CN117143984A

CN117143984A - Primer composition for screening carrier with expansibility of genetic diseases and detection method

Info

Publication number: CN117143984A
Application number: CN202311097845.2A
Authority: CN
Inventors: 邓红辉; 廖大光; 蔡晓然; 卢晨丽; 闫超; 邢阿宝
Original assignee: Guangzhou Aoce Medical Laboratory Co ltd
Current assignee: Guangzhou Aoce Medical Laboratory Co ltd
Priority date: 2023-08-29
Filing date: 2023-08-29
Publication date: 2023-12-01

Abstract

The invention discloses a primer composition for screening a carrier with expansibility of genetic diseases, and the nucleotide sequence of the primer is shown as SEQ ID NO. 1-220. The primer composition can detect the mutation of 420 genes of 435 diseases and the copy number variation of partial diseases. The invention also discloses an expansibility carrier screening method based on high-throughput sequencing. The primer group provided by the invention can be used for effectively, rapidly and accurately detecting all the gene mutation sites at one time, so that the diagnosis process can be simplified, the detection cost can be reduced, and the detection timeliness can be improved.

Description

Primer composition for screening carrier with expansibility of genetic diseases and detection method

Technical Field

The invention relates to the field of gene detection, in particular to a primer composition for screening a carrier with expansibility of genetic diseases and a detection method.

Background

Carrier screening is a genetic testing technique used to identify individuals carrying a particular genetic disease. The expansibility carrier screening is an expansion of single carrier screening, and can screen several, tens or even hundreds of genetic diseases at a time, and the purpose of carrier screening is to detect individuals carrying the genetic diseases. The carrier can know the carrying condition of the pathogenic genes of the carrier before or at early stage of pregnancy in advance so as to take corresponding preventive measures, avoid the occurrence of offspring genetic diseases and avoid the later high economic burden with low screening cost.

Single gene genetic diseases in the genetic disease classification are diseases caused by a single gene defect. By day 1 and 31 of 2023, 6255 distinct monogenic diseases were shown from the OMIM database (www.omim.org/statistics/geneMap). Many monogenic genetic diseases are extremely rare in the human population, but their number is so large that the probability of an individual carrying at least one monogenic disease is also significantly higher. It has been reported that by screening 104 unrelated individuals for 448 childhood severe recessive genetic diseases, all of the individuals were found to carry 2.8 pathogenic mutations. According to World Health Organization (WHO) statistics, the comprehensive incidence rate of single-gene genetic diseases is as high as 1/100. This level has exceeded the down syndrome incidence (1/680) counted in 2012 in our country. The statistics of the report (2012) for preventing and treating birth defects in China shows that the incidence rate of birth defects in China is about 5.6%, and among various reasons causing birth defects, single-gene genetic diseases account for about 22.2%, which indicates that the popularization of screening of extended carriers is very necessary.

Carrier screening may reduce the incidence of genetic disease in a population. For example, thalassemia (thassaemia) has been effectively controlled in recent years in guangdong, guangxi, etc., with the incidence of fetal edema syndrome (severe alpha-poor) falling from 21.7/ten and 44.6/ten-thousandth in 2006 to 1.93/ten and 3.15/ten-thousandth in 2017, respectively, by 91% and 93%.

The prevention of birth defects is classified as a three-stage precaution, whereas carrier screening belongs to the first stage of prevention. The carrier screening of genetic diseases of the couples before pregnancy or early pregnancy can find the carrying condition of pathogenic mutation in time, and can effectively avoid serious genetic diseases by combining with measures such as genetic counseling, prenatal diagnosis, auxiliary reproduction and the like. Treatment of monogenic diseases is very difficult, and thus prophylactic screening is an important means of reducing their incidence. Especially, the screening of the prenatal/prenatal expansibility carrier is a primary prevention means aiming at the birth defect generation source, and can prevent the first occurrence of single-gene genetic diseases, thereby more effectively reducing the birth defects.

