WO2019061199A1 - A primer combination for performing simultaneous amplification of target region and whole genome, gene amplification method and application thereof - Google Patents

Abstract

The present invention provides a primer composition and amplification method and application for simultaneous target region and whole gene amplification, the primer composition comprising: a first set of primer sets, the first primer set comprising a specific primer 1 Primer pool and sequencing universal primer 1; a second set of primer sets including primer pool of specific primer 2, sequencing universal primer 1 and sequencing universal primer 2.

Description

Primer composition and amplification method and application for simultaneous target region and whole gene amplification Technical field

The invention belongs to the field of biotechnology, and relates to a primer composition and an amplification method and application for simultaneously performing target region and whole gene amplification.

Background technique

As one of the important components of genetic diagnosis, prenatal diagnosis is an important diagnostic project for clinical obstetrics. It diagnoses the genetic status of the fetus through physical, biochemical and genetic techniques and methods, with the aim of providing adequate families with genetic risks. Reliable information allows them to make appropriate choices during pregnancy. Prenatal diagnosis methods usually include two categories, traumatic (invasive) methods and non-invasive (non-invasive) methods. The former is mainly obtained by amniocentesis, villus sampling, umbilical vein blood sampling, etc. The genetic material of the fetus is detected, but there is a risk of miscarriage (0.2-3%), chorioamnion, amniotic fluid leakage, bleeding and infection at the puncture site, fetal bradycardia, premature rupture of membranes, etc. Prenatal diagnosis technology is the direction of clinical medical workers and scientific researchers.

In 1997, Lo et al. extracted plasma free DNA (cfDNA) from pregnant women's plasma and serum, and used the specificity of Y chromosome to judge the sex of the fetus. It was confirmed for the first time that there is fetal free DNA in maternal plasma. This important finding is non-invasive. The field of prenatal diagnosis has brought dawn. With the development of next-generation sequencing technology, cfDNA has been successfully applied to the screening of non-invasive prenatal fetal chromosome aneuploidy, and the detection of fetal chromosomes by high-throughput sequencing of free DNA in pregnant women's plasma.

In addition to genetic diseases caused by chromosomal abnormalities, defective genes such as substitutions, deletions, insertions, and frameshift mutations and splicing mutations of genes can also cause genetic diseases. These defective genes are usually derived from the germ cells of parents and are inherited. For the next generation, it is also known as a single genetic disease. There are many types of single-gene genetic diseases. According to incomplete statistics, more than 6,600 single-gene diseases have been discovered, and with the deepening of research, dozens of new single-gene diseases are added every year. Among the single-gene diseases that have been discovered, more than 1,000 diseases have clear pathogenesis and can be applied to clinical tests such as hemophilia, phenylketonuria, progressive muscular dystrophy, and thalassemia. Single-gene diseases can be divided into five types: autosomal dominant genetic diseases (AD, such as short-term symptoms, achondroplasia, etc.), autosomal recessive genetic diseases (AR, such as albinism, etc.), X-linked dominant inheritance Disease (XD, such as anti-vitamin D deficiency disease, etc.), X-linked recessive genetic disease (XR, such as color blindness, etc.), Y-linked genetic disease (YL, such as auricle long hair, etc.). Prenatal diagnosis combined with interventions can now be used to avoid the birth of children with these defects.

Chromosomal aneuploidy screening and single-gene disease screening use different strategies. The staining aneuploidy screening methods mainly include: large-scale parallel shotgun sequencing (MPSS), targeted large-scale parallel sequencing (t-MPS), and SNP-based multiplex PCR targeted sequencing. The most widely used is the large-scale parallel shotgun sequencing (MPSS) method, Huada Gene Dyeing. The NIFTY screening product for color aneuploidy is based on this method. The method obtains the number of nucleic acid fragments distributed on each chromosome by amplifying and sequencing the plasma free DNA of the mother and the fetus isolated from the mother plasma, and analyzing the aneuploidy of the fetal chromosome after counting and comparing, In addition, the method can also be used to detect deletions of large fragments.

For the detection of single-gene genetic diseases, the method of targeted enrichment is generally adopted. This method can effectively use precious sample resources to sequence specific nucleic acids most relevant to the study. Targeted enrichment is the sequencing of specific nucleic acid fragments, rather than the entire genome, which enables sequencing depth and sensitivity much higher than whole-genome sequencing, greatly improving the ability to detect mutations. The commonly used targeted enrichment methods are mainly based on probe capture target sequences, such as Roche's Seqcap, Agilent's Sureselect, etc., but these methods are cumbersome, time consuming, and costly, which limits the clinical promotion of single gene disease detection.

