CN120442775A - Hematopoietic stem cell transplantation ligand and prognosis evaluation kit and using method thereof - Google Patents
Hematopoietic stem cell transplantation ligand and prognosis evaluation kit and using method thereofInfo
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Abstract
The invention relates to a hematopoietic stem cell transplantation and prognosis evaluation kit and a use method thereof. The kit contains probes for capturing target genes, wherein the target genes comprise HLA genes, KIR genes, MICA genes, MICB genes and SUFU rs17114808 sites. The invention can detect all necessary gene loci at one time by selecting a specific exon region and limiting the Tm value, GC content, hairpin free energy, sequence complexity and genome homology of probe design, provides scientific basis for personalized treatment scheme of hematopoietic stem cell transplantation, is beneficial to predicting and preventing graft versus host disease after transplantation, can optimize treatment scheme of transplantation pretreatment and perioperative period, thereby bringing better prognosis for patients, covers a plurality of important genes and loci related to hematopoietic stem cell transplantation, and is beneficial to comprehensively evaluating transplantation risk.
Description
Technical Field
The invention relates to the technical field of gene detection kits, in particular to a hematopoietic stem cell transplantation and prognosis evaluation kit and a use method thereof.
Background
With advances in haplotype-matched hematopoietic stem cell transplantation technology, HLA-type differences between donor and recipient have a significant impact on treatment outcome. While existing PCR-SBT methods are capable of performing both types of HLA typing, such methods do not provide more accurate typing for multiple half-identical siblings to determine the best donor. In addition, other immune-related genes, such as KIR, MICA, and SUFU genes, are also closely related to the immune status of the patient and the risk after transplantation, in addition to HLA genes. Reconstitution of immunity after transplantation is a complex process, where reconstitution of innate immunity is particularly important, which directly affects the defenses against tumors and pathogens. NK cells function depends on education, while the existence of KIR receptor mediates the generation of homoreactivity NK cell activity, thereby influencing the occurrence and development of infection, GVHD and other important complications after transplantation. Detailed genotyping analysis of donors and recipients of hematopoietic stem cell transplants has become particularly critical in order to increase the success rate of the transplantation and reduce the risk of rejection. These include HLA genotyping, KIR genotyping, MICA genotyping and mutation analysis thereof, and SUFU (fusion inhibition) rs17114808 locus genotyping.
The Chinese patent CN102877136B discloses a method and a kit for constructing a DNA library based on genome simplification and second generation sequencing, which are used for full-genome SNP detection and genotyping of species without haplotype map, and are focused on the application of the full-genome SNP detection and the reference genome imperfect species and have weak pertinence with clinical transplantation scenes.
Disclosure of Invention
The first aspect of the invention provides a hematopoietic stem cell transplantation and prognosis evaluation kit, which comprises a probe for capturing a target gene, wherein the target gene comprises an HLA gene, a KIR gene, a MICA gene, a MICB gene and SUFU rs17114808 sites.
The probe information corresponding to the HLA genes comprises chromosome 6, and exon areas are 29942531-29945870、31268748-31272092、31353874-31357179、32578768-32589836、32637405-32654846、32659466-32666657、33064568-33089696 and 32637405-32654846.