Gene detection for screening carriers of genetic diseases, which is internationally early in the consumer market, is mainly based on Polymerase Chain Reaction (PCR), polymorphism chip hybridization, and fluorescence detection of large fragment deletions and duplications. However, these methods can only identify one or several genetic mutations (such as thalassemia, hereditary hearing loss) at a time, are suitable for targeted examination, and are less applicable to diseases, narrow in popularity and low in flux. The advent of high throughput sequencing technology has made it possible to screen hundreds of genetic diseases at a time.

The high-throughput sequencing technology is also called a new generation sequencing technology (nextgeneration sequencing, NGS), allows parallel deep sequencing of millions of different DNA fragment mixtures, has the characteristics of high throughput, large information content and high sensitivity, and brings great breakthrough to diagnosis and treatment. A large amount of variation information such as Single Nucleotide Polymorphism (SNP), copy Number Variation (CNV), inDel (InDel) and the like can be obtained through high-throughput sequencing, the method is a relatively comprehensive genome analysis means, an enrichment primer set is designed for a disease target gene by utilizing a target sequence capturing sequencing technology, sequencing cost and detection period are reduced while disease pathogenic gene loci are detected in a targeted manner, and precious time is reserved for prenatal diagnosis of pregnant women in early pregnancy. Multiple diseases are detected simultaneously through high-throughput sequencing, so that the screening efficiency is higher and the cost is lower.

In view of the foregoing, there is a need for the development of a gene detection primer set capable of simultaneously detecting/screening a plurality of genetic diseases, and a detection method compatible therewith.

Disclosure of Invention

Aiming at the problems of high screening and detecting cost, less applicable diseases, narrow popularization and low flux of the existing carrier, the invention combines a primer group (shown in table 1 and table 2) which is designed by high-flux sequencing and can capture a plurality of areas and is used for high-flux sequencing for detecting a plurality of genes aiming at 435 genetic diseases. Can realize carrier screening of 435 genetic disease genes, help the pre-pregnant couple/early pregnant couple to know the carrying condition of the severe genetic disease pathogenic genes of both parties in advance, accurately predict the risk of the infant suffering from severe genetic disease affecting fertility decision, facilitate the intervention of effective measures such as auxiliary reproduction or prenatal diagnosis in advance, and reduce the birth of the infant suffering from genetic disease.

To achieve the above object, the first aspect of the present invention provides a primer composition for genetic disease expansibility carrier screening based on high throughput sequencing, which detects mutations of 420 genes of related diseases including 435 diseases and copy number variation of a part of diseases.

The second aspect of the invention provides an expansibility carrier screening method based on high-throughput sequencing.

The primer group provided by the invention can effectively, rapidly and accurately detect all the gene mutation sites in the following table by one-time detection, thereby simplifying the diagnosis process, reducing the detection cost and improving the detection timeliness. In addition, the primer composition is used for screening a pre-pregnant carrier, and through high-quality and high-efficiency genetic diagnosis screening, the birth of the infant is reduced, and the quality of the birth population is improved.

435 diseases related by the invention and 420 genes corresponding to the 435 diseases:

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as a further scheme of the invention: the genetic disease carrier related mutant gene cases include detecting the above 444 gene mutation cases.

In order to realize detection of genetic disease related gene mutation, the invention adopts specific primers to carry out multiplex PCR amplification, and high throughput sequencing is carried out after library establishment.

As an alternative embodiment, the gene detection method of the present invention specifically comprises the steps of:

(1) Sample DNA extraction: extracting DNA from a fresh sample to obtain genomic DNA, and measuring the concentration;

(2) Amplification of the target fragment: mixing the extracted genome DNA with a multiplex PCR primer and a PCR amplification reagent, and carrying out multiplex PCR amplification by a specific primer to obtain a target gene DNA fragment to be detected;

(3) Primer elimination: mixing the DNA fragment obtained in the step (2) with a primer elimination reagent for reaction, and removing a primer sequence in the PCR amplified fragment to obtain a DNA fragment without a primer;

(4) And (3) joint connection: mixing the DNA fragment which does not contain the primer in the step (3) with a P1 joint, a specific joint and a connecting reaction reagent for connection reaction to obtain a joined DNA library fragment;

(5) Purifying magnetic beads: performing magnetic bead purification on the spliced DNA fragments, and removing redundant small fragment splices to obtain magnetic bead precipitates containing purified DNA library fragments;