Currently, screening products for hereditary diseases mainly include NIFTY, a detection product for fetal chromosomal abnormality screening based on large-scale parallel sequencing, Hamony based on target region deep sequencing for detection of fetal chromosomal abnormalities, and fetal chromosomes based on target region SNP information. Arise for the detection of abnormal screening products.

The advantage of NIFTY is that it can amplify and sequence all the cfDNA of mother and fetus. It does not need to find the target sequence information, and has significant advantages in fetal chromosome aneuploidy analysis, but its disadvantage is that the amount of data required is large. Mutation detection in some target regions does not achieve good detection; Hamony amplifies the targeted region to detect target chromosomes and monogenic diseases, but the disadvantage is that large repeats may occur for the entire genome. There is no good screening effect, and its complex database construction process and expensive reagent cost limit its clinical application; Natera's detection of chromosomal abnormalities (deletion duplication) is limited by the SNP site on the region, Only target chromosomes and single-gene diseases can be detected, and other non-target chromosomes cannot be detected.

In order to simultaneously detect chromosomal abnormal diseases and single-gene diseases, 201510794535.5 discloses a method for simultaneously performing gene locus, chromosome and linkage analysis, which utilizes whole genome amplification technology combined with high-throughput sequencing to complete multiple comprehensive detections in one step. It avoids the use of multiple methods and multiple steps to detect single-gene genetic disease mutation sites, chromosomal diseases and linkage analysis. However, the method is to separately perform whole genome amplification and amplification of the target gene mutation site, and then mix the two amplification products in a certain ratio, and then build and sequence the same, and cannot use the same reaction system in the same reaction system. The sample completes the amplification of the whole genome and the target gene at the same time, and needs to consume more sample resources, and the product is highly susceptible to contamination during the mixing process, and the operation requirements are high.

Therefore, a method for simultaneously detecting chromosomal abnormalities and single-gene diseases is established, which overcomes the shortcomings of the prior art samples, such as large demand, cumbersome operation, and high detection cost, thereby solving most of the genetic defects, and has the advantages of prenatal and postnatal care in China. Great significance.

Summary of the invention

In view of the deficiencies and practical needs of the prior art, the present invention provides a primer composition and an amplification method and application for simultaneous target region and whole gene amplification, which can perform chromosomal abnormalities at the whole genome level. Screening can also detect mutations in the target targeting area.

To achieve the object of the present invention, the present invention adopts the following technical solutions:

In one aspect, the invention provides a primer composition for simultaneous target region and whole gene amplification, comprising:

a first set of primer sets comprising a primer pool of specific primer 1 and a sequencing universal primer 1, wherein the 3' end of the specific primer 1 comprises a target region-specific forward primer; the sequencing The 3' end of universal primer 1 comprises the complementary sequence of sequencing linker sequence 1 at one end of the target region;

a second set of primer sets comprising a primer pool of specific primer 2, a sequencing universal primer 1 and a sequencing universal primer 2, wherein the 3' end of the specific primer 2 comprises a target region-specific positive Primer, the 5' end comprises the sequence of the sequencing linker sequence 2 at the other end of the target region; the sequencing universal primer 2 comprises the sequence of the sequencing linker sequence 2 at the other end of the target region.

In the present invention, the 5' end of the specific primer 1 in the first set of primer sets contains 2-10 CG bases, and the 2-10 CG bases refer to the total number of C and G 2-10.

Preferably, the 5' end of the specific primer 1 in the first set of primer sets comprises 2 CG bases.

In the present invention, the CG base is used to balance the homogeneity between different primers, such as the TM value, so that the similarity between the different primers is better, and the uniformity of the amplification is better, thereby ensuring the data of each target region. Uniformity and stability, the 5' end of the specific primer 2 in the second set of primers comprising the sequencing linker sequence 2 was used for subsequent sequencing of the universal primer for anchoring.

In the present invention, the primer pool of the specific primer 1 and the primer pool of the specific primer 2 refer to all primers designed for the target region capable of amplifying the target region.

