4) Corresponding amount of 1.5mL low adsorption centrifuge tube or 1.2mL shallow well plate was taken out according to the sample amount. Thebeads 2, which had been equilibrated at room temperature for 30 minutes, were vortexed for 1 minute, mixed well, and 48. mu.L of thebeads 2 were added to each 1.5mL low adsorption centrifuge tube or 1.2mL shallow well plate.

5) After the PCR was completed, the PCR tube was removed and centrifuged for 2 seconds. The PCR product was transferred to step 4) in a 1.5mL low adsorption centrifuge tube or 1.2mL shallow well plate withmagnetic beads 2.

a) Oscillating the mixed solution obtained in the step 5) for 3 seconds, standing for 5 minutes, performing instantaneous centrifugation, placing on a 16-hole magnetic frame, standing for 3 minutes, and removing the supernatant. b) Preparing 80% ethanol according to the sample amount (the 80% ethanol needs to be prepared newly, the preparation amount is 1 mL/sample), and reversing and mixing. c) Adding 500 μ L of newly prepared 80% ethanol, covering the tube cover, rotating the magnetic frame 90 degrees from back to front for 5 times, standing for 5 seconds, and discarding the supernatant. d) Repeating step c), and carrying out a second ethanol washing. The procedure is followed in steps 6) and 7).

6) And (4) sucking away residual ethanol by using a 20-mu-L range pipettor, opening a tube cover, and airing at room temperature until the magnetic beads are dried and cracked. Note that: if the magnetic beads are too wet, the magnetic beads can be placed in a digital display dry heater to be dried at 40 ℃ until the magnetic beads are cracked. Avoiding the magnetic beads from being dried excessively; and if the magnetic beads have cracks, carrying out the next experiment operation in time.

7) Adding 25 mu L of TE buffer solution, fully dissolving, standing for 5 minutes, placing the centrifuge tube or the shallow-hole plate on a magnetic frame, standing until the mixture is clear, and transferring all the supernatant into a new 1.5mL centrifuge tube to obtain the final DNA library. The prepared DNA library can be stored at-30 to-10 ℃.

3. Library quantification

3.1 preparing quantitative standard products S1-S4

Taking out quantitative standard product S1, quantitative standard product S2, quantitative standard product S3 and quantitative standard product S4, melting, fully oscillating, mixing well, and placing on ice box for later use.

3.2 dilution of the test DNA library

1) According to the number of the DNA library samples to be detected in the step 2.4.7), a certain number of 1.5mL centrifuge tubes are taken and marked with sample numbers. 2) 1799. mu.L of 18 M.OMEGA.pure water was pipetted into each centrifuge tube. 3) And (3) uniformly mixing the sample original tubes of the library to be detected by vortex oscillation for 5 seconds, instantly centrifuging, respectively transferring 1 mu L of sample solution into corresponding centrifuge tubes filled with 1799 mu L of 18M omega pure water, carrying out vortex oscillation for 10 seconds, instantly centrifuging to the bottom of the tubes, and placing the tubes on an ice box for later use.

Note that: firstly, the gun head needs to be rinsed 2-3 times when the 18M omega pure water is sucked. ② accurately absorbing the liquid, and fully oscillating and uniformly mixing the diluent.

3.3 preparation of reaction solution

1) A1.5 mL or 2mL centrifuge tube is taken, a tube cover is marked, and tin foil paper is sleeved for avoiding light. 2) The reaction mixture was prepared according to the reaction system in table 5 below, and the system was prepared according to the amount required for 3 repetitions per quantitative standard and 2 repetitions per sample.

TABLE 5

Components	Required dosage (mu L) for single reaction
		Nuclease-free water	7.2
Quantitative amplification reagent	10
		Quantitative amplification primer	0.8
Total amount of reaction system	18

3) After the preparation, the reaction mixture was shaken for 5 seconds, centrifuged instantaneously, and placed on an ice box.

3.4 sample application

1) The reaction plate was removed. 2) The prepared reaction mixture was added to each well of the reaction tube, and 18. mu.L of the reaction mixture was added to each well. Adding 2 mul of quantitative standard substance and diluted sample to be detected into the reaction hole respectively according to the sequence: when adding quantitative standard substance, respectively adding 2 mu L of quantitative standard substance S1-S4 into the reaction holes of the standard substance, and repeating three holes for each gradient quantitative standard substance; mu.L of diluted sample was added to the sample reaction wells, and two wells were repeated for each sample. 3) The reaction plate was sealed using a sealing tool and an optically adhesive film. 4) The reaction plate was centrifuged using a centrifuge at 2000rpm for 1 minute and loaded into a fluorescent quantitative PCR amplifier to prepare for operation.

3.5 set-up program (example is StepOnePlus, ABI)

1) At the program interface, selecting at the block item: 96-well plates (0.2 mL); in the type item selection: standard Curve. 2) In the reagent item selection: SYBR Green Reagent; selecting the mode items: and (5) Standard. The default selection includes Melt cut. Note that: the above options can be selected according to the program interface of the instrument and the actual situation of the experiment. 3) Selecting standard product holes on a Plate Setup interface, wherein the repetition coefficients are all 3; the sample wells were selected with a repetition factor of 2. The concentration of the inputstandard substance 1 is set as follows: 0.135, dilution gradient: 1:10. 4) The program was set up on a fluorescent quantitative PCR amplificator as shown in table 6:

TABLE 6

While the fluorescence signal was collected at the stage set at 72 ℃ for 30 seconds.

5) The reaction system was set to 20. mu.L and the qPCR reaction was started with a single RUN.

3.6 data analysis

1) And (5) after the reaction is finished, carrying out data analysis and judging the reliability of the result. The fitting degree (R2) of the model and the sample quality inspection is in the range of 0.98-1, and the data result that the amplification efficiency (Eff%) is in the range of 85-100% is good. If the data deviation is too large, the quantification is performed again. 2) If the data is reliable, deriving the data, selecting the concentration with the difference of the repetition threshold (CT value) less than 0.5 in the data processing table, taking the average, multiplying the average by 1800 to obtain the quantitative concentration of the library, and recording the concentration of the library. 3) In order to better ensure the detection result, when the concentration of the library is less than 1nmol/L, the library should be reconstructed for sequencing.