(6) Library amplification and sorting: adding the magnetic bead sediment in the step (5) into library amplification reaction liquid and library amplification primers, transferring supernatant on a magnetic rack, performing PCR amplification, and separating and purifying by adopting two rounds of magnetic beads after the amplification to obtain a DNA library containing target fragments;

(7) Library detection: detecting the concentration and fragment distribution of the library in the step (6), and confirming that the library is diluted and mixed in equal quantity after no abnormality;

(8) Template preparation and high throughput sequencing: using the mixed library obtained in the step (7) for template preparation, and carrying out high-throughput sequencing on the obtained sequencing template by adopting an Ion S5 platform;

(9) Sequencing result analysis: and analyzing the sequencing result of the second generation by adopting bioinformatics.

As an alternative embodiment, in step (1), the sample to be tested is blood or oral mucosa cells.

As a further scheme of the invention: the multiplex PCR amplification system in step (2) was two 10. Mu.L systems corresponding to Pool1 and Pool2, respectively. The reaction system comprises the following components:

the reaction conditions for multiplex PCR amplification were: pre-denaturation at 98 ℃ for 45s; 15sec of denaturation at 98 ℃, 30sec of annealing at 60 ℃ and 30sec of extension at 72 ℃, and 12 times of circulation; finally extending for 1min at 72 ℃; incubate at 10℃for up to 16 h.

Because the target targets that are linked to each other on the genome are not compatible in the same multiplex amplification reaction, 2 amplification systems are required to completely cover the sequencing region, and specific amplification primer sequences can be queried in the Guangzhou Aureosciences Corp Guangzhou AoCe Medical Technology Co., ltd. Functional networks (http:// www.gzacute.com/Expandard Carrier screening. Html), where only primer sequences relevant to the present invention are provided.

Table 1: pool1 primer sequences

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Table 2: pool2 primer sequences

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As a further scheme of the invention: in the step (3), the primer elimination Reagent was FuPa Reagent of Thermo Fisher Scientific company, and 10. Mu.L of each of pool1 and pool2 was mixed to 20. Mu.L, and 2. Mu.L of the primer elimination Reagent was added to the 20. Mu.L PCR reaction system.

The reaction conditions for the elimination of the primer are: 20min at 50 ℃; 20min at 55 ℃; 20min at 60 ℃; incubate at 10℃for up to 1 h.

As an alternative embodiment, in step (4), the linker is IonCode of Thermo Fisher Scientific company ^TM A bar code connector.

As an alternative embodiment, in step (4), ionCode ^TM The barcode linker can be formulated by the following tabular system:

component (A)	Per tube reaction volume (μl)
		P1 Adapter	1
Barcode X	1
		Nuclease-Free Water	2
Total amount of reaction system	4

The P1 Adapter and the IonCode ^TM The material concentrations were 10nM.

Specific sequence of the P1 Adapter:

5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3'

5'-ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG-s-T-s-T-3'

the IonCode ^TM Is defined by the specific sequence of (a):

5'-CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGXXXXXXXXXXGGTGAT-3'

5'-ATCACCYYYYYYYYYYCTGAGTCGGAGACACGC-3'

wherein IonCode ^TM The sequences of the XXXXXX and YYYYYYYYY segments are used as linkers to identify the Barcode sequences, the two sequences are reversely complementary, different samples must use different Barcode sequences so as to distinguish between samples, and the specific Barcode sequences can be queried in a Thermo Fisher Scientific functional network.

As a further scheme of the invention: in the step (4), the joint connection system is a30 mu L system, and the components of the reaction system are as follows:

the reaction conditions of the joint connection are as follows: connecting at 22deg.C for 30min; repair of the notch at 68 ℃ for 5min, repair of the notch at 72 ℃ for 5min, and heat preservation at 10 ℃.

As an alternative embodiment: the magnetic beads used in the step (5) and the step (6) are Agencourt ^TM Ampure ^TM XP。

As a further aspect of the present invention, the magnetic bead precipitate after purification in step (5) is not eluted.