In the present invention, by using the first set of primer sets for the first round of amplification, a target region product having a sequencing linker sequence 1 at one end and a specific primer 1 at one end is obtained, and the original two remaining in the system are present. The whole genome product of the adaptor was added to the end; the second round of amplification was carried out with the second set of primers as the template, and the whole genome library was obtained; the specific primer 2 and the target region-specific sequence were combined. Sequencing universal primer 1 and adaptor sequence 1 are combined, and nest-specific amplification is performed to obtain a target region product having a linker sequence at both ends, and then amplified by sequencing universal primer 1 and sequencing universal primer 2 to obtain a target region. library.

According to the present invention, the distance between the specific primer 1 and the specific primer 2 is overlapped by 10 bp to 15 bp apart, that is, the distance between the specific primer 1 and the specific primer 2 is -10 bp to 15 bp, for example, -10bp, -9bp, -8bp, -7bp, -6bp, -5bp, -4bp, -3bp, -2bp, -1bp, 0bp, 1bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp or 15 bp, preferably -5 bp to 10 bp.

According to the invention, the linker sequence 1 and linker sequence 2 are independently selected from the sequencing linker of any of the second generation sequencing platforms of BGI SEQ-100, BGI SEQ-500, Proton or Illumina.

CAACTCCTTGGCTCACA.According to the present invention, the nucleotide sequence of the linker sequence 1 is shown in SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 is as follows: AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA

CAACTCCTTGGCTCACA.

The gray background portion is a 10 bp sequencing tag sequence for distinguishing sequencing samples.

According to the present invention, the nucleotide sequence of the linker sequence 2 is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows: AGCCAAGGTCAGTAACGACATGGCTACGATCCGACTT.

According to the invention, the sequencing universal primer 1 and the sequencing universal primer 2 are independently selected from sequencing universal primers of any of the second generation sequencing platforms of BGI SEQ-500, Proton or Illumina.

According to the present invention, the nucleotide sequence of the universal primer 1 is shown as SEQ ID NO. 3, and the nucleotide sequence shown in SEQ ID NO. 3 is as follows: TTGGAGCCAGGAGGTTG.

According to the present invention, the nucleotide sequence of the sequencing universal primer 2 is shown in SEQ ID NO. 4, and the nucleotide sequence shown in SEQ ID NO. 4 is as follows: GAACGACATGGCTACGACCGT.

In a second aspect, the invention provides a kit for simultaneous target region and whole gene amplification comprising the primer composition of the first aspect.

In a third aspect, the present invention provides a method for simultaneously performing a target region and whole gene amplification, using the primer composition of the first aspect, comprising the steps of:

(1) adding a tagged linker sequence to both ends of the sample to be tested;

(2) Two rounds of PCR amplification were performed. The first round of PCR amplification was performed using the first set of primer sets, and the second round of PCR amplification was performed using the second set of primer sets to construct a sequencing library.

The method of the invention can effectively increase the specificity of the primer by using two rounds of PCR.

According to the invention, the linker sequence is tagged.

According to the present invention, the conditions of the first round of PCR in the step (2) are: pre-denaturation at 95-99 ° C for 1-5 min; pre-denaturation at 95-99 ° C for 5-15 s, extension at 55-65 ° C for 1-5 min, a total of 15 -25 cycles; 70-75 ° C extension for 5 min.

According to the present invention, the conditions of the first round of PCR in the step (2) are: pre-denaturation at 98 ° C for 2 min, pre-denaturation at 98 ° C for 10 s, extension at 62 ° C for 2 min, a total of 20 cycles; and 72 ° C extension for 5 min.

According to the present invention, the conditions of the second round of PCR in the step (2) are: pre-denaturation at 95-99 ° C for 1-5 min; 95-99 ° C pre- Denaturation 5-15 s, extension 55-65 ° C 1-5 min, a total of 10-20 cycles; 70-75 ° C extension 5 min.

According to the present invention, the conditions of the second round of PCR in the step (2) are: 98 ° C 2 min; 98 ° C 10 s, 62 ° C 2 min, 15 cycles; 72 ° C 5 min, 1 cycle.

In the present invention, since the first round of PCR is used to amplify the target library, the second round of PCR is used to amplify the whole genome library, and those skilled in the art can adjust the target library by adjusting the number of cycles of the first round and the second round of PCR. The ratio of the whole genome library to the first round or the second round of the cycle can increase the proportion of the target library. Decreasing the first round and increasing the number of cycles in the second round can reduce the proportion of the target library to meet the difference. The need to detect data.