4. Library hybridization

4.1 hybrid libraries

The computer records were compiled based on sample DNA library concentrations and specificity tags, taking care not to place identically tagged libraries on the same sequencing RUN. According to the Pooling task list, Pooling is carried out on the sample according to the equal molecular number, the total amount of Pooling is based on the amount of hybridized DNA, the library after Pooling is detected by using the Qubit, the amount of hybridized DNA is 800ng, the volume is 3.4ul (the maximum is not more than 4.5ul), and if the volume is overlarge, the sample can be concentrated to the corresponding volume by using a vacuum concentrator.

4.2 hybridization

1) Preparation of hybridization buffer Mix

The hybridization buffer mix was prepared in the corresponding volume at room temperature using a 0.2ml PCR tube according to the following Table 7, mixed well using a vortex shaker and then centrifuged instantaneously.

TABLE 7

Reagent	Amount of 1 hybridization reaction (. mu.L)
		Hybridization buffer #1	25
Hybridization buffer #2	1
		Hybridization buffer #3(-20 ℃ refrigerator storage)	10
Hybridization buffer #4	13
		Total volume	49

Note that: if the hybridization buffer appeared to precipitate, the solution was heated at 65 ℃ for 5 minutes to confirm that the precipitate disappeared and then used.

2) Sample prehybridization library system for preparing enriched target fragments

According to the components in the following table 8, a sample prehybridization library system was prepared in a PCR tube at room temperature using a 0.2ml PCR tube, the PCR tube was closed and mixed well, and centrifuged briefly for use.

TABLE 8

Reagent	1 hybridization dose (μ L)
		Pooling library	800ng, supplement water to 3.4 μL
Block#
	1	2.5
Block#2			2.5
	Block#3	0.6
Total volume			9.0

3) Library prehybridization

The PCR tube containing the prehybridization library system prepared in the previous step was placed in a PCR instrument, which was operated under the following conditions of table 9:

TABLE 9

Temperature of	Time	Number of cycles
			95℃	5minutes	1
65℃	Holding					1

Note that: the use of a hot lid is required to minimize evaporation.

4) Pre-warming hybridization buffer

Maintaining the PCR instrument at 65 ℃, putting the hybridization buffer mix prepared in the step 1) into the PCR instrument, and incubating at 65 ℃ for at least 5 minutes.

5) Preparing probe mixed liquid

Taking out the hybridization probes stored at-80 ℃ according to the number of samples, and putting the hybridization probes on ice for thawing; according to the sample quantity, a probe mixed solution is prepared according to the reaction system shown in the following table 10:

watch 10

Reagent	Volume (μ L)
		Nuclease-free water	4.5
RNase inhibitors	0.5
		Customized probe	2.0
Total volume	7.0

The probe mixture (no air bubbles should be added) prepared in the above manner was added to the bottom of each well in the middle of the wells (no more than 8 per row) of the 96-well PCR plate, covered with an octal tube, and placed on ice. Note that the above operation was performed on ice.

6) Preheating probe mixed liquid

The 96-well PCR plate containing the probe mixture prepared in step 5) was put into a PCR instrument while keeping the PCR plate at 65 ℃ and incubated at 65 ℃ for 2 minutes.

7) Pipetting hybridization

Keeping the PCR plate at 65 ℃, uncovering the hybridization buffer mix and the tube cover of the prehybridization library, quickly transferring the hybridization buffer mix into the PCR tube of the prehybridization library by using a 20-mu-L pipette adjusted to 16-mu-L, blowing and uniformly mixing, covering the tube cover, using a hot cover, and pressing for 1 minute. (ii) a

Keeping the PCR plate at 65 ℃, uncovering an octal tube cover containing the probe, quickly using a 100 mu L liquid transfer machine adjusted to 25 mu L to transfer all the library solution in the PCR tube containing the pre-hybridization library to the hole containing the probe of the PCR plate, and slowly sucking and blowing the library solution up and down for 3 to 4 times by using the liquid transfer machine to avoid the generation of bubbles;

the PCR plate was sealed quickly with a sealing film and the sealing film was compressed with a special doctor blade to ensure complete sealing of all the wells, hot capped for 2 minutes and the second layer of sealing film was resealed using the same method.

The PCR plate was kept at 65 ℃ for hybridization (105 ℃ for thermal cycler lid) for 16-24 hours (care was taken to prevent evaporation).

Note that: hybridization probes were stored directly in a-80 ℃ freezer after shipment.

4.3 elution

1) Preparation of streptavidin magnetic bead T1

a. Violently oscillating the streptavidin magnetic bead T1 by using a vortex mixer until the streptavidin magnetic bead T1 is uniformly mixed; b. 50 mu L of streptavidin magnetic bead T1 is taken for each hybridization reaction and placed in a 1.5mL labeled centrifuge tube; c. adding 200 μ L of capture buffer solution, violently oscillating and mixing uniformly by using a vortex mixer, placing the centrifuge tube on a magnetic frame, and removing clarified liquid after the liquid becomes clear; d. repeating c twice; e. add 200. mu.L of capture buffer to resuspend streptavidin magnetic bead T1 for use.

2) Purification of the hybrid captured fragment of interest

Binding magnetic bead T1

a. After incubation for 16-24 hours, continuously storing the hybridization mixed solution on a PCR instrument, scratching and uncovering a sealing film of a 96-hole PCR plate line by using a blade, quickly transferring all the hybridization mixed solution into a 1.5mL tube containing streptavidin magnetic bead T1 prepared in the step 1) one by using a 100uL pipette adjusted to 32uL, roughly estimating the volume of the hybridization solution, closing a hot cover for 30 seconds after completing a line of reaction, and then continuing. Note that: if the evaporation volume exceeds 8uL, the hybridization experiment is considered to be failed.

b. The centrifuge tube containing the hybridization mixture and streptavidin magnetic bead T1 is symmetrically fixed on a Nutator or similar device to be uniformly mixed by 360 degrees of rotation, and the mixture is incubated for 2 hours at room temperature.

c.Wash 2 was preheated in a 67 ℃ water bath.

