CN109280701A - Probe, genetic chip and preparation method and application for thalassemia detection - Google Patents
Probe, genetic chip and preparation method and application for thalassemia detection Download PDFInfo
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Abstract
This application discloses a kind of probe, genetic chip and preparation method and application for thalassemia detection.The preparation method of the probe for thalassemia detection of the application, including according to different Mediterranean anemia genes, modifier and different mutation, building capture interval library；Capture probe is designed accordingly；Capture interval library includes, alpha gene cluster and all globin genes of beta gene cluster and its important regulating and controlling region full length fragment, the breakpoint mutational site for having record, the SNP site on the poor modifier of SNP site and ground chosen in deletion mutation.The preparation method of the application carries out probe design for the poor all gene clusters in ground and its related gene, probe is enable to cover poor gene mutation allly.Build library and high-flux sequence in conjunction with Tn5, can not only cover all mutation, and it is easy to operate, flux is high, at low cost, be suitable for the research and prediction of poor large-scale crowd diagnosis and screening and the mutation of new unknown gene.
Description
Technical field
This application involves thalassemia detection field, more particularly to a kind of probe for thalassemia detection,
Genetic chip and preparation method and application.
Background technique
Thalassemia is one of most common hereditary disease in the whole world, be distributed mainly on Mediterranean Region, the Middle East, India,
Southeast Asia and Africa.Thalassemia is also that southern china is most common, one of the maximum hereditary disease of harm.Thalassemia is in heat
The disease incidence of band and subtropical zone and southeast China reaches 2.5%-15%.Wherein, it is counted according to the health planning committee of Guangdong Province
Data show, poor gene carrying rate about 16.8% in the household registration population of Guangdong, homotype poor gene Mr. and Mrs account for about 1.7%, often
The poor fetal birth in the ground year with having more than 5000 heavy type α poor and 1500 heavy type β.
Different according to the type of globin gene defect, ground is poor to be broadly divided into α-thalassemia and β-thalassemia.
α-thalassemia is mainly that alpha globin gene (16p13.3) occurs caused by point mutation or copy number variation, wherein 95%
α-thalassemia patient be due to caused by alpha globin gene large fragment deletion, i.e. deletion form α it is poor.It is identified at present
At least 36 kinds of the poor deletion type in α-ground, wherein Chinese account for 8 kinds.Southeast Asia deletion form (-- SEA), rightward deletion (- α 3.7)
It is the most common 3 kinds of deletion forms of Chinese with lefrward deletion (- α 4.2).In addition, can be led there are also 68 kinds of alpha globin gene point mutation
Cause the generation of α-thalassemia.β-thalassemia is mainly master caused by beta globin genes (11p15) mutates
Also there is the report of large fragment deletion based on point mutation and small insertion or missing.At present it has been reported that more than 200 kinds of β-pearl egg
White gene mutation can cause the generation of β-thalassemia.In addition, the polyploid of alpha globin gene repeats, such as α α α anti3.7/
α α, α α α anti4.2/ α α, with also will increase β poor clinical symptoms.And the missing of beta-globin gene to α poor can be presented with
Modification.
Thalassemia is a kind of single gene inheritance disease, if couple for homotype poor gene carry when, children have
1/4 chance is that poor patient, 1/2 chance are poor gene carrier as parent medium and heavyly, and 1/4 is normal
Fetus.In addition, numerously poor gene carrier can not show any clinical symptoms, there are concealments.Therefore, it is pre-marital,
It is pregnant before and it is antenatal carry out poor carrier screening and combination genetic counselling and pre-natal diagnosis, be to reduce poor disease incidence the most
Effective method.