As a further aspect of the present invention, in the step (6), the composition of the library amplification reaction system specifically comprises:

as a further aspect of the present invention, after mixing the library amplification reaction system of step (6), it is necessary to transfer 50. Mu.L of the supernatant on a magnetic rack and subject the supernatant to a PCR amplification reaction.

The reaction conditions for the amplification of the library are: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15sec, annealing at 64℃for 1min, and cycling for 15 times; preserving heat at 10 ℃.

As a further aspect of the invention, in step (6), the ratio of the two rounds of magnetic bead sorting is 0.5X volume to 1.2X volume.

As an alternative embodiment, in step (7), the quality inspection method of the library includes, but is not limited to, qubit4.0 ^TM Fluorometer, qsep capillary nucleic acid electrophoresis apparatus, qPCR library quantification.

As an alternative embodiment, the template preparation of step (8) includes, but is not limited to, using a Ion Chef instrument or a Ion OneTouch 2 instrument, followed by sequencing the prepared template using Ion S5.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a genetic disease carrier screening method based on a high-throughput sequencing technology, which has the following characteristics:

practicality: solves the problem of confirming the carrier by various methodologies in the conventional method.

The simplicity is as follows: there is no need for multiple methodologies to distinguish between normal and carriers.

Commonality: the optimized raw letter analysis flow is adopted, and can be used for screening different genetic diseases.

High flux: multiple samples can be analyzed at one time, so that the detection rate is greatly improved, and the cost performance is higher.

Meanwhile, the method has the following advantages:

various genetic diseases are detected at a time, including high incidence single gene genetic diseases, barren land, hereditary hearing loss and the like.

And the multiplex PCR technology and a special belief analysis algorithm are used, so that the interference of pseudogenes is avoided, and the detection accuracy is improved.

Can make more people know that the pathogenic mutation carrying the genetic disease is a ubiquitous condition, thereby reducing the potential risk of discrimination of the carrier.

In conclusion, the genetic disease carrier screening method disclosed by the invention can be used for rapidly, accurately and high-flux screening of various genetic diseases, and has important clinical application prospect and social significance.

Drawings

FIG. 1 is a flow chart of variation detection and analysis according to an embodiment of the present invention.

FIG. 2 shows the results of the verification of samples M1, M10, M17, M21, consistent with known clinical results.

FIG. 3 shows the results of the verification of samples M2, M5, M11, M14, M20, consistent with the known clinical results.

FIG. 4 shows the results of the validation of samples M39, M42, M45, M53, M56, consistent with known clinical results.

FIGS. 5-9 show the results of a first generation sequencing validation of the detection sites.

FIG. 10 shows that M56 detects the deletion of SMN1, and verifies that the detection is performed using the Siamiron technology human motor neuron survival gene (SMN 1) detection kit.

Detailed Description

The technical scheme in the embodiment of the invention will be described in detail below with reference to specific embodiments. It should be understood that the specific examples described herein are for illustrative purposes only and are not intended to limit the scope of the present invention, and that commercial reagents, consumables and instruments used in the examples may be substituted for those having equivalent functions depending on the actual operating requirements. Based on these embodiments, one of ordinary skill may obtain many more embodiments without undue burden, which are also within the scope of the invention.

In some embodiments, the invention provides a method and primer set capable of simultaneously screening 435 genetic disease genes for a population. By adopting the detection method, the related genetic variation can be detected simultaneously by carrying out DNA extraction, target fragment amplification, library construction and sequencing on the sample and combining with corresponding variation analysis flow, so that carrier screening of 435 genetic diseases based on one method is realized, the detection flow is effectively simplified, the detection flux and the detection accuracy are improved, and the detection cost is reduced compared with carrier screening products of single diseases based on the traditional method.

Example 15 cases of genetic disease expansile Carrier screening

DNA is extracted from a small amount (2 mL) of peripheral blood of a subject, and the condition of carrying a single-gene genetic disease pathogenic gene is screened from mass genetic information of the subject by combining high-throughput target gene sequencing with bioinformatics analysis means, and the specific steps are as follows:

the method is adopted to carry out carrier screening detection on 15 samples with known clinical results.

Specific information for 15 clinically confirmed samples is shown in Table 3.

