Example 616 chromosome instability regions and gene variation regions were constructed as genomic DNA libraries for screening and diagnosing prostate cancer as analytical markers.

The relevant kit in this example was purchased from NEB corporation, cat no: E7645S.

1. Genome fragmentation: 20ng of human genomic DNA (obtained from a clinically confirmed sample) was used to prepare an enzyme digestion reaction system shown in Table 2, and the reaction was carried out according to the procedure shown in Table 3. In this example, the concentration of human genomic DNA was 2 ng/. mu.L, and 10. mu.L was added to the reaction system.

TABLE 2

Components	System/. mu.L
		Genomic DNA	10
Fragmentation mix enzyme	10
		Fragmentation buffer	5
Non-nucleic acid water	Supplement to 50
		General System	50

After shaking, mixing and centrifugation (avoiding air bubbles), the following procedure was run on the PCR instrument:

TABLE 3

2. Terminal addition of A

If the human genome DNA is cell-free DNA, such as cfDNA in blood, the non-modified A is directly carried out without genome fragmentation, 20ng of cfDNA is taken to prepare an enzyme digestion reaction system shown in Table 4, and the reaction is carried out according to the procedure in Table 6. In this example, the concentration of cfDNA was 2 ng/. mu.L, and 10. mu.L was added to the reaction system. The reaction product of the previous step was prepared as shown in Table 5.

TABLE 4

cfDNA end-modification A reaction	System/. mu.L
		cfDNA	10
Non-modified enzyme A	3
		Buffer solution A was not added	7
Nuclease-free water	Supplement to 60
		General System	60

TABLE 5

TABLE 6

3. And (3) joint connection reaction: after adaptor ligase (5. mu.L/sample) and adaptor ligation enhancer (30. mu.L/sample) were mixed by pipetting an appropriate volume based on the number of samples to be tested, 35. mu.L of the mixed solution was pipetted and added to the reaction product in the previous step, 5. mu.L of the mixed adaptor prepared in example 5 was pipetted and added to the reaction solution, and then the mixture was shaken, homogenized and centrifuged. The reaction system in table 7 below was finally formed.

TABLE 7

Components	System/. mu.L
		Last stepReaction product	60
Joint connection premix liquid	30
		Joint connection enhancer	1
Hybrid joint	2.5
		General System	93.5

The system is placed in a PCR instrument, and the program is run: at 20 deg.C, 30min, the hot lid was closed.

4. A purification step after the ligation reaction:

a. taking out the library purified magnetic beads in advance, standing at room temperature for at least 30min, and mixing uniformly before use.

b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with the corresponding number, adding 84 mu L of the resuspended library purified magnetic beads, sucking and beating the library purified magnetic beads for 20 times at a constant speed by using a pipettor with a proper range, and incubating the library for 5min at room temperature.

c. Placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified.

d. To this, 200. mu.L of 80% ethanol was added in a fresh state, and after standing for 30 seconds, the supernatant was discarded.

e. Repeating the step d once.

f. And taking off the centrifugal tube from the magnetic frame, performing instantaneous centrifugation for 3s, putting the centrifugal tube back on the magnetic frame, and removing the residual 80% ethanol by suction, taking care not to suck the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.

g. When the magnetic beads become sub-bright, 17. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.

h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 15. mu.L of the supernatant was collected for the next amplification reaction.

5. And (3) PCR amplification: the corresponding reagents were added to the PCR tubes as in table 8 below:

TABLE 8

Components	System/. mu.L
		Purification of ligation product in the previous step	15
PCR amplification premix solution	25
		P7 end-tag primer	5
P5 end-tag primer	5
		General System	50

Among them, the P7 end-tag primer and the P5 end-tag primer were synthesized by bio (shanghai) corporation. The specific sequence is as follows:

TABLE 9

Numbering	Tag primer sequence 5 '-3'
		P7-01	SEQ ID NO.3
P7-02	SEQ ID NO.4
		P7-03	SEQ ID NO.5
P7-04	SEQ ID NO.6
		P7-05	SEQ ID NO.7
P7-06	SEQ ID NO.8
		P7-07	SEQ ID NO.9
P7-08	SEQ ID NO.10
		P5-01	SEQ ID NO.11
P5-02	SEQ ID NO.12
		P5-03	SEQ ID NO.13
P5-04	SEQ ID NO.14
		P5-05	SEQ ID NO.15
P5-06	SEQ ID NO.16
		P5-07	SEQ ID NO.17
P5-08	SEQ ID NO.18

The mixed PCR tube was placed in a PCR instrument and the following procedure was run:

watch 10

6. PCR product purification reference step 3 purification step, wherein:

the amount of the resuspended library purification magnetic beads in step b was 45. mu.L;

adding 31 mu L of Low TE buffer solution (or nuclease-free water) in the step g;

in step h, 30. mu.L of the supernatant was collected for further processing.