The common method of Screening for Thalassemia has red blood cell and hemoglobin detection method, such as average volume of red blood cells
Method, fragility test, hemoglobin electrophoresis analysis side etc., these method sensitivitys and specificity are poor, and there are biggish
Rate of missed diagnosis and misdiagnosis rate.Currently, the most common screening based on the poor genetic test in ground and diagnostic method are Gap-PCR and PCR-
RDB can detecte somewhere overwhelming majority mutation and deletion type, but all mutation types can not be completely covered, necessary
When also need using Real-Time PCR and sanger sequencing etc. technologies diagnosed, with reduce it is some rarely it is poor mutation or lack
Lose the omission factor of type.But program experimental implementation is complicated, testing cost is higher, flux is low, is not suitable for extensive people
The screening and diagnosis of group.In addition, also some point of use hybridizing methods detect common β point mutation, such as patent application
" for diagnosing beta-thalassemia nucleic acid hybond membrane item and kit " disclosed in CN1718742A, which is only capable of examining
Survey 17 kinds of common mutation types of China；It is disclosed in patent application CN1453363A " for diagnosing the DNA core of thalassemia
Piece and preparation method thereof " is only capable of detecting the mutation in 21 seed types of China's discovery；It is public in patent application CN102115781 A
The detection method opened only detects 27 kinds of beta Thalassemia regions.The detectable spectrum of mutation of these methods is limited, can not cover all
Mutation type, also, be only capable of detecting known mutation type, flux is lower, higher cost, is not suitable for the big rule of thalassemia
Mould crowd diagnosis and screening.
Summary of the invention
The purpose of the application is to provide a kind of new probe, genetic chip and its preparation side for thalassemia detection
Method and application.
The application uses following technical scheme:
The one side of the application discloses a kind of preparation method of probe for thalassemia detection, including according to not
With thalassemia gene, modifier and different mutation, building capture interval library；It is visited according to capture interval library design capture
Needle carries out high quality and efficient capture to capture interval library；
Capturing interval library includes following region,
(a) all globin genes of alpha gene cluster and important regulating and controlling region, including HBA1, HBA2, HBZ, HBQ1, HS-
40 regions and 5 ' HVR, HVR, 3 ' HVR for the above full length gene segment and outward extend 70bp as target acquistion area
Domain；
(b) all globin genes of beta gene cluster and important regulating and controlling region, including HBB, HBD, HBG1, HBG2, HBE1,
LCR HS-1, LCR HS-2, LCR HS-3, LCR HS-4 and LCR HS-5, for the above full length gene segment and outward
Extend 70bp as target acquistion region；
(c) using with having the deletion form alpha of record poor mutation breakpoint and deletion form beta poor mutation breakpoint as mesh
Mark capture region；
(d) using maximum range of loss involved in the deletion form alpha mutation recorded as target, within its scope,
Every 1kb choose 1 SNP site, selected SNP site in the MAF recorded in dbSNP between 0.3-0.5, meanwhile, selected SNP
Sequence is in the genome without other very high homology sequences where site, using selected SNP site as target acquistion region；
(e) using a series of SNP site on the poor modifier in ground as target acquistion region, wherein the poor modifier packet in ground
Include BCL11A, HBS1L, MYB, KLF1, BCL11A and MYB-HBS1L intergenic.
It should be noted that the probe preparation method of the application, for thalassemia alpha gene cluster and beta gene
The ground all globin genes of cluster and important regulating and controlling region and all deletion form alpha poor mutation breakpoint, deletion form beta
Poor mutation breakpoint, the deletion form alpha mutation a series of gene such as SNP site and the poor modifier in ground and region are visited
Needle design enables the probe of preparation to cover known thalassemia gene mutations all at present to the greatest extent；This Shen
The probe that preparation method please obtains, can not only detect all known thalassemia gene mutations, and can also
It is enough in discovery and studies new thalassemia gene mutation, be suitable for the diagnosis of thalassemia large-scale crowd and screening.
It should also be noted that, the preparation method of the application, key is to capture the building of interval library, as probe
Specific design scheme can refer to the design of existing genetic chip middle probe.In order to ensure probe design effect, the application into
In the preferred scheme of one step, probe design is also defined, is described in detail as follows.
Preferably, the length of capture probe is 50-120bp, and the denaturation temperature of capture probe and hybridising region is 60-100
℃；Also, 0.6 or the capture probe lower than 0.3 are higher than for the G/C content of hybridising region, multiplier is greater than 2.Wherein, multiplier is big
Refer in 2, theoretically the overburden depth of designed probe be greater than 2 ×.
It is furthermore preferred that the length of capture probe is 75bp.
It is furthermore preferred that the denaturation temperature of capture probe and hybridising region is 80 DEG C.
The another side of the application discloses the probe that the preparation method of the application obtains.