The probe information corresponding to the KIR gene comprises chromosome 19, exon regions ：54724175-54724616、54725174-54726394、54726433-54728353、54728473-54728833、54728872-54728992、54729197-54730277、54730877-54730997、54731357-54732258、54732557-54733277、54733517-54734237、54735072-54735357、54735393-54735513、54735820-54735872、54735971-54736633、54738245-54738724、54738864-54739016、54739136-54739256、54739357-54739613、54739700-54740576、54740577-54741038、54741380-54742472、54742585-54742857、54742858-54743000、54743060-54743180、54743569-54743689、54743795-54744088、54745419-54745539、54747127-54747385、54747549-54747667、54747936-54748087、54748148-54748268、54748497-54748617、54748960-54749446、54749922-54750042、54750052-54750172、54750313-54750635、54750727-54750847、54751378-54751497、54751649-54751753、54751916-54752165、54752216-54752268、54752367-54753052、54756308-54756764、54757034-54757274、54757280-54757514、54757993-54758113、54758373-54758729、54758833-54759102、54759130-54759335、54759369-54759489、54759490-54759812、54760165-54760325、54760662-54760782、54760926-54761142、54761622-54761742、54763796-54764096、54764353-54764473、54764752-54764872、54764982-54765342、54765555-54765795、54766768-54766888、54766927-54767084、54767418-54767538、54769582-54769942、54770595-54770715、54770782-54770955、54771021-54771262、54771320-54771502、54771622-54772462、54772602-54772822、54772914-54773662、54773940-54774060、54774142-54774537、54774819-54774939、54775165-54775458、54775539-54775779、54775899-54776038、54776642-54776762、54777749-54777869、54778612-54778662、54778705-54778944、54779213-54779333、54779425-54779894、54780237-54780754、54781199-54781563、54781755-54782055、54782065-54782415、54782922-54783023、54783315-54783614、54783637-54784322、54785807-54786167、54786327-54786561、54786567-54787134、54787254-54787374、54787558-54788094、54788168-54788692、54789174-54789400、54789414-54789635、54789691-54789921、54790014-54790327、54803384-54803984、54803985-54806470、54806590-54806710、54807032-54807426、54808144-54808265、54808505-54810160、54810423-54811143、54811383-54812343、54812463-54813781、54813786-54814518、54816022-54816143、54816352-54816467、54816468-54816534、54816814-54816934、54817313-54818753、54818873-54819233、54819353-54820257、54820299-54820419、54820433-54820913、54821110-54821470、54821565-54822233、54822593-54822713、54823816-54823936、54824873-54825353、54825628-54825779、54825867-54825987、54826427-54826548、54826597-54826717、54826827-54826982、54827336-54827456、54827590-54828070、54828093-54828297、54828299-54828493、54828540-54828681、54829053-54829173、54829189-54829309、54829361-54829465、54829652-54829772、54829811-54829980、54830099-54830779、54832707-54832798、54832958-54833097、54833279-54833399、54833510-54833630、54833741-54833861、54833971-54834091、54834177-54834417、54834546-54834666、54834729-54834897、54834950-54835070、54835079-54835114、54835137-54835617、54835840-54836006、54836049-54836401、54836457-54836697、54836937-54837057、54837114-54837417、54837576-54837893、54837950-54838069、54838090-54838617、54839097-54839333、54839410-54839911、54840035-54840155、54842773-54842918、54842954-54843200、54843207-54843327、54843708-54844038、54844268-54844389、54844438-54844633、54844932-54845152、54845410-54845967、54846013-54846133、54846154-54846540、54847047-54847341、54847451-54847595、54847634-54848575、54850207-54850327、54850447-54850567、54851047-54851508、54851566-54851686、54851806-54852526、54852828-54852948、54853327-54853447、54853586-54853706、54853747-54854047、54854066-54854186、54854407-54854887、54858924-54859284、54859447-54859567、54860025-54860385、54860625-54860855、54861074-54861194、54861222-54861342、54861704-54861960、54862064-54862519、54865548-54866508 and 54866522-54867216.
The probe information corresponding to the MICA gene and the MICB gene comprises the chromosome 6, and the exon areas are 31400710-31415315 and 31494880-31511124.
The probe information corresponding to the SUFU rs17114808 locus comprises the information of the chromosome 10 and the exon area 102631527-102631528.
The probe is designed by the following method:
s1, obtaining an exon region of a target gene according to a database;
S2, intercepting 1-2 unitbp of an exon area as a first probe Unit, intercepting (n-1) Unit-Unitbp of the exon area as an nth probe Unit, intercepting unit=60 bp in sequence, and obtaining a probe of the exon area;
S3, evaluating the nucleotide sequence of the probe, wherein the evaluated parameters comprise Tm value of 65+/-2℃, GC content 45% -55%, hairpin free energy, sequence complexity and homology in genome, so as to ensure specific capture of the probe to the target sequence and stable combination of the probes under the condition of fixed temperature in the hybridization process;
and S4, adjusting the probes with scores lower than the threshold value, wherein the Unit value of the probes is 60+/-10 bp when the sequences of the probes are intercepted, and repeating the steps S2 and S3 to optimize the sequences of the probes.
The invention provides a method for using a hematopoietic stem cell transplantation matching and prognosis evaluation kit, which comprises the following steps of 1, DNA sample preparation, 2, pre-library preparation, 3, hybrid capture, 4, library mixing and on-machine sequencing, and 5, bioinformatic flow analysis, wherein gDNA of target cells is taken as a sample for genomic DNA extraction.