7. Library quantitative processing machine

The purified library was analyzed for fragment size using the Qseq Biofragment analysis System

The mass concentration of the library was measured by the dsDNA HS Assay Kit (purchased from Saimer Feishi technologies), and the molar concentration of the library was calculated from the mass concentration and the fragment size, according to the aboveSequencer instructions sequencing was performed using Illumina Hiseq X-ten.

Example 716 chromosome instability regions and Gene variation regions as markers for analysis and screening, diagnosis of prostate cancer

The kit selected in this example was purchased from NEB corporation under the following cargo number: e6310, E7770.

1. Hybridization of the RNA sample to the probe: taking 10-50ng of RNA, taking out probe hybridization buffer solution, melting on an ice box, preparing probe hybridization premix solution according to the probe hybridization buffer solution (3 mu L/sample) and rRNA probe mixed solution (H/M/R) (2 mu L/sample), preparing a reaction system (operating on the ice box) in the table 11, gently blowing and uniformly mixing by using a pipette, and carrying out instantaneous centrifugation.

TABLE 11

X represents the volume of 10-50ng RNA sample (for example: RNA concentration 25 ng/. mu.L, 50ng RNA is added, X-50/25-2. mu.L).

Placed in a PCR instrument and run the following program:

TABLE 12

After hybridization, the sample was immediately removed from the PCR machine and placed on an ice box for further processing.

2. RNase H digestion: 10 XRNase H Buffer was removed from the ice and thawed before preparing RNase H reaction premix according to the system shown in Table 13 below.

Watch 13

Components	System/. mu.L
		10×RNase H Buffer	2
RNase H	2
		Nuclease-free water	1
General System	5

And (3) adding 5 mu L of RNase H reaction premixed solution into the reaction solution in thestep 1 to ensure that the RNase H reaction system reaches 20 mu L, gently blowing and uniformly mixing by a pipette, and carrying out instantaneous centrifugation.

The reaction system is placed in a PCR instrument (the hot cover is more than or equal to 45 ℃) for 30min at 37 ℃ to carry out RNase H reaction. After the digestion of RNase H was completed, the sample was immediately taken out of the PCR machine and placed on an ice box for the next operation.

3. DNase I digestion: the 10 XDNase I Buffer was removed from the ice and thawed, and the DNase I reaction premix was prepared according to the system in Table 14 below.

TABLE 14

Components	System/. mu.L
		10×DNase I Buffer	5
DNase I	2.5
		Nuclease-free water	22.5
General System	30

Adding 30 mu L of DNase I reaction premixed solution into the reaction solution in the step 2 to ensure that the DNase I reaction system reaches 50 mu L, gently blowing and beating the mixed solution by a pipette, and performing instantaneous centrifugation.

The reaction system is placed in a PCR instrument (the hot cover is more than or equal to 45 ℃) for 30min at 37 ℃ for DNase I reaction. After the digestion of DNase I, the samples were immediately removed from the PCR machine and placed on an ice box for immediate purification.

4. RNA purification to remove rRNA:

a. taking out the RNA purified magnetic beads from 2-8 ℃ in advance, standing and balancing for 30min to room temperature, and uniformly mixing by vortex or oscillation before use;

after the DNase I reaction is finished, adding 110 mu L of RNA purified magnetic beads (2.2 x) into each reaction tube, and blowing, beating and uniformly mixing;

c. standing at room temperature for 5min, transferring to magnetic frame for 5min until the solution becomes clear, and carefully removing supernatant;

d. keeping the centrifugal tube on a magnetic frame, adding 200 microliter of 80% ethanol, standing for 30s, and discarding all supernatants;

e. repeating the step d, and washing the magnetic beads with 80% ethanol for 1 time. Thoroughly sucking the residual liquid by using a 10-mu-L gun head;

f. drying the magnetic beads for 2-3min, adding 7 μ L of nuclease-free water after the alcohol is completely volatilized, and blowing, beating and mixing uniformly;

g. standing at room temperature for 2min, mounting on a magnetic frame for 1min, carefully sucking 5 μ L of supernatant into another new centrifuge tube when the solution becomes clear;

h. add 5. mu.L fragmentation buffer to the supernatant tube, blow and mix well, perform RNA disruption according to the following procedure (hot lid 105 ℃ C.) in Table 15 (free RNA does not need this fragmentation, add water directly to 10. mu.L for the next step):

watch 15

When the temperature was lowered to 4 ℃, it was removed, subjected to instantaneous centrifugation and then placed on ice, and immediately subjected to the next first strand cDNA synthesis.