The probe that the another side of the application discloses the application is used for the base of thalassemia detection in Gene Mutation in preparation
Because of the application in chip, kit or detection device.
The application's discloses a kind of genetic chip for thalassemia detection in Gene Mutation on one side again, the gene core
On piece is fixed with the probe of the application.
The application's discloses a kind of kit for thalassemia detection in Gene Mutation on one side again, in the kit
The genetic chip of probe or the application containing the application.
It should be noted that the probe or genetic chip of the application, can carry out high-throughput inspection to thalassemia
It surveys, and experimental implementation is simple, and known thalassemia gene mutations all at present can be covered, especially suitable for extensive
Therefore kit can be made in probe or genetic chip by the screening and diagnosis of crowd, be easy to use.Moreover, the application
Probe or genetic chip, additionally it is possible to for the research and analysis of new gene mutation, it is prominent to can be used as thalassemia gene
Become the tool of research.
The application's discloses a kind of detection method of thalassemia gene mutation on one side again, including uses Tn5 swivel base
Enzyme carries out library construction to the genomic DNA of sample to be tested, and using the probe or genetic chip of the application to constructed text
Library carries out thalassemia gene and its relevant modifications gene, controlling gene or region and is enriched with, then, to enriched product into
Row high-flux sequence.
It should be noted that the detection method of the application, on the one hand, carry out library construction using Tn5 transposase, greatly
Improve the flux of pattern detection and the efficiency of thalassemia genetic test；On the other hand, using the probe of the application or base
Because chip carries out capture enrichment to Mediterranean anemia gene and its related gene and region, so that testing result can not only cover
Known thalassemia gene mutations all at present, additionally it is possible to detect new unknown gene mutation；In another aspect, this Shen
Library and high-flux sequence are built incorporated by reference to Tn5 transposase, the accuracy and detection flux of detection is improved, reduces testing cost,
Also, experimental implementation is simple.Therefore, the detection method of the thalassemia gene mutation of the application, can be applied not only to ground
Middle sea anaemia large-scale crowd diagnosis and screening；And it can also be used to the new unknown thalassemia gene mutation of research and development.
Preferably, using Tn5 transposase carry out library construction joint sequence include Tn5MErev, Tn5ME-A and
Tn5ME-B, Tn5MErev are sequence shown in Seq ID No.1, and Tn5ME-A is sequence shown in Seq ID No.2, and Tn5ME-B is
Sequence shown in Seq ID No.3.
Preferably, the index sequence that high-flux sequence uses is N5 index sequence and N7 index sequence, N5 index sequence packet
Sequence shown in Seq ID No.4 to Seq ID No.33 is included, N7 index sequence includes Seq ID No.34 to Seq ID No.63
Shown sequence.
Preferably, the detection method of the application further includes being detected using known thalassemia gene mutation to all
Gene mutation be labeled；The gene mutation not marked to detecting carries out crowd's frequency annotation and function prediction.
It dashes forward it should be noted that the detection method of the application can not only cover thalassemia genes all at present
Become, but also is capable of detecting when new unknown thalassemia gene mutation；Therefore, prominent to unknown thalassemia gene
Become carry out crowd's frequency annotation and function prediction, in order to find new unknown pathogenic mutation.
The beneficial effects of the present application are as follows:
The probe preparation method of the application is visited for all gene clusters of thalassemia and its related gene and region
Needle design enables the probe of preparation to cover current all thalassemia gene mutations.The probe of the application turns in conjunction with Tn5
Seat enzyme builds library and high-flux sequence detection thalassemia gene mutation, can not only cover all mutation types, and operate
It is simple and convenient, flux is high, at low cost, be suitable for the diagnosis of thalassemia large-scale crowd and screening and new unknown gene
The research and prediction of mutation.
Detailed description of the invention
Fig. 1 is split reads detection schematic diagram in the embodiment of the present application；
Fig. 2 is the CNV detection schematic diagram of sample 1 in the embodiment of the present application；
Fig. 3 is the CNV detection schematic diagram of sample 2 in the embodiment of the present application；
Fig. 4 is the CNV detection schematic diagram of sample 3 in the embodiment of the present application.