According to the present invention, before step (1), the steps of extracting the sample, repairing the end, and adding A, the extraction sample, the end repair and the addition of A are conventional techniques in the art, and those skilled in the art can select according to the needs. This is not a special limitation.

According to the invention, step (1) further comprises a step of purifying, preferably using magnetic beads.

According to the invention, step (2) further comprises the step of data analysis.

According to the present invention, the data analysis specifically includes: original offline data filtering, comparing the reference genome with BWA, and performing target region analysis and genome-wide analysis, respectively. Preferably, the whole genome analysis comprises: removing non-specific amplification products outside the target region caused by specific primers by primer sequences, performing homogeneity analysis on the removed data, and removing non-specific products as needed The genome-wide data is analyzed for specific analysis, such as abnormal chromosome number analysis and chromosome structural variation analysis.

According to the invention, the target region analysis specifically comprises a mutation analysis.

According to the invention, the mutation comprises any one of a point mutation, an insertion deletion or a gene fusion or a combination of at least two types.

According to the present invention, the analysis of the point mutation specifically includes the following steps: counting the reads of the target region, removing the specific primer sequences on the reads, analyzing and counting the reads after removing the specific primers in the target region, using samtools for the mutation Site base type and ratio were analyzed.

According to the present invention, the whole genome analysis specifically comprises: removing non-specific amplification outside the target region caused by the specific primer by the primer sequence, performing homogeneity analysis on the removed data, and removing the non-specific product as needed. The genome-wide data is analyzed for specific analysis, such as abnormal chromosome number analysis and chromosome structural variation analysis.

In a fourth aspect, the present invention provides a system for simultaneously performing target region and whole gene amplification, comprising:

(1) Sample processing module: for extracting a sample, and adding a labeled link sequence to both ends of the sample;

(2) PCR amplification module: connected to the sample processing module for performing two rounds of PCR amplification, the first round of PCR amplification uses the first set of primer sets, and the second round of PCR amplification uses the second set of primer sets to construct Sequencing library.

Preferably, the system further comprises a data analysis module: connected to the PCR amplification module for sequencing the constructed library and analyzing the data.

In a fifth aspect, the present invention provides the use of the primer composition of the first aspect for detecting an abnormality of a sample to be tested and an abnormality in the number of chromosomes.

Compared with the prior art, the present application has the following beneficial effects:

(1) The primer composition of the invention can simultaneously achieve whole genome amplification and multiple target region amplification, requires a small sample size, reduces cost, is simple to operate, and obtains stable target data, and the target region data can be used for multiple Detection of mutation types, and target region amplification and whole genome amplification do not interfere with each other;

(2) The method of the present invention can complete the construction of the whole genome and the target region with only one sample. The whole genome library and the target library are sequenced to obtain different depth sequencing data, and the whole genome data is low depth full coverage. It can be used for the detection of chromosomal data and structural abnormalities within the genome. The data of the target region is high-depth and can be used for the detection of various types of mutations such as point mutations and small insertions and deletions;

(3) The method of the invention has great application value in the field of non-invasive prenatal detection.

DRAWINGS

Figure 1 is a schematic diagram showing the design of specific primer 1 and specific primer 2 of the present application;

2 is a flow chart of a method for simultaneously performing target region amplification and whole gene amplification in the present application;

Figure 3 is a flow chart showing the construction of the target region and the whole genome library of the present application;

4 is a diagram showing the results of detection of the sequencing library 2100 in the embodiment of the present application;

FIG. 5 is a flowchart of information analysis of an embodiment of the present application;

Figure 6 is a diagram showing the results of in-depth analysis of the target region of the FGFR3 gene of the present application;

Figure 7 is a diagram showing the results of amplification stability analysis of different samples of the FGFR3 gene of the present application;

8 is a diagram showing the results of data distribution on the whole genome of the method of the present application, wherein the white histogram is the reference chromosome size, and the black histogram is the only number of reads on the modified chromosome after deduplication;

Figure 9 is a graph showing the stability analysis of the method on the whole genome of the genome of the present application.

Detailed ways

In order to further explain the technical means and effects of the present invention, the present invention will be further described below in conjunction with the embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The specific techniques or conditions are not indicated in the examples, according to the techniques or conditions described in the literature in the art, or in accordance with the product specifications. If the reagents or instruments used do not indicate the manufacturer, they are all commercially available through formal channels. Regulatory products.