Washing magnetic bead T1

a. Taking the sample off the blending device, and performing instantaneous centrifugation to ensure that no liquid remains on the tube cover; b. transferring the centrifugal tube to a magnetic frame, standing for 1-2 minutes until the liquid in the tube is clear, and removing the supernatant; c. keeping the centrifuge tube on a magnetic frame, adding 500 mu L of cleaningsolution 1 for resuspending magnetic beads, oscillating on a vortex mixer for 5 seconds to uniformly mix the sample, and incubating the sample for 15 minutes at normal temperature; d. transferring the centrifugal tube to a magnetic frame, standing for 1-2 minutes until the centrifugal tube is clear, and removing supernatant; e. keeping the centrifuge tube on a magnetic frame, adding 500 mu L of 67℃ cleaning solution 2 for resuspending magnetic beads, and oscillating on a vortex mixer for 5 seconds to uniformly mix the sample; f. putting the sample into a dry bath kettle to incubate for 10 minutes at 67 ℃; g. inverting the centrifuge tube to mix the sample, centrifuging for 3 seconds instantaneously, and removing the supernatant; h. transferring the centrifugal tube to a magnetic frame, standing for 1-2 minutes until the centrifugal tube is clear, and removing supernatant; i. repeating the step e to the step h for 2 times, and washing for 3 times in total;j. wash 2 was removed as clean as possible with a 10. mu.L pipette.

Elution of DNA captured by hybridization

a. Add 36.5. mu.L of nuclease free water and shake on vortex mixer for 5 seconds to resuspend the beads, incubate for 10 minutes at room temperature; b. the 36.5. mu.L solution was transferred in its entirety to a 0.2ml PCR tube together with magnetic beads for subsequent PCR.

3) Post-hybridization PCR and purification

The post-hybridization PCR reaction system was configured as follows:

TABLE 11

Reagent	Volume (μ L)
		DNA (together with T1 magnetic beads, obtained in the previous step)	36.5
5 Xfusion PCR buffer	10
		dNTPs(25mM each)	0.5
Q1 primer (10. mu.M)	1
		Q2 primer (10. mu.M)	1
Fusion DNA polymerase	1
		Total volume	50

The procedure as shown in table 12 below was set up on a PCR instrument:

TABLE 12

PCR product purification

a. Themagnetic beads 2 were removed from the 4 ℃ freezer and equilibrated at room temperature for 30 minutes. b. And (3) placing the sample on a magnetic frame, standing for 1-2 minutes until the sample is clear, transferring the solution into a centrifuge tube with 65 mu L of magnetic beads B2, uniformly mixing, and standing for 5 minutes at room temperature. c. After 3 seconds of instantaneous centrifugation, the centrifuge tubes were placed on a magnetic rack until the liquid was clear, which took approximately 3 minutes. d. The supernatant was carefully removed with a pipette (taking care not to attract the beads). e. The tube was kept in the magnetic rack, 500. mu.L of 80% ethanol (care was taken not to blow the beads) was added, the tube was covered and the rack was turned upside down to mix seven times, and the supernatant was removed. f. Repeat step e (this step takes care to remove the ethanol on the tube cap when removing the supernatant). g. The centrifuge tube was left uncapped and dried at 37 ℃ for approximately 5-10 minutes (until the beads dried apart, it was necessary to avoid over-drying). After the magnetic beads are dried, the centrifuge tube is placed in a magnetic rack. h. 50 μ LLow TE was added, the mixture was blown by a gun to dissolve the beads sufficiently, and the mixture was allowed to stand at room temperature for 5 minutes. i. The centrifuge tube was placed on a magnetic stand until the liquid was clear, which took approximately 2 to 3 minutes. j. 50. mu.L of the eluted DNA was transferred to a new 1.5ml centrifuge tube containing 60. mu.L of magnetic bead B2, mixed well and allowed to stand at room temperature for 5 minutes. k. After 3s of instantaneous centrifugation, the centrifuge tubes were placed on a magnetic rack until the liquid was clear, which took approximately 3 minutes. l, carefully remove the supernatant with a pipette (care not to attract beads). m, keeping the centrifuge tube on a magnetic frame, adding 500 μ L of 80% ethanol (care not to blow the magnetic beads), covering the centrifuge tube, turning the magnetic frame upside down, mixing for seven times, and removing the supernatant. n, repeat step m (this step takes care to remove the ethanol on the tube cap when removing the supernatant). And o, opening the centrifuge tube and drying at 37 ℃ for about 5-10 minutes (until the magnetic beads are dried and excessive drying needs to be avoided). After the magnetic beads are dried, the centrifuge tube is placed in a magnetic rack. p, adding 20 mu L of Low TE, blowing and uniformly mixing by using a gun to fully dissolve the magnetic beads, and standing for 5 minutes at room temperature. q, placing the centrifugal tube on a magnetic frame until the liquid is clear, and taking about 2-3 minutes. r, transferring 20 mu L of the eluted DNA into a new 1.5ml centrifuge tube to obtain the required library.

4.4 library quality testing

The library was quantified using Qubit.

5. Sequencing template preparation

The procedures were carried out in accordance with the instructions of the general kit for sequencing reactions (semiconductor method) (S10010).

5.1 hybrid libraries and dilutions thereof

1) The computer records were compiled based on sample DNA library concentrations and specificity tags, taking care not to place identically tagged libraries on the same sequencing RUN. The recommended range of the computer concentration on the library is as follows: 25 to 40pmol/L, preferably 33pmol/L (converted from the quantitative value of the Qubit), and can be adjusted in accordance with the case. The dilution factor of the sample library was calculated according to the following formula (1). Formula (1): dilution factor ═ mixed library concentration (nM) × 1000 ÷ computer concentration (pM)

2) Oscillating the sample library for 2 seconds, centrifuging the sample library to the bottom of the tube instantaneously, and sequencing the processed sample library according to a record table; according to the calculated sampling volume, the library is mixed into a new marked 1.5mL centrifuge tube one by one, after the mixing is finished, the vibration is carried out for 5 seconds, and the mixture is instantaneously centrifuged to the bottom of the tube. And taking another new 1.5mL centrifuge tube, diluting the mixed library to the required concentration on the computer by using nuclease-free water, shaking for 2 seconds after dilution, instantly centrifuging to the bottom of the tube, and placing the tube on an ice box for later use.

5.2 amplification

Note that: the step can also be carried out by selecting other suitable models, the following method is a method when the One Touch2 is used for operation, and if instruments of other models are selected, please refer to an instrument operation instruction.