Specific embodiment
The thalassemia detection method of gene mutation of the application is to propose on the basis of the probe of the application, and combine
Tn5 transposase builds library and high-flux sequence detection.Wherein, the preparation method of probe includes, to all gene clusters of thalassemia
And its related gene and region carry out probe design, it is all known to ensure that designed probe can cover to the greatest extent with this
Thalassemia gene mutation.Tn5 transposase builds library, can greatly improve pattern detection flux and thalassemia gene
Detection efficiency.High-flux sequence, can be simple and effective, and accurately detects to all gene mutations, including it is all
The gene mutation and some unknown gene mutations known, this is conducive to the detection and knowledge of novel unknown thalassemia gene mutation
Not.
Gene mutation is detected using high-flux sequence, mainly by carrying out analysis of biological information to sequencing result
It realizes, sequencing information is analyzed and is studied, to obtain single nucleotide variations (abbreviation SNV), a small number of alkali of related gene
The hereditary variations letter such as the insertion of base and missing (abbreviation InDel), DNA copy number variation (abbreviation CNV), structure variation (abbreviation SV)
Breath.Principle is as follows:
CNV testing principle: using the copy number analysis of variance based on region reads sequencing depth.Except inherently gene
In group other than duplicate sequence, after one section of region on genome occurs missing or repeats, it can be generated in sequencing depth bright
Aobvious variation, for example, about N/2 times when the sequence depth that n times are repeated can become normal two copies, and heterozygous deletion and pure
Conjunction missing then becomes about 1/2 times or respectively close to 0, according to this characteristic, after the signal to sequencing depth carries out GC amendment,
The depth correlation between sample is recycled, the section that depth signal can be abnormal identifies, and makees corresponding copy
Number prediction and reliability assessment.
Structure variation testing principle: when reads itself covers the breakpoint that SV occurs, the sequence of reads itself is with disconnected
Point is that boundary is divided into two sections, and for the same SV, when there is the reads in different disconnections to support in the same breakpoint
On, for example, for deletion situation shown in A figure in Fig. 1, in the reads that experiment sequencing obtains in comparison process, in this way
Read can be significantly identified as two parts, wherein main a part is denoted as the part correctly compared, second segment it is another
What a part cannot usually compare, but mass value with higher, it is denoted as the part soft clip, such portion
Divide the generation for usually just corresponding to the SV such as translocation, deletion, shown in B figure as shown in figure 1；By soft clip's
Independent analysis is taken out in part, is being based on soft clip reads length, under sequencing quality etc. filter condition, is picking out height
Believable clip reads, the gap that it is compared again compare position and the breakpoint location progress of original reads
Analysis and statistics, so that it may which the concrete condition for detecting SV generation can be direct for the detection of missing shown in C figure as shown in figure 1
Accurate position and length are provided, for repeating to combine the judgement of CNV result that can accurately judge that duplicate length, position occurs
And repeat type.
The application is described in further detail below by specific embodiment.Following embodiment only to the application carry out into
One step explanation, should not be construed as the limitation to the application.
Embodiment
This example devises the probe for thalassemia detection in Gene Mutation first, and is prepared for corresponding gene core
Piece；Library and high-flux sequence are built then in conjunction with Tn5 transposase, to 2 patients with thalassemia and 1 carrying HBB pathogenic mutation
The individual of compound alpha deletion form carries out genetic test.It is specific as follows:
One, probe and genetic chip preparation
This example is according to different Mediterranean anemia genes, modifier and different mutation, building capture interval library；According to capture
Interval library designs capture probe, carries out high quality and efficient capture to capture interval library；
Capturing interval library includes following region,
(a) all globin genes of alpha gene cluster and important regulating and controlling region, including HBA1, HBA2, HBZ, HBQ1, HS-
40 regions and 5 ' HVR, HVR, 3 ' HVR for the above full length gene segment and outward extend 70bp as target acquistion area
Domain；
(b) all globin genes of beta gene cluster and important regulating and controlling region, including HBB, HBD, HBG1, HBG2, HBE1,
LCR HS-1, LCR HS-2, LCR HS-3, LCR HS-4 and LCR HS-5, for the above full length gene segment and outward
Extend 70bp as target acquistion region；
(c) using with having the deletion form alpha of record poor mutation breakpoint and deletion form beta poor mutation breakpoint as mesh
Mark capture region；
(d) using maximum range of loss involved in the deletion form alpha mutation recorded as target, within its scope,
Every 1kb choose 1 SNP site, selected SNP site in the MAF recorded in dbSNP between 0.3-0.5, meanwhile, selected SNP
Sequence is in the genome without other very high homology sequences where site, using selected SNP site as target acquistion region；
(e) using a series of SNP site on the poor modifier in ground as target acquistion region, wherein the poor modifier packet in ground
Include BCL11A, HBS1L, MYB, KLF1, BCL11A and MYB-HBS1L intergenic.