The target cells include bone marrow blood cells or peripheral blood leukocytes.
The preparation of the pre-library comprises the steps of sequentially carrying out genome DNA fragmentation, linker ligation and purification, PCR amplification and purification.
The hybridization capture comprises labeling the probe with biotin and hybridizing with a pre-library, and then using streptavidin magnetic beads to bind the probe so as to capture the target region.
The PCR amplification and purification further comprises library quantification, library quality detection and pre-library quality control.
Advantageous effects
1. The invention selects a specific exon region and limits the Tm value, GC content, hairpin free energy, sequence complexity and genome homology of the probe design, can detect all necessary gene loci at one time, and provides scientific basis for personalized treatment scheme of hematopoietic stem cell transplantation.
2. The kit can detect HLA genes, KIR genes, MICA genes, MICB genes and SUFU rs17114808 sites of hematopoietic stem cell transplantation donors and recipients simultaneously, covers a plurality of important genes and sites related to hematopoietic stem cell transplantation, and is beneficial to comprehensively evaluating the transplantation risk.
3. The kit can obtain all exon sequences of HLA, KIR, MICA, MICB genes, and has more accurate typing and higher precision.
4. The kit disclosed by the invention is not only beneficial to predicting and preventing graft-versus-host diseases after transplantation, but also can optimize treatment schemes of pretreatment and perioperative period of transplantation, thereby bringing better prognosis to patients.
5. Compared with the traditional complex operation of detecting a plurality of genes respectively, the kit provided by the invention only needs one-time detection, simplifies the detection flow and improves the detection efficiency.
6. In the use process of the kit, the required sample size is smaller, and the donor can obtain the DNA sample size required by detection without drawing blood and only by providing an oral swab.
Detailed Description
Examples
A kit for hematopoietic stem cell transplantation and prognosis evaluation contains a probe for capturing target genes, wherein the target genes comprise HLA genes, KIR genes, MICA genes, MICB genes and SUFU rs17114808 sites.
The probe information corresponding to the HLA genes is located on chromosome 6, and the exon areas are 29942531-29945870、31268748-31272092、31353874-31357179、32578768-32589836、32637405-32654846、32659466-32666657、33064568-33089696 and 32637405-32654846.
The KIR gene corresponds to probe information, namely, the 19 # chromosome, exon areas ：54724175-54724616、54725174-54726394、54726433-54728353、54728473-54728833、54728872-54728992、54729197-54730277、54730877-54730997、54731357-54732258、54732557-54733277、54733517-54734237、54735072-54735357、54735393-54735513、54735820-54735872、54735971-54736633、54738245-54738724、54738864-54739016、54739136-54739256、54739357-54739613、54739700-54740576、54740577-54741038、54741380-54742472、54742585-54742857、54742858-54743000、54743060-54743180、54743569-54743689、54743795-54744088、54745419-54745539、54747127-54747385、54747549-54747667、54747936-54748087、54748148-54748268、54748497-54748617、54748960-54749446、54749922-54750042、54750052-54750172、54750313-54750635、54750727-54750847、54751378-54751497、54751649-54751753、54751916-54752165、54752216-54752268、54752367-54753052、54756308-54756764、54757034-54757274、54757280-54757514、54757993-54758113、54758373-54758729、54758833-54759102、54759130-54759335、54759369-54759489、54759490-54759812、54760165-54760325、54760662-54760782、54760926-54761142、54761622-54761742、54763796-54764096、54764353-54764473、54764752-54764872、54764982-54765342、54765555-54765795、54766768-54766888、54766927-54767084、54767418-54767538、54769582-54769942、54770595-54770715、54770782-54770955、54771021-54771262、54771320-54771502、54771622-54772462、54772602-54772822、54772914-54773662、54773940-54774060、54774142-54774537、54774819-54774939、54775165-54775458、54775539-54775779、54775899-54776038、54776642-54776762、54777749-54777869、54778612-54778662、54778705-54778944、54779213-54779333、54779425-54779894、54780237-54780754、54781199-54781563、54781755-54782055、54782065-54782415、54782922-54783023、54783315-54783614、54783637-54784322、54785807-54786167、54786327-54786561、54786567-54787134、54787254-54787374、54787558-54788094、54788168-54788692、54789174-54789