5. First strand cDNA synthesis: taking out the RT chain specific reagent to melt at room temperature, preparing the system shown in the table 16 on ice, lightly blowing and uniformly mixing by a pipette, and performing instantaneous centrifugation.

TABLE 16

Components	System/. mu.L
		Post-disrupted mRNA	10
RT chainspecific reagents	8
		First Strand synthetase mixture	2
General System	20

Placed in a PCR instrument and run the following program:

TABLE 17

6. Second strand cDNA synthesis: the reaction system was prepared on the ice box according to the following table, gently blown and beaten by a pipette, mixed well, and centrifuged instantaneously.

Watch 18

Components	System of
		First Strand cDNA	20
Second StrandSynthesis reaction buffer	8
		Second Strand synthetase mixture	4
Enzyme-free water	48
		General System	80

The PCR was run in a PCR machine at 16 ℃ for 60min with the hot lid closed.

Immediately after the reaction, purification was carried out by the following steps:

b. 144 μ L of RNA purified magnetic beads (2.2X) were added to each reaction tube, and the mixture was pipetted and mixed;

f. drying the magnetic beads for 2-3min, adding 39 μ L of nuclease-free water after the alcohol is completely volatilized, and blowing, beating and uniformly mixing;

g. after standing at room temperature for 2min and on a magnetic stand for 1min, after the solution became clear, 37. mu.L of the supernatant was carefully pipetted into another new centrifuge tube.

7. End repair plus a: taking out the unmodified buffer solution in advance, melting on an ice box, preparing a premixed solution according to the unmodified buffer solution (10 mu L/sample) and the unmodified enzyme (3 mu L/sample), adding 13 mu L of the premixed solution into the purified product in the previous step, blowing and mixing by a pipette, and performing instantaneous centrifugation (operation on the ice box). The concrete system is as follows:

watch 19

Components	System/. mu.L
		Double-stranded cDNA purification product	37
Buffer for final repair	10
		Un-modified enzyme	3
General System	50

Placed in a PCR instrument and run the following program:

watch 20

8. Connecting a joint: after the ligation buffer (16.5. mu.L/sample) and ligase (3. mu.L/sample) were mixed by pipetting an appropriate volume in accordance with the number of samples to be detected, 19.5. mu.L of the mixed solution was pipetted and added to the reaction product of the previous step, 2.5. mu.L of the prepared mixed adaptor was pipetted and added to the above reaction solution, and the mixture was shaken, mixed and centrifuged instantaneously. The reaction system of Table 21 below was finally formed.

TABLE 21

Components	System/. mu.L
		Reaction product of the last step	50
Ligation buffer	16.5
		Ligase	3
Joint	2.5
		General System	72

Place in PCR, run at 22 deg.C for 15min, and close the hot lid.

Purifying a joint connection product:

a. taking out the RNA purification magnetic beads from 2-8 ℃ in advance, standing at room temperature for at least 30min, and mixing uniformly before use.

b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with the corresponding number, adding 56 mu L of resuspended magnetic beads, uniformly sucking and stirring the mixture by using a pipettor with a proper range at a constant speed, and incubating the mixture for 5min at room temperature.

e. Repeating the step d once.

f. And taking off the centrifugal tube from the magnetic frame, performing instantaneous centrifugation for 3s, putting the centrifugal tube back on the magnetic frame, and sucking away the residual 80% ethanol without sucking the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.

g. When the magnetic beads become sub-bright, 21. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.

h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 20. mu.L of the supernatant was collected for the next amplification reaction.

9. And (3) PCR amplification: the reaction system was prepared according to the following table 22, mixed well and centrifuged instantaneously.

TABLE 22

Components	System/. mu.L
		Purification of ligation product in theprevious step	20
PCR amplification premix solution	25
		P7 end-tag primer	2.5
P5 end-tag primer	2.5
		General System	50

Put into a PCR instrument and run the following program:

TABLE 23

And (3) PCR product purification:

a. taking out RNA purified magnetic beads from 2-8 ℃ in advance, standing at room temperature for at least 30min, and mixing uniformly before use.

b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with a corresponding number, adding 40 mu L of resuspended magnetic beads, uniformly sucking and stirring the mixture by using a pipettor with a proper range at a constant speed, and incubating the mixture for 5min at room temperature.

e. Repeating the step d once.

g. When the magnetic beads become sub-bright, 42. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.

h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 40. mu.L of the supernatant was collected for the next amplification reaction.

