE 6

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The PCR instrument parameters were set as follows:

TABLE 7

5. Using IGT ^TM The amplicon liquid of step 4 was purified by Pure Beads or Agencourt AMPure XP Beads to give 14 μl of eluate.

6. Performing nested PCR second round amplification by using the inner primer pair, adding the outer mixed primer pair, the targeted amplification reagent and a primer Anchor seq which is specifically complementary to the UMI-containing adaptor into a reaction system ^TM Anchor Primer (for Illumina), the target region was subjected to a second round of nested PCR amplification.

The inner primer pair is a sequence with the sequence number of 2n+1 in the nucleotide sequences shown in SEQ ID No. 1-300, and n is an integer more than or equal to 0

The following reaction system is prepared:

TABLE 8

Reagent(s)	Volume of
		Step 5 sample after completion of the reaction	11μL
AnchorSeq ^TM PCR Master Mix	15μL
		AnchorSeq ^TM Anchored Primer	2μL
Inner mixed primer pair	2μL
		Total volume of	30μL

The parameters of the PCR instrument are set as follows

TABLE 9

7. Using IGT ^TM Pure Beads purified the amplicon liquid of step 6 to give 15. Mu.L of eluate.

8. And (3) adding library amplification buffer solution, an I5 label, an I7 label and double distilled water into the amplicon liquid obtained in the step (7), and carrying out PCR reaction to enable sequencing adapter sequences to be carried on two sides of the amplicon.

The following reaction system is prepared:

table 10

Reagent(s)	Volume of
		Step 7 sample after the reaction	13μL
AnchorSeq ^TM PCR Master Mix	15μL
		AnchorSeq ^TM UDI Primer	2μL
Total volume of	30μL

The PCR instrument parameters were set as follows:

TABLE 11

9. Using IGT ^TM Pure Beads purified the amplicon liquid of step 8 to give 52. Mu.L of eluate. mu.L of the supernatant was pipetted into a new PCR tube and labeled for sequencing. 1 μl of library was taken and library concentration was sequenced using Qubit d sDNA HS Assay Kit and recorded. 1 μl of the library was used for fragment length measurement using a fragment analyzer.

10. After the detection is qualified, the Huada MGISEQ-2000 is subjected to machine sequencing.

The detection data of known mutation standards according to the technical flow show that the coverage (coverage rate) of each primer is 100%.

The test results are shown in Table 12.

Table 12

Continuous table 12

Continuous table 12

Continuous table 12

Continuous table 12

Table 12 is the right data of Table 12.

The meanings of the indices in table 12 are as follows:

sequencing data statistics:

(1) Raw reads (M): the total sequence number of the original data is M;

(2) Raw bases (Mb): total base number of the original data, wherein the unit is Mb;

(3) UMI reads (M): extracting the total sequence number of the molecular tag, wherein the unit is M;

(4) UMI bases (Mb): extracting total base number of the molecular tag, wherein the unit is Mb;

(5) The polling rate (%): filtration ratio, 1-UMI bases (Mb)/Raw bases (Mb)

(6) Clear reads (M): extracting the total sequence number of the filtered data after molecular tag extraction, wherein the unit is Mb;

(7) Clear bases (Mb): extracting the total number of bases remained after filtering the data after molecular tag extraction, wherein the unit is Mb;

(8) QC rate (%): clear ratio, clear bases (Mb)/UMI bases (Mb)

Library quality statistics:

(1) Sample: sample names, commonly filled in company numbers;

(2) Average read length: sequencing average read length (average read length after quality control is slightly shorter than original data read length);

(3) Average base quality: sequencing base average homogeneity value, typically > =30;

(4) Average insert size: the average library construction length is more than 200 when the reading length is 150;

(5) Alignment rate: alignment rate, read/clean reads aligned to the reference genome

(6) Target size: the size of the target area to be captured is measured in base units;

(7) Target covered size: the size of the target area actually covered;

(8) Coverage rate (%): coverage rate, revealing whether the Target area covers good indicators, target covered size/Target size;

(9) Total effffective bases (Mb): total number of effective bases;