TABLE 3 Table 3

The detection procedure is as follows.

(1) Sample DNA extraction: genomic DNA extraction was performed on 16 clinically confirmed peripheral blood samples, respectively. The extraction kit was used for the extraction according to the procedure of the kit instruction, using MagPure Fast Blood DNA LQ Kit (manufacturer: meiyi, cat# MD 5111-03). The obtained sample DNA was subjected to concentration measurement using the Qubit4.0 and dsDNA HS Assay Kit kit (manufacturer: the next holy organism; product number: 12642ES 76).

All sample DNA concentrations >20 ng/. Mu.l.

Taking 5 μl of extracted genomic DNA sample, performing 1% agarose gel electrophoresis, and verifying the integrity of the sample, to confirm that the used sample template has no obvious degradation.

(2) Amplification of the target fragment: ion Ampliseq Panel primers were synthesized using the multiplex primer tables of SEQ ID NO. 1-SEQ ID NO. 11.

(1) All primers were synthesized in equimolar amounts, and after dilution, the primers were mixed in equal amounts into two primer pools (pool) according to the two primer tables, respectively.

(2) Each sample of the above-mentioned 15 genomic DNAs was subjected to multiplex PCR amplification by configuring two multiplex amplification systems (manufacturer: thermo Fisher; cat No. 4475345) with different pool, respectively, and the whole process required to be performed on ice:

note that: x is the volume required for the sample DNA to reach 30ng, and is no more than 3. Mu.L at maximum.

(3) After the reaction system is prepared, an 8-joint tube cover is covered, the mixture is gently shaken and mixed uniformly (without too violent shaking), and the mixture is subjected to instantaneous centrifugation (note that no bubbles exist in the tube). The PCR reaction tube was loaded into a PCR instrument and then the multiplex PCR amplification reaction procedure was run: pre-denaturation at 98 ℃ for 45s; 15sec of denaturation at 98 ℃, 30sec of annealing at 60 ℃ and 30sec of extension at 72 ℃, and 12 times of circulation; finally extending for 1min at 72 ℃; incubate at 10℃for up to 16 h.

(3) Primer elimination

(4) Transiently centrifuging the multiple amplified products, combining the two products of the same sample in a new 0.2ml PCR tube, and marking; 2. Mu.l of primer elimination reagent FuPaReagent (manufacturer: thermo Fisher; product number: 4475345) was added to each mixed 20. Mu.l of multiplex PCR amplification product to make the total system of each sample reach 22. Mu.l, vortexing, and instantaneous centrifugation; and (3) injection: the entire configuration process requires an on-ice operation. Before using, the FuPa Reagent needs to be subjected to instantaneous centrifugation after being evenly mixed;

(5) the tube containing the reaction system was placed in a PCR instrument and a primer elimination reaction program was run: 20min at 50 ℃; 20min at 55 ℃; 20min at 60 ℃; incubate at 10℃for up to 1 h.

(4) Joint connection

(6) IonCode ^TM Joint (factory)And (3) family: thermo Fisher; cargo number: a29747 Switch Solution, DNA enzyme (manufacturer: thermo Fisher; cargo number: 4475345 Placing on ice for thawing, if the solution or tube cover which is opened after thawing has visible sediment, slightly shaking and mixing by using vortex under the room temperature condition before pipetting, or blowing and mixing up and down by a pipetting gun, and after re-suspending the reagent, centrifuging instantaneously;

(7) the reaction system after the primer elimination is subjected to instantaneous centrifugation, and in 22 mu l of products, the reaction reagent is sequentially added to 30 mu l according to the component sequence in the form joint connection system;

joint connection system:

note that: ensure that the DNA ligase is added last. Specific IonCode ^TM Adapters Barcode is:

(8) gently shaking and uniformly mixing the joint connection reaction system, and performing instantaneous centrifugation (if bubbles exist, performing instantaneous separation again after the cartridge is light); the tube containing the reaction system was placed in a PCR instrument and the linker ligation reaction program was run: connecting at 22deg.C for 30min; repair of the notch at 68℃for 5min, repair of the notch at 72℃for 5min, and incubation at 10℃for up to 24 h.