400、54789414-54789635、54789691-54789921、54790014-54790327、54803384-54803984、54803985-54806470、54806590-54806710、54807032-54807426、54808144-54808265、54808505-54810160、54810423-54811143、54811383-54812343、54812463-54813781、54813786-54814518、54816022-54816143、54816352-54816467、54816468-54816534、54816814-54816934、54817313-54818753、54818873-54819233、54819353-54820257、54820299-54820419、54820433-54820913、54821110-54821470、54821565-54822233、54822593-54822713、54823816-54823936、54824873-54825353、54825628-54825779、54825867-54825987、54826427-54826548、54826597-54826717、54826827-54826982、54827336-54827456、54827590-54828070、54828093-54828297、54828299-54828493、54828540-54828681、54829053-54829173、54829189-54829309、54829361-54829465、54829652-54829772、54829811-54829980、54830099-54830779、54832707-54832798、54832958-54833097、54833279-54833399、54833510-54833630、54833741-54833861、54833971-54834091、54834177-54834417、54834546-54834666、54834729-54834897、54834950-54835070、54835079-54835114、54835137-54835617、54835840-54836006、54836049-54836401、54836457-54836697、54836937-54837057、54837114-54837417、54837576-54837893、54837950-54838069、54838090-54838617、54839097-54839333、54839410-54839911、54840035-54840155、54842773-54842918、54842954-54843200、54843207-54843327、54843708-54844038、54844268-54844389、54844438-54844633、54844932-54845152、54845410-54845967、54846013-54846133、54846154-54846540、54847047-54847341、54847451-54847595、54847634-54848575、54850207-54850327、54850447-54850567、54851047-54851508、54851566-54851686、54851806-54852526、54852828-54852948、54853327-54853447、54853586-54853706、54853747-54854047、54854066-54854186、54854407-54854887、54858924-54859284、54859447-54859567、54860025-54860385、54860625-54860855、54861074-54861194、54861222-54861342、54861704-54861960、54862064-54862519、54865548-54866508 and 54866522-54867216.
The probe information corresponding to the MICA gene and the MICB gene is located on chromosome 6, and the exon areas are 31400710-31415315 and 31494880-31511124.
The probe information corresponding to SUFU rs17114808 locus is 102631527-102631528 in the exon region located on chromosome 10.
The probe is designed by the following method:
s1, obtaining an exon region of a target gene according to a database;
S2, intercepting 1-2 unitbp of an exon area as a first probe Unit, intercepting (n-1) Unit-Unitbp of the exon area as an nth probe Unit, intercepting unit=60 bp in sequence, and obtaining a probe of the exon area;
s3, evaluating the nucleotide sequence of the probe, wherein the evaluated parameters comprise Tm value 65+/-2℃, GC content 45% -55%, hairpin free energy, sequence complexity and homology in genome;
and S4, adjusting the probes with scores lower than the threshold value, wherein the Unit value of the probes is 60+/-10 bp when the sequences of the probes are intercepted, and repeating the steps S2 and S3 to optimize the sequences of the probes.
The application method of the hematopoietic stem cell transplantation matching and prognosis evaluation kit comprises the following steps:
1. DNA sample preparation, namely taking a peripheral blood leukocyte gDNA sample, extracting genome DNA by referring to the standard operation procedure for extracting blood genome DNA, eluting with 100 mu L of ddH 2 O (double deionized water), and preserving after eluting to obtain a template for subsequent experiments.
2. The pre-library is prepared by adopting QuarPrep enzyme-cutting DNA library-building kit.
2.1 Genome DNA fragmentation
The Frag/AT buffer and the Frag/AT enzyme in the kit are taken out, thawed, fully mixed, collected by short centrifugation to the bottom of a tube, placed on ice for standby, all the following steps are operated on ice, and prepared in a sterilized PCR tube for reaction as shown in the following table 1:
TABLE 1
| Reagent(s) | Content of |
| InputDNA (template obtained in step 1) | 100ng |
| Frag/AT buffer | 4μL |
| Frag/AT enzyme | 6μL |
| ddH2O | Dissolving InputDNA to 40 μl |
The reaction solution was collected to the bottom of the tube by pipetting or shaking and briefly centrifuged, and immediately placed in a PCR apparatus (Agielnt 8800) to perform the following reactions in Table 2:
TABLE 2
| Temperature (temperature) | Time of |
| Thermal cover 105 DEG C | 0 |
| 37°C | 18min |
| 65°C | 30min |
| 4°C | / |
When the thermocycler program is over and the sample module returns to 4 ℃, the sample is removed from the template, placed on ice, and immediately subjected to subsequent steps.