1) Starting an One Touch2 Instrument, and selecting a Clean Instrument program; ensuring that at least 10mL of reaction oil and 3mL of resuscitation solution are contained in the reagent tube; ensuring that the old amplification plate and the cleaning joint are installed correctly; emptying the waste liquid bottle, placing a bottle cap or other waste liquid collecting container under the needle head, and collecting waste liquid; each step is confirmed again on the screen, clicking "Next" until the wash program starts running, with a wash time of 14 minutes. 2) After the One Touch2 is cleaned, unloading the old amplification plate; installing a new amplification plate, a collection pipe and a collection bridge; 150 mu L of demulsifying agent is added into each collecting tube respectively, the reaction oil and the solution in the recovery liquid bottle are reversed for 3 times and mixed evenly, the reaction oil is supplemented to the position of thereagent tube 1/2, and the recovery liquid is supplemented to the position of thereagent tube 1/4. 3)

Preparation of reagents: melting the emulsion PCR buffer solution at room temperature, carrying out vortex oscillation for 30 seconds, carrying out instantaneous centrifugation to the bottom of the tube, and placing at room temperature for later use; instantly centrifuging the emulsion PCR enzyme mixed solution, and placing the emulsion PCR enzyme mixed solution on an ice box for later use; the microbead solution is placed at room temperature, shaken for 1 minute before use, fully mixed, and instantaneously centrifuged to the bottom of the tube. Note that: if the emulsion PCR buffer is not used on the same day after freezing and thawing, the emulsion PCR buffer needs to be stored at 4 ℃. 4) Reagents were added to the emulsion PCR buffer tubes in sequence at room temperature according to Table 13 below, and mixed by vortexing for 5 seconds. Note that: the reaction solution is prepared at room temperature (15-30 ℃). ② the prepared amplification reaction liquid needs to be reacted on an One Touch2 instrument within 15 minutes.

Watch 13

Components	Volume (μ L)
		Emulsion PCR buffer	2000
Nuclease-free water	80
		Emulsion PCR enzyme mixture	120
Microbead solution	100
		Mixed libraries	100
Total volume of the system	2400

5) A new reaction filter was placed on the shelf with the wells facing up. And (3) carrying out vortex oscillation on the amplification reaction solution for 5 seconds, carrying out instantaneous centrifugation to the bottom of the tube, immediately adjusting to 800 mu L by using a 1000 mu L pipette, blowing, sucking and uniformly mixing for 3 times, and vertically and slowly adding the amplification reaction solution into the filter from the sample adding hole in three times. 200. mu.L of the spacer oil was aspirated and slowly added vertically all over the well to the filter. 6) The cleaning joint on One Touch2 was removed, and the reaction filter in the previous step was slowly inverted until the sample addition well was facing downward and mounted on OneTouch 2. 7) Click "Run" on the One Touch2 screen, select the corresponding program: ion PI^TMHi-Q OT 2200 Kit, select "Assisted" againDetermining each step, clicking 'Next' until the program runs, wherein the running time of the program is about 5 hours; note that: before the RUN was initiated, 150 μ L of emulsion breaker was added to each collection tube. 8)

At the end of the program run, click "Final spin" on the screen and centrifuge the collection tube for 10 minutes. Immediately carrying out the following steps after centrifugation; note that: the program run should not exceed 16 hours from the start of the program run to the completion of the centrifugation. 9)

Ending the centrifugation of the One Touch2, clicking the open lid on the screen, and opening the cover; the collection bridge on the rotor was removed, the two collection tubes were removed, and the supernatant carefully removed by a 1000. mu.L pipette with the tip directed at the non-sediment, and approximately 150. mu.L of solution remained in each tube, against the liquid surface. 10) Repeatedly blowing the residual solution in the suction pipe by using a liquid transfer machine, fully and uniformly mixing, and transferring all the liquid in the two collection pipes into a new 1.5mL low-adsorption centrifuge pipe. 11) Add 200. mu.L nuclease free water to each collection tube and blow and suck the mixture, and transfer all the nuclease free water to the 1.5mL low adsorption centrifuge tube in the previous step. 12) Repeat step 11) once. 13)

Vortexing the solution obtained in the step 12) for 30 seconds, and centrifuging 15500g for 8 minutes. The tip carefully pipetted the supernatant against the non-pellet leaving about 100. mu.L of solution in the tube. 14) Adding 900 mu L of nuclease-free water into the 1.5mL low-adsorption centrifuge tube in the previous step until the total volume reaches 1000 mu L; note that: firstly, the sample microbead solution which completes the PCR amplification of the emulsion in the step can be stored for 3 days at most at the temperature of 2-8 ℃; ② if the sample microbead solution was stored at 2-8 ℃ then proceed to step 15)

15) Vortexing and shaking the sample microbead solution obtained in the step 14) for 30 seconds, and centrifuging at 15500g for 8 minutes. The tip was pointed against the non-pellet and the supernatant carefully aspirated off, leaving approximately 20. mu.L of solution in the tube. 16) Adding 80 mu L of template resuspension into the sample microbead solution obtained in the previous step to enable the total volume in the tube to reach 100 mu L, carrying out vortex oscillation for 30 seconds, and carrying out instantaneous centrifugation to the bottom of the tube. Note that: the sample microbead solution obtained in this step is subjected to ES operation on the same day. 17) And cleaning the One Touch2 instrument, and turning off the power supply if the instrument is not used for a long time.

5.3 enrichment of template sequences

1) Streptavidin C1 magnetic beads were equilibrated for 30 minutes at room temperature. 2) 1M NaOH was prepared in a 1.5mL centrifuge tube (preparation method: 0.1mL of 10M NaOH and 0.9mL of nuclease-free water mixed together). Note that: the 1M NaOH solution is used within 7 days, for example, more than 7 days, and is reconstituted. 3) The reagents were added sequentially to 1.5mL centrifuge tubes according to table 14 below to formulate eluents, vortexed for 10 seconds, and centrifuged instantaneously. The eluent is used for the day.

TABLE 14

Components	Volume (μ L)
		Tween solution	280
1M NaOH	40
		Total volume	320

4) The streptavidin C1 magnetic beads were vortexed for 2 minutes and mixed well. Adding 100 mu L of the solution into a new 1.5mL low adsorption centrifuge tube; the low adsorption centrifuge tube was placed on a magnetic rack, allowed to stand for 2 minutes for adsorption, and the supernatant carefully removed. 5) Adding 1000 mu L C1 magnetic bead cleaning solution into a low-adsorption centrifuge tube, performing vortex oscillation for 30 seconds, and performing instantaneous centrifugation; the low adsorption centrifuge tube was placed on a magnetic rack, allowed to stand for 2 minutes for adsorption, and the supernatant carefully removed. 6) Add 130. mu. L C1 magnetic bead capture solution to the low adsorption centrifuge tube, vortex for 30 seconds, and centrifuge instantaneously. 7) An 8-well reaction strip was placed on the laboratory table in the left-right circle direction, and the reaction solutions were added in the order shown in Table 15 below.