(5) Magnetic bead purification

(9) Will make Agenecurt ^TM Ampure ^TM XP (manufacturer: beckman Coulter; product number: A63880) magnetic beads are balanced to room temperature (balanced for half an hour at room temperature), and the magnetic beads are fully vortex, vibrated, uniformly mixed and dispersed before use, and then are subjected to instantaneous centrifugation; preparing fresh 70% ethanol; carefully pipette 100% ethanol to ensure that the correct volume is added;

the library after the end of the linker reaction was subjected to transient centrifugation,the seal was carefully opened and then 45 μl (1.5 x sample volume) of agancourt was added to each library ^TM Ampure ^TM XP magnetic beads. Blowing up and down for 5 times (or firstly adding magnetic beads and then blowing up and down for 5 times by a row gun) to fully mix the magnetic bead suspension and DNA; incubating for 5min at room temperature after instantaneous centrifugation;

after the incubation is completed, the PCR tube is placed on a magnetic rack, and the mixture is left to stand for incubation for 2 minutes until the solution is clear. Then the gun was adjusted to 75 μl and the supernatant was removed, taking care not to remove the beads;

150. Mu.L of freshly prepared 70% ethanol was added, and the PCR tube was then moved 4-5 times left and right at two positions on the magnet rack to wash the beads. Then the supernatant is sucked and removed, and the magnetic beads are not required to be sucked;

repeating the steps and cleaning with 70% ethanol once again;

remove from the magnetic rack, spin briefly, place on the magnetic rack and blot all ethanol liquid from the tube holes with a 10 μl pipette. Keeping the test tube cover open, and drying the ethanol at room temperature for 5 minutes; care is taken not to overdry;

(6) Library amplification and sorting

Adding the magnetic bead precipitate purified in the step (5) into library amplification reaction liquid and library amplification primers, transferring supernatant on a magnetic rack, performing PCR amplification, and separating and purifying by adopting two rounds of magnetic beads after the amplification to obtain a DNA library containing target fragments;

the library amplification Mix was prepared by proportioning 1X Library Amp Mix and 25X Library Amp Primers (manufacturer: thermo Fisher; cat. No. 4475345) in advance.

The PCR tube containing the magnetic bead pellet was removed from the magnet rack and then 52. Mu.L of library amplification Mix was added to each sample.

Adjusting the pipette to 30 mu L, blowing up and down, mixing uniformly, covering a cover, slightly vortex shaking, mixing uniformly, flicking off bubbles, and then performing instantaneous centrifugation;

the PCR tube was returned to the magnet rack for at least 2 minutes until the reaction solution was clear, and then 50. Mu.L of supernatant was carefully transferred from each well to a new PCR tube, taking care not to suck the beads;

cover, transiently centrifuge, load into PCR instrument, and then run library amplification program: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15sec, annealing at 64℃for 1min, and cycling for 15 times; preserving heat at 10 ℃.

Using Agenecurt ^TM Ampure ^TM XP reagent was purified in two rounds

First round purification, agenemy ^TM Ampure ^TM After the XP reagent is balanced to the room temperature, the magnetic beads are uniformly dispersed by full vortex vibration, and then the magnetic beads are collected by short centrifugation;

fresh 70% ethanol was prepared, 100% ethanol was carefully aspirated to ensure accurate volume;

gently shaking the PCR tube with library products on a hard and flat table top, and carefully opening a sealing cover after short centrifugation; mu.L (0.5 Xvolume of 50. Mu.L sample) of Agenemy was added to each well containing 50. Mu.L sample ^TM Ampure ^TM XP reagent (magnetic bead) is blown up and down for 5 times to fully and evenly mix the magnetic bead suspension and DNA;

after removing the air bubbles, the mixture was centrifuged briefly and incubated at room temperature for 5 minutes. The PCR tube was placed on a magnet rack for at least 5 minutes, or until the solution became clear.

Carefully transfer 75 μl of supernatant from each well separately into a new PCR tube, taking care not to suck the beads.