(10) Target effffective bases (Mb): total number of bases covered by the target region;

(11) Bases capture rate (%): base capture rate, revealing an index of the sample data utilization rate, target effff ectivebases/Accurate mapped bases;

(12) Target average depth: average depth of Target region Target effffective bases/Target size;

(13) 1000X coverage rate (%): depth > = target area ratio of 1000X, 1000X coverage;

(14) 2000X coverage rate (%): depth > = target area ratio of 2000X, 2000X coverage;

(15) 5000X coverage rate (%): depth > = target area ratio of 5000X, 5000X coverage;

(16) 10000X coverage rate (%): depth > = target area ratio of 10000X, 10000X coverage;

(17) 5%X mean depth coverage rate (%): depth > = target area ratio of average depth 5%X (assuming average depth 20000 layers, ratio of 1000 layers or more);

(18) 10%X mean depth coverage rate (%): depth > = target area ratio of average depth 10% x;

(19) 20%X mean depth coverage rate (%): depth > = target area ratio of average depth 20% x;

(20) 30%X mean depth coverage rate (%): depth > = target area ratio of average depth 30% x;

(21) 40%X mean depth coverage rate (%): depth > = target area ratio of 40% x of average depth;

(22) 50%X mean depth coverage rate (%): depth > = target area ratio of average depth 50% x;

(23) Flank 10%X mean depth coverage rate (%): target area ratio of 10% depth on both sides of the average depth (assuming average depth 10000 layers, i.e., 9000-11000 layers);

(24) Average tag family size average tag family size, when the start and end positions of a fragment are identical to the tag, it is considered a family;

(25) SSCS effffective reads (M) number of valid reads before single ended correction;

(26) SSCS after reads (M), the number of valid reads after single ended correction;

(27) SSCS effffective rate (%) single ended correction ratio;

(28) SSCS Target average depth average depth of target area after single-ended correction;

(29) Again alignment rate (%) is the re-alignment rate after double-ended correction;

(30) DCS Target average depth average depth of target region after double-ended correction;

(31) Panel name: target area name.

The data for known mutation sites are as follows:

TABLE 13

Example 2

Endometrial exfoliated cells were collected from three Hunan elegans, genomic DNA extraction was performed using the Tiangen blood/cell/tissue genome kit (accession number DP 304), genomic DNA concentration was measured by Quibt, and sample processing and on-machine detection were performed according to the procedure of example 1.

The test data of the samples are shown in Table 14.

TABLE 14

Continuous meter 14

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Continuous meter 14

Continuous meter 14

The following table 14 is the right data of table 14.

As can be seen, alignment rate refers to the read/clean reads aligned to the reference genome, and over 98% of the short sequences can be aligned to the upper genome, with Coverage (Coverage rate) of 100% revealing good Coverage of the target region, demonstrating the high specificity of multiplex PCR sequence enrichment. And the average sequencing depth of the target region reaches about 1 ten thousand X.

Example 3

At least 3 genes are exemplified, and specific positions on the chromosome of the gene fragments are detected corresponding to the inner/outer primers of the nested PCR.

In order to improve the accuracy of mutation detection, 2 pairs of nested PCR primers are designed according to the initial sites of amplified fragments, and the following is an example of 3 fragments.

TABLE 15

Example 4

The invention is optimized from methodology, ovary mutation covers the 9330 locus of 18 genes, 3 pairs of primer pairs are designed in each round of amplified gene fragment 2 rounds of nest type schemes, 3 pairing schemes are designed, F end and R end are matched in multiple PCR steps, and the primer with coverage rate (coverage rate,%) reaching the optimal (100%) is selected to determine as the preferable primer pair. The following are examples of more than 3 gene embodiments for an optimization scheme.

Table 16

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In one embodiment, the method of the present invention is a highly efficient, reliable method of enriching high throughput sequencing sequences. The method has the advantages of high detection flux, strong specificity, wide sample sources, difficult pollution, high safety and the like for 9330 mutation sites of 18 genes of ovarian cancer, and the detection result has good repeatability and accuracy.