The probe design principle of this example is as follows:
(1) length of probe is 50-120bp, and this example is preferably 75bp；
(2) probe being capable of each target area of specific recognition；
(3) specific recognition G/C content is higher than the probe in 0.6 or the region lower than 0.3, and multiplier is greater than 2；
(4) melting temperature of probe and target sequence is 60-100 degrees Celsius, and this example is preferably 80 degrees Celsius；
(5) probe does not include hairpin structure；
(6) probe is matched with reference at most 2 sites on genome；
(7) window sliding size when probe selects is 10bp.
This example designs probe for the relevant gene of thalassemia or region, and gene and region include that the poor albumen in α-ground is compiled
Code gene: HBA1, HBA2, HBQ1, HBZ；The poor regulatory region in α-ground: 5'HVR, HS40, HVR, 3'HVR；α-ground is poor relevant
CNV:SEA, 3.7,4.2, THAI, FIL etc.；The poor protein coding gene in β-ground: HBB, HBD, HBE1, HBG1, HBG2；The poor tune in α-ground
Control region: LCR HS-1 to HS-5；The poor relevant CNV:Chinese in α-ground, (SEA)-HPFH etc.；The poor modifier in ground and change
It is different: KLF1, BCL11A, HBS1L, MYB, 16 SNP of 12 SNP of BCL11A gene, MYB-HBS1L intergenic.It visits
Needle quantity is 5571 total, and genetic chip is made by Hua Da intelligence and synthesized.
Two, Tn5 transposase builds library
1. sample DNA extracts
To the peripheral blood for being derived from 2 patients with thalassemia and 1 carrying HBB individual, genomic DNA is carried out respectively and is mentioned
It takes, this example extracts genomic DNA using paramagnetic particle method, and carries out quality testing to the genomic DNA of acquisition using electrophoresis and OD.3
The results are shown in Table 1 for the electrophoresis and OD of a DNA sample.
The electrophoresis observation result and OD testing result of 1 DNA sample of table
| Sample | Concentration (ng/ μ L) | Volume (μ L) | OD260/280 | OD260/230 | Sample integrity |
| Sample 1 | 28.86 | 50 | 1.44 | 0.66 | Slight degradation |
| Sample 2 | 32.58 | 50 | 1.46 | 0.62 | Slight degradation |
| Sample 3 | 38.1 | 50 | 1.48 | 0.68 | Slight degradation |
In table 1, sample 1 and sample 2 are the peripheral blood DNAs for being taken respectively from 2 patients with thalassemia, and sample 3 is taken from 1
Example carries the peripheral blood DNA of HBB individual, and sample integrity is obtained by electrophoresis observation, although the results show that three samples
There is slight degradation, but DNA concentration is preferable, can be used for follow-up test.
2.Tn5 prepared by transposase library
(1) prepared by connector (Adapter Mix)
It designs and synthesizes Tn5 and builds connector needed for library, and butt joint is mixed, prepare Adapter Mix.The Tn5 of this example
Building library connector includes Tn5MErev, Tn5ME-A and Tn5ME-B, and Tn5MErev is sequence shown in Seq ID No.1,5 ' end tools
There is phosphorylation modification, Tn5ME-A is sequence shown in Seq ID No.2, and Tn5ME-B is sequence shown in Seq ID No.3.