Second round of purification, 60. Mu.L (1.2 Xvolume of 50ul of original sample) of Agenemy was added to the supernatant of the above step ^TM Ampure ^TM XP reagent (magnetic beads). Blowing up and down for 5 times to fully mix the magnetic bead suspension and DNA; (the number of times of blowing is not limited until the mixture is completely mixed

Incubating the mixture at room temperature for 5 minutes; the PCR tube was placed on a magnet rack for 3 minutes or until the solution became clear. Adjusting the pipette to 130 mu L to suck out the supernatant, sucking out the supernatant, and then sucking up the supernatant by using a small 10 mu L gun head, and retaining the magnetic beads; (Note: amplification products are bound to the magnetic beads. Do not discard the magnetic beads);

150. Mu.L of freshly prepared 70% ethanol was added to each well, and the PCR tube was then moved left and right 4-5 times on a magnetic rack to wash the beads. Standing on a magnetic rack until the solution is clear, and then sucking the supernatant, taking care that the magnetic beads are not required to be sucked;

repeating the steps and cleaning with 70% ethanol once again;

ensure that all ethanol was blotted dry. The PCR tube was placed on a magnetic rack and dried at room temperature for 2-5 minutes. Note that: excessive drying is not required;

removing the PCR tube from the magnetic frame, and then adding 50 mu L of Low TE to the magnetic beads to disperse the magnetic beads; adjusting the pipette to 40 mu L to perform up-down pipetting for at least 5 times or covering a cover, vibrating for uniform mixing for a short time, and then performing instantaneous centrifugation; incubating for at least 2 minutes at room temperature;

placing the PCR tube on a magnetic rack for at least 2 minutes until the solution is clear, and transferring 38 mu L of supernatant to a new PCR tube for library preservation;

(7) Library detection

Using Qubit4.0 ^TM And (3) performing concentration and fragment quality inspection on the library concentration by using a fluorometer and a Qsep1 capillary electrophoresis apparatus.

Library concentration quality inspection: using Qubit4.0 with Qubit for the library obtained ^TM The concentration was determined using a 1X dsDNA kit (manufacturer: thermo Fisher; cat. No. Q33230) and tested according to the kit instructions. The concentration of the library product substance control requirement is more than or equal to 300-1500ng/mL;

library fragment quality inspection: the obtained library was subjected to fragment distribution measurement using Qsep1 and standard clips (S2) (manufacturer: ding Bio; product number: C104250), and was tested according to the procedure of the instrument instructions. The main peak of fragment distribution should be located at 340bp;

after confirming that the library concentration and the fragments were not abnormal, the library was diluted to a final concentration of 100pM (average amplicon size of 300bp, mass concentration of 19.5 ng/mL) according to the measured library concentration, the diluted library was mixed in equal amounts, the mixed library was quantified using a fluorescent quantitative PCR instrument, diluted to 65pM according to the quantitative value, and then subjected to template preparation by Chef.

For example: the library concentration was 450ng/mL, the dilution factor to 100pM was 450 ng/mL/19.5=23, and 5. Mu.L of library was taken and 110. Mu.L of Low TE (1:23) was added.

Mixing an equal volume: all libraries were mixed by shaking at 5. Mu.L each.

The library after mixing was quantified using Ion universal library quantification kit (manufacturer: thermo Fisher; cat No. A26117), and template preparation was performed after dilution adjustment to 65pM according to the quantitative value.

(8) Template preparation and high throughput sequencing

And (3) using the mixed library obtained in the step (7) for template preparation, and carrying out high-throughput sequencing on the obtained sequencing template by adopting an Ion S5 platform.

Using Ion 540 ^TM Kit-Chef Kit (manufacturer: thermo Fisher; product number: A30011) template preparation and sequencing were performed in Ion Chef and Ion S5 sequencers, and the operations were performed according to the instrument instructions.

(9) Sequencing result analysis: and analyzing the second generation sequencing result.

By analyzing the sequencing result of the Ion S5 sequencing platform, the sequencing degree of all samples reaches or exceeds 200X, the uniformity is 85% on average, and the data size of each sequencing reaction sample is uniformly distributed.

The test results are shown in Table 4.

Table 4:

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note that: homogeneity is the area ratio of the target area sequencing depth to 20% of the average sequencing depth of the sample.