Example 1 Primer Composition of FGFR3 Gene

Primers designed according to the FGFR3 gene, as shown in Figure 1, primers covering the FGFR3 gene and ACH-related hotspot mutations, the primer pool of the specific primer 1 designed as shown in SEQ ID NO. 5-22, as shown in Table 1, the specificity of the design Primer 2 The primer pools of SEQ ID NOS. 23-40 are shown in Table 2, for a total of 18 pairs, as follows:

Table 1 Specific primer 1

Primer number Primer sequence (5'-3') SEQ ID NO. 5 CGTGGCCCCTGAGCGTCATC SEQ ID NO.6 CGAGCCGAGGAGGAGCTGGT SEQ ID NO.7 CGGGCTTCTTCCTGTTCATCC SEQ ID NO.8 CGGAGATGGAGATGATGAAGATGATC SEQ ID NO.9 CGCCTTCCCCAGTGCATCCA SEQ ID NO.10 CGCCCAGCAGTGGGGGCTCG SEQ ID NO.11 CGGGCCCCTGAGCGTCATCT SEQ ID NO.12 CGTGCTGGGCAGCGACGTGG SEQ ID NO.13 CGCCGGGACGTGCACAACCT SEQ ID NO.14 CGACGAGGCGGGCAGTGTGT SEQ ID NO.15 CGCCGGGACGTGCACAACCT SEQ ID NO.16 CGCGCCAGGCCTCCTGGAGC SEQ ID NO.17 CGACGCGTCCATGAGCTCCAACAC SEQ ID NO.18 CGTGCTCCTGGGACGGGTGTAT SEQ ID NO.19 CGTTTGCACACTCATGGTCCCTC SEQ ID NO.20 CGCTGAGGAGCCCGTGTCCCCAG SEQ ID NO. 21 CGGCATCCTCAGCTACGGGGTG SEQ ID NO.22 CGTGTCTGAGATGGAGATGATGAAG

Among them, all the specific primers 1 were mixed at an equimolar concentration (10 μM) to obtain a primer pool of the specific primer 1, and the final concentration of the primer pool was 10 μM.

Table 2 Specific primers 2

Primer number Primer sequence (5'-3')

SEQ ID NO.23 GAACGACATGGCTACGATCCGACTTAGCGTCATCTGCCCCCAC SEQ ID NO.24 GAACGACATGGCTACGATCCGACTTGTGGAGGCTGACGAGGCG SEQ ID NO.25 GAACGACATGGCTACGATCCGACTTTGTTCATCCTGGTGGTGG SEQ ID NO.26 GAACGACATGGCTACGATCCGACTTGAAGATGATCGGGAAACACAAAAAC SEQ ID NO.27 GAACGACATGGCTACGATCCGACTTTGCATCCACAGGGACCTGG SEQ ID NO.28 GAACGACATGGCTACGATCCGACTTGTGGGGGCTCGCGGACGT SEQ ID NO.29 GAACGACATGGCTACGATCCGACTTTGCCCCCACAGAGCGCTC SEQ ID NO.30 GAACGACATGGCTACGATCCGACTTGGAGTTCCACTGCAAGGTGT SEQ ID NO.31 GAACGACATGGCTACGATCCGACTTGTGCACAACCTCGACTACTACAAG SEQ ID NO.32 GAACGACATGGCTACGATCCGACTTTATGCAGGCATCCTCAGCTAC SEQ ID NO.33 GAACGACATGGCTACGATCCGACTTGTGCACAACCTCGACTACTACAAG SEQ ID NO.34 GAACGACATGGCTACGATCCGACTTCCACCTCGGCCCACGCTGG SEQ ID NO.35 GAACGACATGGCTACGATCCGACTTACTGGTGCGCATCGCAAGGCT SEQ ID NO.36 GAACGACATGGCTACGATCCGACTTGCCCCTCTCAAGGTGCCCTG SEQ ID NO.37 GAACGACATGGCTACGATCCGACTTCTCCACTGCCAGGCTGACCCT SEQ ID NO.38 GAACGACATGGCTACGATCCGACTTCCTGTACGTGCTGGTGGAGTA SEQ ID NO.39 GAACGACATGGCTACGATCCGACTTGGCTTCTTCCTGTTCATCCT SEQ ID NO.40 GAACGACATGGCTACGATCCGACTTTGATCGGGAAACACAAAAACATCATC

Among them, the crude is a linker sequence, and all the specific primers 2 were mixed at an equimolar concentration (10 μM) to obtain a primer pool of the specific primer 2, and the final concentration of the primer pool was 10 μM.