2.2, Linker ligation and purification, the end of the end repair product is fragmented in the last step is ligated with a linker.
The universal joint was removed from-20 ℃, thawed, thoroughly mixed, collected by short centrifugation to the bottom of the tube, and placed on ice for use, and the reaction system was prepared according to the following table 3:
TABLE 3 Table 3
| Reagent(s) | Volume (mu L) |
| The product of the last step (2.1 steps) | 50 |
| LigationMasterMix | 20 |
| DNAAdapterX | 5 |
| Totals to | 75 |
The reaction mixture was collected to the bottom of the tube by gentle pipetting (mixing without shaking) and brief centrifugation, and the PCR tube was placed in a PCR apparatus to carry out the following reaction in Table 4:
TABLE 4 Table 4
| Temperature (temperature) | Time of |
| Thermal cover 105 DEG C | 0 |
| 20°C | 15min |
| 4°C | / |
The reaction product was purified using magnetic beads, which were equilibrated to room temperature (30 min) and vortexed to mix the beads. 60. Mu.L of the beads were pipetted into 75. Mu LAdapter Ligation (adaptor-ligated) products, vortexed or gently beaten 10 times with a pipette and thoroughly mixed. Incubate at room temperature for 5min. The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, and after the solution was clarified (about 5 min), the supernatant was removed. The PCR tube was kept always in the magnetic rack, 180. Mu.L of freshly prepared 80% ethanol was added to rinse the beads, incubated at room temperature for 30s, and the supernatant was removed. Step 5 was repeated for a total of two rinses. The PCR tube is kept to be always placed in the magnetic frame, and the magnetic beads are air-dried after being uncapped for 5-10min until no ethanol remains. The PCR tube is taken out of the magnetic frame, 17 mu L of ddH 2 O is added for elution, vortex oscillation or gentle blowing and full mixing are carried out by using a pipettor, the PCR tube is placed at room temperature for 2min, the PCR tube is briefly centrifuged and placed in the magnetic frame for standing, after the solution is clarified (about 5 min), 15 mu L of supernatant is removed to a new EP tube, and the magnetic beads are not touched.
2.3 PCR amplification and purification
The UDI primers in the kit, the Equinox library amplification mix (2 x) was thawed and mixed upside down and prepared in a sterile PCR tube for the following table 5 reaction:
TABLE 5
The mixture was gently stirred with a pipette (stirring without shaking) and the reaction mixture was collected to the bottom of the tube by brief centrifugation. Placing the PCR tube in a PCR instrument, and starting PCR program, wherein the temperature of the hot cover is 105 ℃, 98 ℃,45s, 98 ℃,15s, 60 ℃,30s, 72 ℃ and 1min, and the PCR tube is circulated for 8 times and stored at 4 ℃
Purifying the reaction product, namely, after the magnetic beads are balanced to room temperature, vortex oscillation and uniform mixing of the magnetic beads are carried out. 50ul (1 x) of magnetic beads were pipetted into the 50. Mu L LibraryAmplification product, vortexed or gently beaten 10 times with a pipette and thoroughly mixed. Incubate at room temperature for 5min. The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, after which the solution was clarified (about 5 min) and the supernatant carefully removed. The PCR tube was kept always in a magnetic rack, the beads were rinsed with 180 μl of freshly prepared 80% ethanol, incubated at room temperature for 30s, and the supernatant carefully removed. Step 5 was repeated for a total of two rinses. The PCR tube is kept to be always placed in the magnetic frame, and the magnetic beads are air-dried after being uncapped for 5-10min until no ethanol remains. The PCR tube was removed from the magnetic rack, 65. Mu.L ddH2O was added for elution, vortexing or gentle pipetting with a pipettor was performed to mix thoroughly, standing for 2min at room temperature, briefly centrifuging the PCR tube and standing in the magnetic rack, after clarification of the solution (about 5 min), carefully pipetting 65. Mu.L supernatant into the fresh EP tube, and the supernatant in the tube was the prepared pre-library.
2.4 Library quantitation 1. Mu.L library was measured for library concentration using Qubit 2.0Fluorometer (Qubit DSDNAHS ASSAY KIT) and library concentration was recorded.