The first generation sequencing verification result of the detected site is shown in fig. 5-9.

The deletion of HBA1/HBA2 detected by M1, M0, M17 and M21 is positive site, which has been verified by Gap-PCR, and the result is shown in FIG. 2.

M56 detected the absence of SMN1 and was validated using the Siamitraz technology human motor neuron survival gene (SMN 1) detection kit (cat# P124). The results are shown in FIG. 10, with reference to the kit instructions.

In the example, 15 fresh samples are selected, genomic gDNA is extracted, library construction and sequencing are carried out by adopting the method, the obtained sequencing result is compared with the conventional method, the coincidence rate of positive site results is 100%, and newly reported disease carrying sites are verified by the conventional method, and the coincidence rate of the results is 100%. The invention can well complete the identification of the monogenic disease gene carrier, and the embodiment can be used as the basis of carrier screening.

Repeatability assessment protocol

Embodiment two: inter-batch repeatability

15 samples in example 1 were selected, and libraries were built under the same experimental conditions and in the same manner to obtain 3 duplicate libraries, which were repeated 3 times on the machine. The report sites for each sample were obtained, containing the sequencing results of example one, with 100% identity for four replicates.

Table 5: repeating two of the batches

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Table 6: repeating three of the batches

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Table 7: repeating four times between batches

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The sequencing method detects an in-vitro sample, and judges whether the detected sample has a pathogenic gene according to a detection result. If the detected result is the disease genotype, the obtained intermediate reference result is usually combined with various data such as clinical symptoms, past medical history, family genetic history and the like of the subject to prepare and judge the disease condition of the subject, so the method is not a diagnosis and treatment method of the disease.

Claims

1. The primer composition for screening the carrier with the expansibility of the genetic diseases is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO. 1-220.

2. A kit comprising the primer composition for genetic disease carrier screening of claim 1.

3. The high-throughput sequencing-based expansibility carrier screening method is characterized by comprising the following steps of:

(2) Amplification of the target fragment: multiplex PCR amplification is carried out on the extracted genome DNA and the primer group of claim 1, so as to obtain a target gene DNA fragment to be detected;

(6) Library amplification and sorting: adding the magnetic bead sediment in the step (5) into library amplification reaction liquid and library amplification primers, transferring supernatant on a magnetic frame, performing PCR amplification, and separating and purifying by adopting two rounds of magnetic beads after the amplification to obtain a DNA library containing target fragments;

4. The method according to claim 3, wherein in the step (1), the sample to be tested is blood or oral mucosa cells.

5. The method according to claim 3, wherein the primer elimination Reagent in the step (3) is FuPa Reagent of Thermo Fisher Scientific company, 10. Mu.L of pool1 and 2 are mixed to 20. Mu.L, and 2. Mu.L of primer elimination Reagent is added to 20. Mu.L of PCR reaction system.

6. The high throughput sequencing-based expansibility carrier screening method according to claim 3, wherein the reaction conditions for primer elimination are: 20min at 50 ℃; 20min at 55 ℃; 20min at 60 ℃; preserving heat at 10 ℃.

7. The high throughput sequencing-based expansibility carrier screening method according to claim 3, wherein in the step (4), the linker is ion code of Thermo Fisher Scientific company ^TM Bar code connectorA head.

8. The high throughput sequencing-based expansibility carrier screening method according to claim 3, wherein the magnetic beads used in step (5) and step (6) are Agencourt ^TM Ampure ^TM XP。

9. The high throughput sequencing-based expansibility carrier screening method according to claim 8, wherein the magnetic bead pellet purified in step (5) is not eluted; after the library amplification reaction system of the step (6) is mixed, transferring 50 mu L of supernatant on a magnetic rack and carrying out PCR amplification reaction on the supernatant; the reaction conditions for the amplification of the library are: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15sec, annealing at 64℃for 1min, and cycling for 15 times; preserving heat at 10 ℃.

10. The high throughput sequencing-based expansile carrier screening method of claim 3 wherein in step (6) the ratio of two rounds of magnetic bead sorting is 0.5X volume to 1.2X volume.