Seq ID No.1:5 '-[phos] CTGTCTCTTATACACATCT-3 '
Seq ID No.2:5 '-TCGTCG GCAGCGTCAGATGTGTATAAGAGACAG-3 '
Seq ID No.3:5 '-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3 '
Tn5MErev, Tn5ME-A, Tn5ME-B are dissolved respectively to 100 μM of final concentration using Annealing Buffer, and
Following system is respectively configured:
10 μ L of Tn5MErev, 100 μM of the 10 μ L of Tn5ME-A that one: 100 μM of system amount to 20 μ L；
10 μ L of Tn5MErev, 100 μM of the 10 μ L of Tn5ME-B that two: 100 μM of system amount to 20 μ L；
Respectively the solution of two systems is vortexed to shake and be mixed well, and of short duration centrifugation makes solution return to tube bottom, is placed in PCR
In instrument, following response procedures: 75 DEG C of 15min, 60 DEG C of 10min, 50 DEG C of 10min, 40 DEG C of 10min, 25 DEG C of 30min are carried out.Reaction
After, by system one and the second-class volume mixture of system, mixes, be named as Adapter Mix, saved backup in -20 DEG C.
(2) Adapter Mix is embedded
Each reactive component: the Tn5 of 12.5 μ L of Adapter Mix, concentration 0.75mg/mL is successively added in sterilizing PCR pipe
87.5 μ L of transposase；It is gently blown and beaten, is mixed well using pipettor.Reaction system is placed in 25 DEG C of reaction 60min.Reaction product
It is named as Tagment Enzyme Advanced Mix V5S, -20 DEG C is placed in and saves backup.
3.DNA fragmentation
Thaw 5 × TAPS-DMF buffer at room temperature, spare after mixing of turning upside down；Confirm at 0.2%SDS
In room temperature, and flicks tube wall and confirm that whether there is or not precipitatings.If any precipitating, it is instant that precipitating is mixed well in 37 DEG C of heating and acutely concussion
Solution.
Each reactive component: 5 × TAPS-DMF buffer, 4 μ L, 1 μ L of genomic DNA is successively added in sterilizing PCR pipe
About 50ng, 0.3 μ L of Tagment Enzyme Advanced Mix V5S, finally with deionized water polishing to 20 μ L.
After reaction system prepares, in 55 DEG C of warm bath 7min, 5 μ L 0.1%SDS room temperature 7min are then added, are terminated anti-
It answers, is placed on ice, 20 μ L of total volume is reacted for subsequent PCR.
4. fragmentation products PCR amplification
Design index sequence simultaneously merges, and this example specifically uses N5 index sequence and N7 index sequence, partial index sequence
Particular sequence it is as shown in table 2.
The partial sequence of table 2 N5 index sequence and N7 index sequence
Sterilizing PCR pipe is placed in ice bath, each reactive component shown in table 3 is successively added.
Table 3 builds library PCR reaction system
It is gently blown and beaten 10 times and is mixed well using pipettor.PCR pipe is placed in PCR instrument, following response procedures are set:
105 DEG C of heat lids are opened, and 72 DEG C of 3min, 95 DEG C of 3min are recycled subsequently into 6: 98 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 20s, circulation
After 72 DEG C of 5min, 4 DEG C are standby.
Above-mentioned amplified production pillar or magnetic bead type purifying, that is, obtain the Tn5 transposase library of single sample.
Three, gene chip hybridization captures
This example carries out ground to Tn5 transposase library using the genetic chip synthesized in " one, probe and genetic chip preparation "
Middle sea anemia gene and its related gene and region carry out hybrid capture.Specific steps and operation are used referring to NimbleGen to be said
Bright book progress, hybridization elution product, that is, thalassemia gene and its related gene and region.
PCR amplification enrichment is carried out to hybridization elution product, is subsequently used for high-flux sequence.
PCR amplification is enriched with reaction system: 20 μ L, the 2 × KAPA HIFI Hotstart Ready of DNA of hybrid capture
Mixr 2.5 μ L, 10 μM of Flowcell primers F 2.5 μ L, 10 μM of Flowcell primers R2.5 μ L, supplement
Sterile purified water is to 30 μ L.
PCR response procedures are 98 DEG C of initial denaturation 45s, are recycled subsequently into 14: 98 DEG C of 15s, 65 DEG C of 30s, 72 DEG C of 30s；
72 DEG C of extension 60s after circulation terminates, 4 DEG C standby.
Four, upper machine sequencing
This example carries out machine sequencing using hiseq4000PE101+8+101 program.