In one embodiment, the invention provides a primer pair, a kit and a method for detecting ovarian cancer polygenic mutation based on molecular tag nested multiplex PCR (polymerase chain reaction) high-throughput targeted sequencing. The invention specifically discloses a multiplex PCR primer shown in SEQ ID No. 1-SEQ ID No.308, a kit containing the primer, and a method for detecting a gene mutation site by combining 2 rounds of nested PCR amplification reaction with the primer or the kit and carrying out targeted enrichment on an ovarian cancer oncogene fragment.

In one embodiment, the method of the invention is a highly efficient and reliable method that allows for the enrichment of mutant features. The multiplex PCR primer, the kit and the detection method can detect the gene mutation sites in a plurality of areas of 18 ovarian cancer oncogenes at the same time, directly reflect the specific mutation sites of related genes, effectively eliminate false positive caused in the PCR process by adopting the molecular tag nested multiplex PCR, improve the detection limit of a detection low-frequency mutation sample, and detect 0.1% of low-frequency mutation. Can efficiently and sensitively detect the mutation condition of the ovarian cancer oncogene, and simultaneously reduces the cost and simplifies the operation steps. Can be used for screening ovarian cancer, early diagnosis, auxiliary diagnosis, monitoring after cancer healing, targeted drug screening and the like.

In one embodiment, the invention provides a primer pair, a kit and a method for detecting ovarian cancer polygene by high-throughput targeted sequencing based on molecular tag nested multiplex PCR.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A primer combination for targeted amplification of a site in at least one of the following genes: PTEN, TP53, PIK3CA, PIK3R1, KRAS, CTNNB1, FGFR2, RNF43, tele, PPP2R1A, FBXW7, AKT1, APC, BRAF, CDKN2A, EGFR, NRAS, MAPK1.

2. The primer combination of claim 1, wherein the primer combination comprises an outer primer, an inner primer;

preferably, the outer primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, wherein the sequence number is 2n, and n is an integer more than or equal to 0;

preferably, the inner primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, wherein the sequence number is 2n+1, and n is an integer not less than 0;

preferably, the primer combination is used for detection of ovarian cancer.

3. A kit comprising the primer combination of claim 1 or 2.

4. The kit of claim 3, further comprising at least one of a molecular tagged linker, library pretreatment reagent, targeted amplification reagent, library amplification reagent, I5 tag, I7 tag, purification reagent.

5. The kit of claim 4, wherein the library pretreatment reagent comprises at least one of a terminal addition "a" reagent, a linker ligation reagent;

preferably, the purification reagent comprises magnetic beads.

6. A method of library construction comprising:

7. The method according to claim 6, wherein the outer primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, wherein the sequence number is 2n, and n is an integer not less than 0;

preferably, the inner primer comprises at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 300, wherein the sequence number is 2n+1, and n is an integer not less than 0.

8. The method of library construction according to claim 6, wherein in the first round of amplification step of nested PCR, the reaction system contains a primer that is complementary to at least a portion of the sequence-specific reverse complement of the molecular tagged linker;

preferably, in the first round of amplification step of the nested PCR, the reaction system also contains a target amplification reagent;

preferably, in the second round of amplification step of the nested PCR, the reaction system contains a primer which is in specific reverse complementary pairing with at least part of the sequence in the adaptor with the molecular tag;

preferably, in the first round of amplification step of the nested PCR, the reaction system also contains a targeted amplification reagent.

9. The library construction method of claim 6, further comprising a purification step comprising subjecting the second round of amplification products to magnetic bead purification to obtain purified products;

preferably, the method further comprises a library amplification step, comprising amplifying the purified product to obtain a library amplification product;

preferably, in the library amplification step, the reaction system contains at least one of library amplification reagent, I5 tag and I7 tag;

preferably, the method further comprises a secondary purification step, wherein the secondary purification step comprises magnetic bead purification of the amplified products of the library to obtain a library which can be used for on-machine sequencing;

preferably, in the adaptor-ligation step, the nucleic acid sample is a sample subjected to an end repair and an "A" addition reaction.

10. Library prepared by the library construction method according to any one of claims 6 to 9.