10. Performing quality inspection on the library: use of the purified library

The dsDNA HS Assay Kit measures the library mass concentration, the Qseq biological fragment analysis system analyzes the fragment size, the library molar concentration is calculated according to the mass concentration and the fragment size, and the Illumina Hiseq X-ten is used for sequencing according to the instruction of the sequencer.

Results analysis discussion:

the sequencing data from the machine was analyzed to obtain chromosome instability and genetic variation profiles, as shown in FIGS. 1-4.

The chromosome instability analysis result consists of three parts: chromosome number, chromosome partitioning, and chromosome sequencing depth profile. The dots in the chromosome sequencing depth distribution region are the distribution of the copy number of the chromosome small region, and are judged to have no instability when the score value of the vertical axis is between-3 and 3, are judged to be amplified when the score value of the vertical axis is larger than 3, are judged to be deleted when the score value of the vertical axis is smaller than-3, and are distinguished from the normal region by using a gray background for the region in which amplification or deletion occurs. And checking whether the chromosome instability occurs or not according to the analysis result, and judging the chromosome instability to be positive if the chromosome instability occurs or judging the chromosome instability to be negative if the chromosome instability does not occur.

Analysis of gene variation: the PCA3 gene is overexpressed, the relative reads number of PCA3 and PSA is firstly calculated, then the PCA3 score is calculated to be (PCA3 relative reads number/PSA relative reads number) multiplied by 1000, and when the PCA3 score is higher than 25, the PCA3 gene is overexpressed; and (3) ERG gene fusion, wherein the screened sequencing data is compared with the whole genome sequence, the ERG gene is positioned at 21q22, and if the comparison result is at other positions, the ERG gene fusion is judged. A schematic representation of ERG gene fusion is shown in FIG. 5.

The second generation analysis results were compared with the pathological results, and the sensitivity and specificity of the analysis method were calculated to obtain the ROC curve, as shown in fig. 6. The AUC area of the ROC curve of the method reaches 0.918, which shows that the 16 chromosome instability regions and the gene variation regions in the method are used as analysis marks, and the diagnosis of the prostate cancer by the second-generation sequencing has high sensitivity and specificity.

Sequence listing

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Claims

1. A set of combinations of chromosomal instability regions and genetic variation regions, wherein the chromosomal instability regions and genetic variation regions comprise: 2q, 3q, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 16q, 18q, PCA3, ERG.

2. The combination of a chromosomal instability region and a genetic variation region according to claim 1, wherein the chromosomal instability comprises deletion or amplification of the entire chromosome or a copy of a chromosomal segment; the gene variation comprises PCA3 gene overexpression and ERG gene fusion.

3. A library adaptor having a sequence selected from SEQ ID No.1 and/or SEQ ID No.2 for use in detecting a combination of a chromosomal instability region and a genetic variation region according to claim 1.

4. Use of the library adaptor of claim 3 in the construction of a gene library.

5. A method for constructing a gene library, which comprises constructing the library adaptor according to claim 3.

6. Use of a combination of a chromosomal instability region and a genetic variation region according to any of claims 1-2 and/or a library adaptor according to claim 3 and/or a method of constructing a genetic library according to claim 5 for the preparation of a reagent or kit for screening and/or diagnosing and/or prognostically assessing prostate cancer.

7. A reagent or kit for screening and/or diagnosis and/or prognostic evaluation of prostate cancer, wherein the reagent is used for detecting the chromosome instability region and the gene variation region according to any one of claims 1 to 2; the kit comprises a reagent for detecting the combination of the chromosome instability region and the gene variation region according to any one of claims 1 to 2.

8. The kit of claim 7, further comprising the library adaptor of claim 3.

9. The kit of claim 7, further comprising one or more of a positive reference, a negative reference, a buffer, an enzyme, a detectable label, a nucleic acid extraction reagent, and a nucleic acid purification reagent; the enzyme comprises one or more of a breaking enzyme, a DNA polymerase, a DNA ligase, an RNA digestive enzyme, a DNA digestive enzyme and a reverse transcriptase.

10. The kit of claim 7, wherein the positive reference is a cell line mixed with 16 region variants of claim 1; the negative reference substance is a cell line without chromosome variation.