Watch 15

Note that: the sample microbead solution is shaken for 30 seconds before being added into the reaction hole, and is instantly centrifuged to the bottom of the tube and then added.

8) The strip was placed in the ES channel, the ES pipette tip was attached to the arm, a new 0.2ml PCR tube was placed at the collection well, the "Start/Stop" button was pressed, and the machine was run for approximately 40 minutes. 9) After the ES operation is finished, checking the volume of the liquid in the PCR tube, if the volume of the liquid is more than 250 mu L or obviously less than 200 mu L, carrying out template amplification again; if the liquid volume is not less than 200 mu L, the next experiment is continued. After the ES run is over, the sample rest time must not exceed 15 minutes. 10) The 0.2mL PCR tube containing the sample bead solution was removed from the ES, the sample information was marked on the tube cap, centrifuged at 15500g for 5 minutes with the tip facing the non-pellet, and the supernatant carefully aspirated away, leaving 10. mu.L of solution in the tube. 11) Add 200. mu.L nuclease-free water, vortex for 60 seconds, centrifuge at 15500g for 5 minutes, and check the bottom of 0.2mL PCR tube for brown residual magnetic beads.

Firstly, if the bottom of a 0.2mL PCR tube has no brown residual magnetic beads: the following step 12) is then performed. If brown residual magnetic beads exist at the bottom of the 0.2ml PCR tube: after sucking away 20. mu.L of the supernatant, the sample microbead solution was mixed by pipetting 10 times, the PCR tube was placed on a magnetic stand and left for 4 minutes, and the supernatant was transferred to a new 0.2mL PCR tube. Adding 20 mu L of nuclease-free water into the original PCR tube, blowing and beating for 10 times by using a pipette, uniformly mixing, transferring all the supernatant into the previous new 0.2mL PCR tube after the magnetic beads are clarified, and centrifuging for 5 minutes at 15500 g.

12) The tip was brought to the non-pellet site, the supernatant carefully aspirated, 10. mu.L of liquid was retained in the tube, 90. mu.L of nuclease-free water was added, vortexed for 60 seconds, and centrifuged instantaneously for further use. Note that: the sample micro-bead solution can be preserved for no more than 3 days at the temperature of 2-8 ℃.

6. Sequencing on machine

The procedures were carried out in accordance with the instructions of the general kit for sequencing reactions (semiconductor method) (S10010). Note: the sequencing on the machine adopts a Bioelectronseq 4000 gene sequencer.

6.1 Instrument cleaning

The nitrogen cylinder was opened and the pressure was adjusted to 30 psi. Starting the sequencer, clicking 'cleaning' on the main interface, and selecting a water washing or chlorine washing program according to the use condition of the sequencer: the machine is stopped for 72 hours, and water washing is selected; the machine was stopped for more than 72 hours and a chlorine wash was selected.

1) Washing with water: the bottles were emptied from C1 and C2 and washed twice with 18M Ω pure water. 100mL of 18M. omega. pure water was added to each of the C1 and C2 wash bottles and mounted at the C1 and C2 positions. And selecting a 'cleaning' option on a sequencer screen, installing a chip, clicking 'next' according to screen prompt until a water washing program starts to run, wherein the cleaning time is about 18 minutes.

2) Chlorine washing: the two chlorine bottles were emptied and the two chlorine bottles and the glass chlorine bottle were washed with 18M Ω pure water. To a glass chlorine bottle containing 1L of 18 M.OMEGA.pure water, the chlorine tablets were added, dissolved for 10 minutes, and 1mL of 1M NaOH was added and mixed by inversion. 100mL of the wash solution was filtered into the chlorine wash bottles with 0.45 μm filter heads, the two chlorine wash bottles were mounted at the C1 and C2 positions, the "wash" option was selected on the sequencer screen, the chip was mounted, and the "next" step was clicked according to the screen prompt until the chlorine wash program started running for about 18 minutes. After the chlorine washing, the chip was replaced and washed once.

6.2 applicable method for Instrument initialization (selection of step 1) or 2 below according to the sequencing kit Specification)

1) The specification of the kit is 4C 4R/set and 8C 8R/set

(1) The W2 reagent bottle was emptied and washed three times with 18M omega pure water. 1920mL of 18M omega pure water was added, and the entire bottle of sequencing solution 2(125 mL/bottle) was added, capped, and mixed by inversion 4-8 times. Two new 140mL reagent tubes were removed, labeled as vial W1 and vial W3, respectively. Adding 32 mu L of 1M NaOH into a W1 bottle, adding 40-50 mL ofsequencing solution 3 into a W3 bottle, and covering the bottle with a bottle cap. Note that: sequencingsolution 2 was stored protected from light.

(2) The sequencer screen is selected to be "initialization" and "Ion P1 Hi-Q Sequencing 200 Kit", pipettes at positions W1, W2 and W3 are replaced, the reagent bottles are mounted at corresponding positions, and the mouthpiece covers are screwed down. The chip for initialization is installed, the state of the instrument is determined according to the screen prompt, the next step is clicked until the program starts to run, and the first stage takes about 40 minutes.

Note that: firstly, when a new straw is replaced, new gloves are needed to be replaced, and the gloves can not touch the straw body when the straw is installed. Except the chip washed by chlorine, both the chip washed by water and the chip initialized can be used as initialization chips.

(3) dGTP, dCTP, dATP and dTTP were taken out in advance, placed on an ice box, thawed, shaken for 10 seconds, and centrifuged for 2 seconds.

(4) After the first phase initialization was complete, 4 new 140mL reagent tubes were labeled "G, C, A, T" and 70. mu.L of the corresponding dNTP solution was added. The pipette at the dNTP position is replaced, and the reagent tube is attached to the corresponding position and tightened. And according to the screen prompt, continuously completing initialization, and taking about 40 minutes. And returning to the main interface after the initialization is completed.

Note that: when a new straw is replaced, a new glove needs to be replaced, and the glove cannot touch the straw body during installation.

2) The specification of the kit is 4C 8R/set

(1) One initialization is completed according to the step 1), and two times of sequencing can be continuously carried out. That is, before the second RUN, the instrument initialization step is not performed, and after the first RUN completes the sequencing, the following operations can be directly performed on the two RUNs.