2.5, Library quality detection, namely, carrying out library fragment length and purity measurement by using Qsep100 full-automatic nucleic acid protein analysis system, wherein the target fragment distribution interval of a normal library is 300bp-400 bp.
2.6, Pre-library quality control, namely, taking 1 mu L of pre-library, carrying out library concentration measurement by using Qubit DSDNAHS ASSAY KIT, recording the pre-library concentration, wherein the pre-library concentration needs to be more than 25 ng/. Mu.L, taking 1 mu L of pre-library, carrying out library fragment distribution quality inspection by using a nucleic acid analyzer such as Agilent 2100Bioanalyzer System, qsep100 (Bioptic) and the like, and the average length of the pre-library is between 250 and 350 bp.
3. The hybrid capture uses Ai Jitai kang TARGETSEQKit.
3.1, Taking out Hyb Human Block, adapter Block, rnase Block and Target Probe from the refrigerator in advance, putting the Hyb Human Block, adapter Block, rnase Block and Target Probe on an ice box for melting, and uniformly mixing the materials on ice or at 4 ℃ for later use after melting;
3.2, hybridization was performed by mixing a plurality of libraries, and labeling was performed by adding an equal amount of each library to a PCR tube in a total library amount of about 1200 ng. Putting the PCR tube into a vacuum concentration centrifuge, opening a PCR tube cover, starting the centrifuge, opening a vacuum pump switch, starting concentration to a dry state, and adding the following hybridization reaction system in table 6 after library concentration is completed:
TABLE 6
| Reagent | Volume(μL) |
| TargetSeqOneHybBuffer | 13 |
| HybHumanBlock | 5 |
| AdapterBlocker (selection according to library type) | 2 |
| RnaseBlock | 5 |
| NucleaseFreeWater | 3 |
| TargetProbe (Probe) | 1 |
| Totalvolume | 30 |
Gently sucking and beating, mixing, and centrifuging briefly. The PCR tube was placed in a PCR apparatus and incubated overnight at a hot lid temperature of 85 ℃, 80 ℃,5min, 50 ℃
And 3.3, preparing to capture the magnetic beads, wherein the magnetic beads used for capturing are required to be Cap beads.
Taking out the captured magnetic beads (T1) from the magnetic rack, standing at room temperature for 30min, performing vortex vibration and resuspension, adding 50 mu L of magnetic beads into a new PCR tube, standing on a magnetic rack for 1min until the solution is clear, removing the supernatant, taking the PCR tube off the magnetic rack, adding 180 mu LBinding Buffer, gently sucking and beating for several times, uniformly mixing, resuspension the magnetic beads, standing on the magnetic rack for 1min, removing the supernatant, repeating the steps 3-4 for 3 times, taking the PCR tube off the magnetic rack, adding 180 mu LBinding Buffer, gently sucking and beating, resuspension the magnetic beads, and uniformly mixing for later use.
3.4 Capturing the DNA library of the target region
When the target area is captured, the resuspended captured magnetic beads are added into the PCR tube, and the PCR tube is kept on the PCR instrument in the process.
The probe and the magnetic beads are combined and incubated for 30min at room temperature, a mixing instrument is used for avoiding sedimentation of the magnetic beads, the revolution is not too high, and the speed is not more than 10 revolutions per min.
After the capture is completed, the magnetic beads are not discarded, and the next step is PCR reaction with the magnetic beads in the system.
If precipitation is found in Wash Buffer 1 stored at room temperature, the Wash Buffer 1 is placed in a water bath kettle at 37 ℃ to be heated, and the Wash Buffer is used after the reagent is completely dissolved.