Table 3 Sequencing universal primers and linker sequences

The bold in SEQ ID NO. 1 is a sequencing 10 bp tag sequence for distinguishing different sequencing samples for parallel sequencing.

Example 2 FGFR3 gene mutation and chromosome number abnormality simultaneous detection

The FGFR3 gene mutation and the abnormal chromosome number are simultaneously detected, and the detection flow chart is shown in FIG. 2, which specifically includes the following steps:

(1) Sample extraction:

10 cases of maternal plasma (from Beijing 301 Hospital, informed consent has been signed with patients, the sample can be used for scientific research), five cases of men and women, 12-14 weeks of gestational age, including 1 case of FGFR3 gene mutation (c.1138G >C, one case of 18 trisomy, one case of 21 trisomy, the rest of the samples are normal, and the sample information is shown in Table 4).

Table 4: Sample Information

10 samples of plasma were extracted using QIAGEN free DNA extraction kit (Ziagen, Germany, Cat. No. 180023), and the obtained cfDNA was dissolved in 40 μl of AE solution, and the obtained cfDNA was subjected to library preparation according to the following procedure ( The library was prepared by the joint probe anchor polymerization and sequencing method of Huada Gene Co., Ltd., the product number BOX3);

(2) End repair and add A

End repair of cf DNA, and base A at the 3 end of the DNA strand, end repair plus A reaction system Use the joint probe anchor polymerization and sequencing method of Huada Gene Company to build the library kit (Cat. No. BOX3), as shown in Table 5:

Table 5: End repair and A reaction system

Reaction conditions: 37 ° C, 15 min, 65 ° C, 15 min;

(3) Adding a linker, the system for the ligation reaction of the linker is constructed by the Joint Probe Anchor Polymerization Sequencing Method of the Huada Gene Company (Cat. No. BOX3), as shown in Table 6:

Table 6: Joint connection reaction system

Reaction conditions: 23 ° C, 30 min;

After completion of the reaction, 60 μl of AXYGEN purified magnetic beads (MAG-FRAG-I-50, Corning, USA) was added for purification, and the obtained DNA was dissolved in 20 μl of distilled water.

(4) performing two rounds of PCR amplification, as shown in FIG. 3, the first round of PCR, the primer pool of the specific primer 1 is as shown in Table 1 of the first embodiment, and the first round of the PCR system is jointly explored by the Huada Gene Company. Needle-anchored polymerization sequencing method to build a library kit (item number BOX3), as shown in Table 7:

Table 7: PCR system

Note: Primer pool for specific primer 1 (10 μM): The sum of all primer concentrations was 10 μM.

The reaction conditions were as follows: pre-denaturation at 98 ° C for 2 min; pre-denaturation at 98 ° C for 10 s, extension at 62 ° C for 2 min, a total of 20 cycles; 72 ° C extension for 5 min;

After the reaction was completed, 60 μl of AXYGEN purified magnetic beads were added for purification, and the obtained DNA was dissolved in 20 μl of distilled water;

For the second round of PCR, the primer pool of the specific primer 2 is as shown in Table 2 of the first embodiment, and the second round of the PCR system uses the joint probe anchor polymerization sequencing method of the Huada Gene Company to build the library kit (Cat. No. BOX3), as shown in Table 8 shows:

Table 8: PCR system

Note: Primer pool for specific primer 2 (10 μM): The sum of all primer concentrations was 10 μM.

The reaction conditions are as follows: 98 ° C 2 min; 98 ° C 10 s, 62 ° C 2 min, 15 cycles; 72 ° C 5 min, 1 cycle;

After the reaction was completed, 60 μl of AXYGEN purified magnetic beads were added for purification, and the obtained DNA was dissolved in 20 μl of distilled water.

The quality of the library is shown in Figure 4. The size of the library and the plasma free DNA size are close to the target region and the whole genome region. The main peak is 245 bp, and there are two small peaks of 400 bp and 577 and plasma free size anastomosis (plasma free DNA). Size plus the 84 bp linker sequence of BGISEQ-500), sequenced on BGISEQ500 after sequencing, and the sequencing type was double-ended 50 bp.