6.3 setting Plan

1) As shown in FIG. 4, the sequencer server is logged in and a single click selects "Plan" - "Templates". 2) Select "white Genome" in the left column "Favorites", click on the top right corner "Plan New run" or "Add New Template" to create a Template. (if there is a Template, choose directly in the "Template Name" list, click "Plan Run" 3 of the drop-down list.) enter the Run setup interface, there are 6 tabs: 4) "DNA" and "white Genome" were selected in "Application", one click "Next". 5) The kit information is selected according to table 16 below, the rest left blank, and then click "Next".

TABLE 16

Options for	Selection type
		Library Kit Type	Ion Plus Fragment Library Kit
Templating Kit	Ion PI Hi-Q OT2 200 Kit
		Sequence Kit	Ion PI Hi-Q 200 Sequence Kit
chip Type	Ion PI Chip
		Barcode Set	IonXpress
Base Calibration Mode	Default Calibration
		Flow	400

6) Plugins insert: and (5) checking a corresponding option (SEA _ plugin) according to the actual sequencing requirement, and clicking 'Next'.

7) Project data form options are default values, and are not modified; or newly creating a folder and selecting the folder, and clicking 'Next'.

8) In the Plan interface, a Run Name is input in a Run Plan Name, the Number of codes is input after the Number of codes, and a rear key is clicked. The Sample number is input in the Sample Name column, and the corresponding barcode number is selected from a pull-down menu. After inputting, whether the sample number and the Barcode number are correct or not needs to be checked.

9) Click "Plan Run" to complete setting Plan.

6.4 preparing the library on the computer

1) And taking out the quality control microbead solution, the sequencing primer and the sequencing polymerase in advance and placing the solutions on an ice box. 2) The library was prepared on-board near the end of instrument initialization. Oscillating the quality control microbead solution for 30 seconds, uniformly mixing, and performing instantaneous centrifugation; adding 5 mu L of the solution into the sample microbead solution, performing vortex oscillation for 30 seconds, and centrifuging 15500g for 5 minutes; the tip was pointed at the non-pellet and the supernatant carefully aspirated, leaving 10. mu.L of solution. 3) Add 15. mu.L of annealing buffer to the sample bead solution from the previous step in a total volume of 25. mu.L. 4) After thawing the sequencing primers on ice, vortexing for 10 seconds, mixing, and centrifuging for 2 seconds. Add 20. mu.L of sequencing primer to the bead sample solution from the previous step to ensure that the total volume is 45. mu.L. And (3) vortexing and oscillating the sample microbead solution for 60 seconds, mixing uniformly, and performing instantaneous centrifugation. 5) Annealing the sample bead solution on a PCR instrument according to the following procedure: 95 ℃ for 2 minutes, 37 ℃ for 2 minutes, 20 ℃.6) And after annealing, adding 10 mu L of loading buffer solution into the sample microbead solution, performing vortex oscillation for 10 seconds, uniformly mixing, performing instantaneous centrifugation, and standing at room temperature.

6.5 Loading and sequencing

1) The chip is unpacked, taken out and placed in the hanging basket, and the notch of the chip and the notch with the larger radian of the hanging basket are positioned on the same side. And sucking 55 mu L of the sample microbead solution obtained in the previous step, and injecting the sample microbead solution into a sample inlet hole of the chip. 2) The chip is placed on a centrifuge with the chip notch facing outward and is trimmed to the old chip. Centrifuge for 10 minutes. 3) During centrifugation, the following reagents were prepared: adding 0.5mL of annealing buffer solution and 0.5mL of nuclease-free water into a 1.5mL centrifuge tube respectively, and mixing uniformly to prepare 50% annealing buffer solution. The solution can be used within 7 days after preparation. ② 0.5mL of 100 percent isopropanol solution and 0.5mL of annealing buffer solution are respectively added into a 1.5mL centrifuge tube and mixed evenly to prepare 50 percent flushing fluid. The solution was prepared on the day of use. ③ 60 mu L of 50 percent annealing buffer solution and 6 mu L of sequencing polymerase are respectively added into a 1.5mL centrifuge tube and mixed evenly to prepare enzyme reaction solution. The solution was prepared and placed on an ice box. And fourthly, respectively adding 49 mu L of 50% annealing buffer solution and 1 mu L of foaming solution into a 1.5mL centrifuge tube, and uniformly mixing to prepare the foaming agent. Note that: 4.9mL of 50% annealing buffer solution and 100. mu.L of foaming solution can be added into a 5mL tube respectively and mixed evenly, and the mixture is stored at 4 ℃ and can be reused within 6 months.

4) 100 microliter of air is pumped into the foaming agent by a 100 microliter pipette, and the liquid is blown for 5 seconds rapidly and repeatedly to change large bubbles into small bubbles. 5) The operation of operation 4) is repeated once to make the blowing agent into dense small bubbles. Note that: the foam volume was around 250 μ L to avoid injecting excess air. 6) After the centrifugation is finished, taking out the chip, injecting 100 mu L of fine bubbles into the sample adding hole at a constant speed, and sucking away liquid overflowing from the sample outlet groove; add 55. mu.L of 50% annealing buffer to the well and place the chip back in the centrifuge and centrifuge for 30 seconds. 7) Repeating the operation of the step 6) once. 8) 100 μ L of the washing solution was slowly added vertically to the well of the chip, and the discharged solution was aspirated off the other well. This operation was repeated once more. 9) Add 100 μ L of 50% annealing buffer vertically and slowly to the sample well of the chip to avoid the formation of air bubbles, and suck the drained liquid off in the other well. The operation is repeated once more. 10) 65. mu.L of the enzyme reaction solution was slowly added vertically to the well of the chip to prevent the formation of bubbles. The drained liquid is sucked away in another hole. 11) After incubation for 5 minutes in the dark at room temperature, the chip was mounted on the sequencer chip slot, the program was run, a Plan set in advance was selected, and after checking, sequencing was started with a running time of about 2.5 hours. 12) And returning to the main menu within 72 hours after the program operation is finished, and washing with water. If the time exceeds 72 hours, the apparatus is washed with chlorine and then with water. After the water washing is finished, the instrument is closed on the main menu, and the 4 dNTP holes are sleeved with tubes for dust prevention. If the nitrogen cylinder is not used for a long time, the nitrogen cylinder gas valve is closed.