Will TARGETSEQPreheating Wash Buffer at 50deg.C in advance by 1) holding hybridization product on PCR instrument, adding 180 μL Cap beads after 6 th step in 6.3.2 into hybridization product, pipetting with pipettor, mixing, standing on rotary mixer at room temperature for 30min, standing PCR tube on magnetic rack for 2min to clarify solution, removing supernatant, 3) adding 150 μL Wash Buffer 1 into hybridization product, gently pipetting, mixing, standing on rotary mixer for 15min, centrifuging briefly, standing PCR tube on magnetic rack for 2min to clarify solution, removing supernatant, and 4) adding 150 μL TARGETSEQ preheated at 50deg.CWash Buffer, gently sucking and beating, mixing, centrifuging briefly, placing on a constant temperature shaking mixer, incubating at 50deg.C for 10min, 5) centrifuging briefly, placing PCR tube on a magnetic rack for 2min to clarify the solution, and removing supernatant. 6) Repeating the steps 4-5 twice, washing the magnetic beads for 3 times, 7) keeping the sample on a magnetic frame, adding 150 mu L of 80% ethanol into the PCR tube, standing for 30s, thoroughly removing the ethanol solution (the residue can be removed by a 10 mu L pipette), airing at room temperature, 8) adding 24 mu LNuclease-FREE WATER into the PCR tube, taking the PCR tube off the magnetic frame, and lightly sucking and beating the re-suspended magnetic beads by a pipette for standby.
3.5 Post Capture PCR amplification
Taking out Post PCR MASTER Mix and Post PCRPrimer from a-20 ℃ refrigerator, placing on an ice box for melting, uniformly mixing the melted materials on ice or at 4 ℃ for standby, carrying out PCR amplification on a DNA library after capturing, and preparing a reaction system according to the following table 7:
TABLE 7
| Reagent | Volume(μL) |
| Sample after completion of the previous reaction | 24 |
| PostPCRMasterMix | 25 |
| PostPCRPrimer | 1 |
| Totalvolume | 50 |
The pipette is adjusted to 40. Mu.L, gently sucked and stirred for 6 times and then immediately placed on a PCR instrument, a PCR tube is placed in the PCR instrument, and the procedures are 95 ℃ and 1min, 98 ℃ and 20s, 60 ℃ and 30s, 72 ℃ and 5min, and 4 ℃ are stored after circulation.
Adding 55 mu LAgencourtAMPure XP magnetic beads into a sample after the PCR is finished, carrying out vortex vibration mixing or suction beating mixing uniformly, standing for 5min at room temperature, carrying out short centrifugation, placing a PCR tube on a magnetic frame for 3min until the solution is clear, keeping the PCR tube on the magnetic frame, removing supernatant, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, keeping the PCR tube on the magnetic frame, removing supernatant, adding 180 mu L of 80% ethanol solution into the PCR tube again, standing for 30s, thoroughly removing supernatant, standing for 3-5min at room temperature, thoroughly volatilizing residual ethanol, adding 25 mu L of Nuclease-FREE WATER, taking the PCR tube off the magnetic frame, carrying out vibration mixing or suction beating mixing uniformly for 10 times, standing for 2min at room temperature, carrying out short centrifugation, placing the PCR tube on the magnetic frame for 2min until the solution is clear, sucking 23 mu L of supernatant into a liquid shifter, and transferring to 1.5mL of sample information.
3.6, Library quantitation
1. Mu.L of library was used for library concentration determination using Qubit 2.0Fluorometer (Qubit DSDNAHS ASSAY KIT), library concentration was recorded, and library fragment length and purity measurement was performed using a Qsep100 fully automated nucleic acid protein analysis system, with normal library main peak lengths ranging from about 330bp to 450 bp.
4. Library mixing and on-machine sequencing
Each final library was diluted to 4 ng/. Mu.L, and the corresponding volume of final library was added to a new 1.5mL EP tube according to the data amount of each item, and the library concentration was measured after thoroughly shaking and mixing. The amount of sequencing data was 1G, and the appropriate on-machine sequencing was selected according to the final expected amount of data.
5 Bioinformatics flow analysis
The raw sequencing data was first processed using Fastp software to remove sequencing Adapter and low quality Reads. The filtered Reads was aligned to the human reference genome (hg 38) using BWA-mem software. Next, the aligned bam files were ranked, deduplicated, and base quality corrected using GATK software. And then, comparing the biological informatics genotyping software with an IPD-IMGT/HLA database to obtain a high-resolution HLA genotyping result.
Performance test method
1. Verification of HLA genotyping and KIR genotyping tests 29 samples of known HLA and KIR genotyping results (from Shanghai Sitede medical test laboratory) were tested using the method of the examples, 29 samples were tested for HLA genotyping as shown in tables 8-10, and all samples were tested for all HLA loci uniformly and once for HLA genotyping as shown in tables 11 and 12, wherein "1" indicated that the sample contained the gene, "0" indicated that the sample did not have the gene, and 29 samples were tested for control results as shown in PCR-SSP. To sum up, the kit has accurate typing.