(5) Data analysis, the flow is shown in Figure 5. The data is filtered and compared with bwa. The readings of the target area are analyzed and statistically analyzed, and then the mutation sites are analyzed by a specific algorithm; Sex primer Non-specific amplification outside the target region, and the obtained data is subjected to genome-wide analysis, including chromosomal abnormalities, fetal sex, fetal free DNA concentration, and the like;

The data of the machine is shown in Table 9:

Table 9: Lower machine data

Sequencing type BGISEQ-500PE50+10, the original off-machine data of each sample is above 10M, the comparison rate is between 91.8-94.6%, the target area data is between 1.1-1.6%, and the target area average depth is 5002-7576× The average depth outside the target area (wide genome range) is between 0.23-0.27×.

(1') Analysis of target area data

(a) Performance assessment of the target area

The performance of the target area is evaluated. The results are shown in Figure 6-7. As can be seen from Figure 6, the depth distribution of each amplified region, where the 0.1× average depth value reaches 94.3%, the uniformity of amplification is good, from the figure. 7 can be seen that the stability of different sample amplification: the depth comparison of each amplified region obtained by two experiments in the same sample, where R2=0.9934, the stability is good;

(b) Target region mutation analysis

The results of FGFR3 positive samples are shown in Table 10:

Table 10: FGFR3 positive sample test results

A total of 10 samples, of which sample 2 was a point mutation positive sample on exon 16 of FGFR3 gene (c.1138G>C), the experiment can correctly detect the site, and the mutation detection G accounts for the proportion of the entire base at the position. At 5.2%, the remaining samples did not detect mutations on the FGFR3 gene.

(2') Whole genome data analysis

(a) Distribution of data on each chromosome

The results of data distribution on each chromosome are shown in Figure 8. It can be seen that one of the samples (sample 1) is selected and analyzed for the distribution of data on the whole genome (on each chromosome). The number of reads on the chromosome is basically consistent with the size of the reference chromosome.

(b) Analysis of data stability on chromosomes

The data stability analysis on the chromosome is shown in Figure 9. The data on the chromosomes between the two samples is statistically proportional. The abscissa is the proportion of each chromosome in one sample, and the ordinate is the chromosomal data in the other sample. The ratio, where R ² =0.9999, is good.

(c) Chromosome abnormality detection

Three-body detection, according to the specific algorithm (Z-Score, Z-Score algorithm reference Fan Jiang, Jinghui Ren, Fang Chen: Noninvasive Fetal Trisomy (NIFTY) test: an advanced noninvasive prenatal diagnosis method for fetal autosomal and sex chromosomal aneuploidies. BMC Medical Genomics 2012 5:57) Calculate whether the chromosome is abnormal. The test results are shown in Table 8:

Table 11: Three-body test results

* Non-invasive prenatal test results are the results obtained using the NGFTY test of Huada Gene.

In this experiment, 10 samples were shared, of which 7/8 samples were T18 trisomy and T21 trisomy positive samples, our method can detect the chromosomal abnormality correctly, and the other samples did not detect chromosomal abnormalities; fetal free DNA concentration and The non-invasive prenatal results were compared with the error of ±0.4%; the fetal gender judgment was consistent with the non-invasive prenatal test results, and the method was consistent with the non-invasive prenatal test results.

In summary, the method of the present application can simultaneously amplify the whole genome and multiple target regions, and obtain low-depth whole genome data and high-depth target region data. For multiple target areas, there is good uniformity and stability. High-depth target area data can detect multiple mutation types in the target area. For whole genome data, it also has good uniformity and stability, low depth. Whole genome data can be used to detect chromosome number and structural abnormalities, and amplification of the target region does not affect the results of genome-wide chromosome detection.

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims (15)