7. Sequencing data analysis

When a semiconductor high-throughput sequencing platform is used for sequencing, a DNA chain is fixed in a micropore of a semiconductor chip, DNA polymerase takes the single-chain DNA as a template to synthesize a complementary DNA chain according to the base complementary principle, when the DNA chain extends for one base, a proton is released to cause local pH change, a system detects the pH change, a chemical signal is converted into a digital signal, and the base is interpreted in real time. Through the analysis of all sequencing sequences, the quality filtration and the comparison judgment of reference positions of DNA sequence fragments are realized; and (3) performing variation analysis and statistics on the number of sequences of the disease-related sites by combining a bioinformatics analysis method, and comparing the statistical result with reference data formed by normal samples to obtain whether the target disease in the detection sample has a high risk result.

1) Sequence alignment and filtration

The sequence comparison is the basis of noninvasive detection of new mutation of dominant genetic disease of fetus by high-throughput sequencing analysis based on free DNA of peripheral blood of pregnant women. Selecting proper alignment software (BWA) to align the raw sequence data (FASTQ file or BAM file) obtained by a sequencing-obtaining semiconductor sequencer with the human genome reference sequence (such as GRCh37/hg19 version), and then filtering sequences with larger influence on the system variation coefficient, such as ummapped, low-ratio-to-quality and multiple-repetition alignment (duplication) and the like to obtain effective sequencing data (unique aligned sequences).

2) Statistics of target region sequencing

And obtaining quality control information such as effective sequencing coverage depth, average sequencing background and the like of the target region through statistics of the sequence captured by the probe region. In this embodiment, the target region is selected according to the constructed reference database, so as to detect the high-risk newly-mutated region.

3) Analysis of variant sites

And (3) performing mutation site analysis on the sequence captured by the probe region by utilizing GATK, SAMtools and the like to obtain information such as sequencing coverage depth, mutation reliability and the like of the mutation site of the target region.

The variant sites are required to meet the following conditions:

depth of sequencing	seq_depth>100
		Base quality value	base_quality>15
Number of cover sequence fragments of variant sequences	alt_depth>2
		Variant sequence ratio (alt _ depth/seq _ depth)	>0.01

4) Disease risk analysis

Analyzing the pathogenicity grade of the variant locus according to ACMG guidelines, wherein if a known and definite pathogenicity locus is obtained, the disease risk grade is high risk; if no variant site is detected or the detected relevant variant sites are all benign variants/suspected benign variants, the disease is at low risk; if a new clinically unknown mutation site is detected, further assessment of risk is suggested in conjunction with the results of the fetal relative detection. Further, this example combines the correspondence between the new mutation and the phenotype in the reference database to perform disease risk assessment.

Specifically, the process of establishing the reference database in step S1 includes:

tracking and recording the newborns corresponding to the samples suspected of the new mutations, carrying out genetic analysis on the newborns with abnormal phenotype to confirm whether the new mutations occur, carrying out reliability evaluation on the new mutation identification bioinformatics method in the verification process according to the confirmation result, and finally recording the reliable new mutations in a basic database in combination with the known corresponding phenotypes; the phenotype comprises the collection of corresponding clinical data of the sample through an existing medical data sharing network, and the clinical data comprises a prenatal B-mode ultrasound result, a pregnant woman pregnancy outcome, a birth follow-up result and the like. The invention is to trace 2016 the pregnant and born newborn baby. Further, corresponding data are recalled from a database formed by the detection data of the free DNA of the fetus in the peripheral blood of the historical pregnant woman, and the corresponding data are retested and verified through clinical data to improve the accuracy of the reference database. The bioinformatics identification may have errors, and analysis results with high accuracy can be further screened through reliability evaluation, so that the data in the reference database has very high referenceability, and the accuracy of a subsequent detection method based on the reference database is improved conveniently.

Specifically, in the process of establishing the reference database, after a reliable new mutation is obtained, the harmfulness is classified according to the known corresponding abnormal phenotype, the existing database including the OMIM database and the existing bioinformatics tool, and the classified new mutation is registered in the reference database. By means of the harmfulness classification, the method is favorable for rapidly screening new mutations with serious influences, and when the number of the detected target gene regions is limited, the new mutations can be preferentially detected so as to realize targeted prevention and control.

Specifically, based on the existing historical maternal peripheral blood fetal free DNA detection data, an analysis model for identifying sequencing errors is established and applied to correct the sequencing results in step S4. In the prior art, low-abundance mutation and sequencing error are difficult to distinguish, but the method can establish an analysis model for effectively identifying the sequencing error based on a database with huge data, so that the sequencing error or the low-abundance mutation is identified and verified, and the accuracy of the detection method is improved.

Specifically, high-throughput sequencing of the target gene region is achieved in step S3 by SNP-based fetal concentration detection technology and DNA hybridization capture technology. The principle of predicting fetal DNA concentration by peripheral blood SNP is shown in FIG. 2. The prediction is more accurate through the application file than the conventional common prediction technology, and the effect of predicting the fetal concentration through various conventional algorithms is shown in figure 3.

Specifically, step S3 specifically includes;

Specifically, after the mutation site is obtained in step S34, a pathogenicity analysis procedure for the mutation site is established based on prior art data. The inventor also establishes a pathogenicity analysis process of the mutation sites based on public databases, documents and databases accumulated by teams.

Specifically, after the detection is completed, a standard sample is further constructed to test the accuracy of the processes from the step S3 to the step S4, and the processes from the step S3 to the step S4 are optimized by continuously feeding back the test result, wherein the standard sample is a plurality of simulated blood plasma formed by constructing fetal DNA and maternal DNA in different proportions and different gradients, and the test content comprises sensitivity, specificity, repeatability and detection success rate. The standard sample is artificially constructed, and the DNA sample of the infant patient and the DNA sample of the female parent are prepared into simulated plasma samples with different gradients according to different proportions. Evaluating the proportion of different fetal DNA, and testing the performance of the detection method, such as sensitivity, specificity, repeatability, detection success rate and the like. And the detection method is optimized in a targeted manner to achieve an optimal state.

Specifically, the accuracy and the application range of the detection method are verified through prospective queue research. The method is applied to prospective queue research to verify the detection method, and is beneficial to timely feedback and optimization so as to obtain the detection method with better accuracy.