2. Quality control test 29 samples are subjected to quality control detection by using the method of the embodiment, and as shown in the table 13, the quality control results all meet the quality control requirements that the average sequencing depth is more than or equal to 1000, the target loading rate is more than or equal to 40%, the sequencing comparison rate is more than 80% and the uniformity is more than 60%, so that the performance of the kit is good. Meanwhile, sequencing results of 29 MICA, MICB and SUFU rs171148083 specific sites are analyzed by using IGV software, the sequencing depth of the related sites meets the analysis requirement (more than or equal to 1000), and the results are reliable and have no abnormality.
3. And (3) testing the sample injection amount (detection limit), namely using 2 detection limit samples (samples with known content), and constructing libraries with sample injection amounts of 5ng, 10ng, 20ng, 50ng and 100ng respectively. As shown in table 14, according to the example method, four samples of 5ng and 10ng failed to be pooled. The detection of 20ng, 50ng and 100ng of library-building sample-injection amount samples can meet the quality control standard, and the detection limit can reach 20ng.
Performance test data
TABLE 8
TABLE 9
Table 10
TABLE 11
Table 12
| KIR2DL5 | KIR3DS1 | KIR3DL1 | KIR3DL2 | KIR3DL3 | KIR2DP1 | KIR3DP1 | KIR2DL23 | |
| Sample 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 |
| Sample 2 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 3 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 4 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 5 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 6 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 7 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 8 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 9 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 10 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 11 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 12 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 13 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 14 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 15 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 16 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 17 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 18 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 19 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 20 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 21 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 22 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 23 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 24 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 25 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 26 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 27 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 28 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
| Sample 29 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 |
TABLE 13
| Average sequencing depth | Target loading rate | Sequencing alignment | Uniformity of | |
| Sample 1 | 3213.54 | 52.60% | 89.14% | 68.72% |
| Sample 2 | 2723.08 | 53.54% | 88.86% | 68.40% |
| Sample 3 | 2905.77 | 56.07% | 86.66% | 66.39% |
| Sample 4 | 2989.04 | 54.27% | 88.16% | 68.37% |
| Sample 5 | 3081.23 | 55.53% | 87.97% | 67.26% |
| Sample 6 | 3229.6 | 50.77% | 85.18% | 67.24% |
| Sample 7 | 2355.58 | 56.02% | 87.08% | 66.97% |
| Sample 8 | 3745.88 | 57.87% | 87.07% | 68.30% |
| Sample 9 | 3048.69 | 54.45% | 87.88% | 67.03% |
| Sample 10 | 3172.85 | 55.33% | 85.73% | 64.94% |
| Sample 11 | 3147.88 | 54.44% | 86.29% | 65.76% |
| Sample 12 | 2941.01 | 53.86% | 88.54% | 67.47% |
| Sample 13 | 1713.45 | 57.31% | 86.42% | 68.27% |
| Sample 14 | 3296.87 | 57.09% | 87.92% | 68.20% |
| Sample 15 | 2860.73 | 54.21% | 86.28% | 63.68% |
| Sample 16 | 2611.13 | 53.55% | 86.69% | 67.62% |
| Sample 17 | 3621.18 | 54.46% | 85.35% | 65.03% |
| Sample 18 | 2810.36 | 51.09% | 85.12% | 64.21% |
| Sample 19 | 1002.86 | 42.49% | 87.67% | 68.85% |
| Sample 20 | 1825.13 | 48.23% | 87.78% | 68.71% |
| Sample 21 | 1808.06 | 49.21% | 89.93% | 67.64% |
| Sample 22 | 2705.3 | 53.72% | 86.79% | 67.50% |
| Sample 23 | 2339.27 | 51.92% | 87.92% | 67.17% |
| Sample 24 | 2864.67 | 54.79% | 87.27% | 68.77% |
| Sample 25 | 3004.89 | 53.43% | 86.56% | 66.49% |
| Sample 26 | 2323.5 | 52.08% | 85.72% | 67.17% |
| Sample 27 | 2169.27 | 52.64% | 86.75% | 67.02% |
| Sample 28 | 3388.61 | 58.22% | 87.21% | 67.26% |
| Sample 29 | 2666.09 | 55.75% | 84.57% | 65.12% |
TABLE 14
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