A primer composition for simultaneously performing target region and whole gene amplification, comprising: a first set of primer sets comprising a primer pool of specific primer 1 and a sequencing universal primer 1, wherein the 3' end of the specific primer 1 comprises a target region-specific forward primer; the sequencing The 3' end of universal primer 1 comprises the complementary sequence of sequencing linker sequence 1 at one end of the target region; a second set of primer sets comprising a primer pool of specific primer 2, a sequencing universal primer 1 and a sequencing universal primer 2, wherein the 3' end of the specific primer 2 comprises a target region-specific positive Primer, the 5' end comprises the sequence of the sequencing linker sequence 2 at the other end of the target region; the sequencing universal primer 2 comprises the sequence of the sequencing linker sequence 2 at the other end of the target region. The primer composition according to claim 1, wherein the 5' end of the specific primer 1 in the first set of primer sets contains 2 to 10 CG bases. The primer composition according to claim 1 or 2, wherein the specific primer 1 and the specific primer 2 have a distance of -10 bp to 15 bp. The primer composition according to any one of claims 1 to 3, wherein the 5' end of the specific primer 1 in the first set of primer sets comprises 2 CG bases; Preferably, the distance between the specific primer 1 and the specific primer 2 is -5 bp to 10 bp; Preferably, the nucleotide sequence of the linker sequence 1 is as shown in SEQ ID NO. Preferably, the nucleotide sequence of said linker sequence 2 is set forth in SEQ ID NO. The primer composition according to claim 1 or 2, wherein the sequencing universal primer 1 and the sequencing universal primer 2 are independently selected from a sequencing universal primer of any one of the second generation sequencing platforms; Preferably, the nucleotide sequence of the sequencing universal primer 1 is as shown in SEQ ID NO. 3; Preferably, the nucleotide sequence of the sequencing universal primer 2 is as shown in SEQ ID NO. A kit for simultaneously performing a target region and whole gene amplification, characterized in that it comprises the primer composition according to any one of claims 1 to 5. A method of simultaneously performing a target region and whole-gene amplification, characterized in that the primer composition according to any one of claims 1 to 5 comprises the following steps: (1) adding a linker sequence to both ends of the sample to be tested; (2) Two rounds of PCR amplification were performed. The first round of PCR amplification was performed using the first set of primer sets, and the second round of PCR amplification was performed using the second set of primer sets to construct a sequencing library. The method of claim 7 wherein said linker sequence is tagged. The method according to claim 7 or 8, wherein the condition of the first round of PCR in step (2) is: 95-99 ° C pre-denaturation 1-5 min; 95-99 ° C pre-denaturation 5-15 s, 55-65 ° C extension 1-5 min, a total of 15-25 cycles; 70-75 ° C extension 5 min; Preferably, the conditions of the first round of PCR in step (2) are: pre-denaturation at 98 ° C for 2 min; pre-denaturation at 98 ° C for 10 s, extension at 62 ° C for 2 min, a total of 20 cycles; and 72 ° C extension for 5 min. The method according to any one of claims 7-9, wherein the condition of the second round of PCR in step (2) is: pre-denaturation at 95-99 ° C for 1-5 min; pre-denaturation at 95-99 ° C 5- 15s, 55-65 ° C extension 1-5min, a total of 10-20 cycles; 70-75 ° C extension 5min; Preferably, the conditions of the second round of PCR in step (2) are: 98 ° C 2 min; 98 ° C 10 s, 62 ° C 2 min, 15 cycles; 72 ° C 5 min, 1 cycle. The method according to any one of claims 7 to 10, further comprising the steps of extracting a sample, repairing the end, and adding A before step (1); Preferably, step (1) further comprises a step of purifying, preferably using magnetic beads for purification. The method according to any one of claims 7-11, characterized in that after step (2), the step of data analysis is further included; Preferably, the data analysis specifically comprises: filtering the original offline data, comparing the reference genome with the BWA, performing target region analysis and genome-wide analysis respectively; Preferably, the target region analysis comprises a mutation analysis; Preferably, the mutation comprises any one of a point mutation, an insertion deletion or a gene fusion or a combination of at least two types; Preferably, the analysis of the point mutation specifically comprises the following steps: counting the reads of the target region, removing the specific primer sequences from the reads, analyzing and counting the specific primers after the target region is removed, and using the samtools pair of mutations. Point base type and ratio for analysis; Preferably, the whole genome analysis comprises: removing non-specific amplification outside the target region caused by the specific primer by the primer sequence, performing homogeneity analysis on the removed data, and removing the non-specific product as needed Whole genome data for specific analysis. A system for simultaneous target region and whole gene amplification, comprising: (1) Sample processing module: for extracting a sample, and adding a labeled link sequence to both ends of the sample; (2) PCR amplification module: connected to the sample processing module for performing two rounds of PCR amplification, the first round of PCR amplification uses the first set of primer sets, and the second round of PCR amplification uses the second set of primer sets to construct Sequencing library. The system of claim 13 wherein said system further comprises a data analysis module: and PCR Amplification modules are connected for sequencing and data analysis of the constructed library. Use of a primer composition according to any one of claims 1 to 5 for detecting mutations and chromosome numbers or structural abnormalities of a sample to be tested.

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