Example 2

The embodiment discloses an application of a noninvasive gene detection method for new mutations of a fetal dominant hereditary disease in detection of the first mutations of the fetal dominant hereditary disease, which can realize rapid and accurate detection of the new mutations of the fetal dominant hereditary disease, can detect a plurality of new mutations each time, is efficient and comprehensive, is suitable for clinical general screening, and can be widely applied to realize birth defect prevention and control.

Example 3

The implementation discloses a noninvasive gene detection system for new mutation of dominant genetic disease of fetus, which comprises: a reference database, a high-throughput sequencing platform and a detection platform; the reference database comprises corresponding information of new fetal mutation genotypes and phenotypes; the high-throughput sequencing platform is used for carrying out high-throughput sequencing on a gene region of the whole sample DNA or the local sample DNA; the detection platform is connected with the reference database, the high-throughput sequencing platform and the existing new mutation related database through data interfaces; the detection platform determines a target gene region with high mutation risk according to a reference database and an existing new mutation related database, and performs high-throughput sequencing on the sample DNA by a high-throughput sequencing platform aiming at the target gene region; the detection platform obtains high-throughput sequencing results, corrects and identifies new mutations that occur in the target gene region. The sudden change high risk is a high risk area which is easy to have sudden change.

The detection platform is connected with a detection database containing historical pregnant woman peripheral blood fetus free DNA detection data, establishes an analysis model for identifying sequencing errors based on the detection database, and corrects a sequencing result in a new mutation identification bioinformatics method by applying the analysis model. Specifically, the detection platform utilizes a high-throughput sequencing platform to realize high-throughput sequencing of a target gene region through an SNP-based fetal concentration detection technology and a DNA hybridization capture technology. More specifically, the process of the detection platform using the high-throughput sequencing platform to perform high-throughput sequencing on the target gene region and identifying new mutations according to the high-throughput sequencing result comprises: obtaining a reliable sequencing sequence through a sequencing quality filtering program after obtaining the fetal DNA of an object to be detected; obtaining a target gene region in a sequencing sequence based on a DNA hybridization capture technology, comparing the target gene region with a genome, screening out sequences which are not the only comparison, and obtaining an analysis sequence for subsequent analysis; carrying out statistics and sequencing preference correction on each site in the analysis sequence, and carrying out statistics on polymorphism information of each site to obtain SNP (single nucleotide polymorphism) sites for evaluating the concentration of the DNA of the fetus; and comprehensively calculating the DNA concentration of the fetus according to the obtained SNP loci, and simultaneously acquiring the aneuploidy information of the chromosome of the fetus and the corresponding genetic background of the fetus according to the SNP locus information to identify new mutation occurring in a target gene region.

The system also comprises a pathogenicity analysis module, and after the detection platform acquires the mutation site, the pathogenicity analysis process of the mutation site is established through the pathogenicity analysis module based on the existing data; the system also comprises an accuracy testing module which is used for carrying out accuracy testing and feedback optimization on the detection platform; the detection platform detects the constructed standard sample, and the accuracy testing module determines the accuracy of the standard sample according to a detection result and feeds back data to optimize the detection process of the detection platform; the standard sample is a plurality of simulated blood plasmas formed by constructing fetal DNA and maternal DNA according to different proportions and different gradients, and the test contents comprise sensitivity, specificity, repeatability and detection success rate.

Specifically, the detection method described inembodiment 1 or the detection method described inembodiment 2 is implemented by applying the present system.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.

Claims

1. A noninvasive gene detection method for new mutation of a fetal dominant genetic disease is characterized by comprising the following steps:

s1, establishing a reference database corresponding to the new fetal mutation genotype and phenotype;

s2, confirming the mutation-high-risk target gene region according to the reference database established by the S1 and the related database of the new mutation in the prior art;

s3, obtaining the fetal DNA of the object to be detected, carrying out high-throughput sequencing on the target gene region, correcting the sequencing result and identifying new mutation in the target gene region.

2. The method for noninvasive gene detection of new mutation of fetal dominant genetic disease as claimed in claim 1, wherein the step of creating reference database in step S1 comprises the steps of:

tracking and recording the newborns corresponding to the samples suspected of the new mutations, carrying out genetic analysis on the newborns with abnormal phenotype to confirm whether the new mutations occur, carrying out reliability evaluation on the new mutation identification bioinformatics method in the verification process according to the confirmation result, and finally recording the reliable new mutations in a reference database in combination with the known corresponding phenotypes; the phenotype comprises the collection of sample corresponding clinical data through an existing medical data sharing network, and the clinical data comprises a prenatal B-mode ultrasound result, a pregnant woman pregnancy outcome and a birth follow-up result.

3. The method of claim 2, wherein after reliable new mutations are obtained, the method further comprises classifying the harmfulness of the new mutations according to known corresponding abnormal phenotypes, existing databases including OMIM database, and existing bioinformatics tools, and then registering the classified new mutations in the reference database.

4. The noninvasive gene detection method for new mutations of fetal dominant genetic diseases of claim 2, wherein an analysis model for identifying sequencing errors is established based on existing historical maternal peripheral blood fetal free DNA detection data and applied to bioinformatics methods for identifying new mutations to correct sequencing results.

5. The method of claim 1, wherein the step S3 of high throughput sequencing of the target gene region is achieved by SNP-based fetal concentration detection and DNA hybridization capture.

6. The method for noninvasive gene detection of new mutation of fetal dominant inheritance disease as claimed in claim 5, wherein the step S3 specifically comprises;

7. The noninvasive gene detection method for new mutation of fetal dominant hereditary disease of claim 6, wherein after the mutation site is obtained in step S34, the pathogenicity analysis process of the mutation site is established based on the prior art data.

8. The noninvasive gene detection method for new mutations of fetal dominant hereditary diseases of claim 1, wherein after detection, standard samples are constructed to test the accuracy of the processes of steps S3-S4, and the processes of steps S3-S4 are optimized according to the continuous feedback of the test results, wherein the standard samples are a plurality of simulated plasma formed by constructing fetal DNA and maternal DNA according to different proportions and different gradients, and the test contents include sensitivity, specificity, repeatability and detection success rate.

9. The noninvasive gene detection method for new mutations of the dominant genetic diseases of fetuses according to any one of claims 1-8, characterized in that the accuracy and the application range of the detection method are verified by prospective cohort studies.

10. The noninvasive gene detection method for new mutations of fetal dominant genetic diseases as claimed in any one of claims 1-9, which is applied to identification of new mutations of fetal dominant genetic diseases.