Five, information analysis and result
Sequenator obtains original short sequence；Remove the connector and low quality data in sequencing data；Short sequence is navigated to
On the corresponding position of human genome data；Sequencing result information is counted, short sequence quantity, is averaged at target area covering size
Depth etc. is sequenced；Filter the mononucleotide of low quality value and low cover degree；Determine gene, coordinate, amino that mutational site occurs
Acid change etc.；Carry out CNV analysis and the analysis of breaking point.
The results show that the target area coverage of three samples is as shown in table 4.
4 target area coverage of table
Target area capture is counted, by the result of table 4 as it can be seen that the average 10 × covering in three sample object regions
For degree 98% or more, 20 × coverage shows that designed probe can effectively capture target area 98% or more, meet into
The requirement of row analysis.
Testing result shows that there are heterozygous deletions between Chr11:5191114-5270053 for 1 sample of Sample, is related to
HBB gene, as shown in Figure 2.There are 11 split reads supports to have in the section chr11:5191128-5270051 simultaneously
The missing of 78939bp.It is roughly the same that Split supports that breakpoint and Chinese type missing record breakpoint, prompts the sample to be
Chinese type heterozygous deletion.It is c.126_129delCTTT (hemizygous that HBB gene common mutations have also been detected in the sample simultaneously
Type), mutation allele depth be 88 ×.I.e. in the sample with having detected beta poor relevant Chinese type missing with
C.126_129delCTTT compound heterozygous mutations.
For 2 sample of Sample, testing result shows the newly observed Chr16:216336- on No. 16 chromosomes
There is heterozygous deletion between 234626, is related to HBM, HBA2, HBA2, HBQ1 gene, as shown in Figure 3.There are 9 split reads simultaneously
Support that there are 19304bp missings in the section Chr16:215395-234700, breakpoint is identical as SEA type missing, prompts the sample also
There are SEA type heterozygous deletions.In addition, there is the homozygosis of about 3.7kb in the section Chr16:223303-226996 for the sample
Missing, consistent with 3.7 deletion form ranges, prompting the gene, there are 3.7kb homozygous deletions.The above analysis sample exists
Alpha poor relevant SEA and 3.7 compound heterozygous mutations.
For 3 sample of Sample, testing result shows the newly observed Chr16:216334- on No. 16 chromosomes
There is heterozygous deletion between 234628, is related to HBM, HBA2, HBA2, HBQ1 gene, as shown in Figure 4.There are 13 split reads simultaneously
Support that there are 19304bp missings in the section Chr16:215395-234700, breakpoint is identical as SEA type missing, prompts the sample also
There are SEA type heterozygous deletions.In addition, testing result prompts the specimen sample, there are .316-197C > T heterozygosis to dash forward in HBB gene
Become, for a kind of common HBB mutation.
As it can be seen that the probe and thalassemia detection method of gene mutation of the application, can effectively to three samples into
Row detection, and testing result is consistent with expection.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection of the application
Range.
SEQUENCE LISTING
<110>Shenzhen Hua Da gene limited liability company
Nanfang Medical Univ
<120>probe, genetic chip and the preparation method and application for thalassemia detection
<130> 17I24036
<160> 63
<170> PatentIn version 3.3
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gtctcgtggg ctcggagatg tgtataagag acag 34
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aatgatacgg cgaccaccga gatctacacg gaacagttcg tcggcagcgt c 51
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aatgatacgg cgaccaccga gatctacacg gaggaagtcg tcggcagcgt c 51
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aatgatacgg cgaccaccga gatctacacg gtgttattcg tcggcagcgt c 51
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aatgatacgg cgaccaccga gatctacacg tcgagagtcg tcggcagcgt c 51
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aatgatacgg cgaccaccga gatctacacg tgcgtagtcg tcggcagcgt c 51
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caagcagaag acggcatacg agataatgat gagtctcgtg ggctcgg 47
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caagcagaag acggcatacg agatacaagc ctgtctcgtg ggctcgg 47
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caagcagaag acggcatacg agatacagga gcgtctcgtg ggctcgg 47
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Claims (10)
1. a kind of preparation method of the probe for thalassemia detection, it is characterised in that: including poor according to different Mediterranean
Blood gene, modifier and different mutation, building capture interval library；Capture probe is designed according to the capture interval library, to institute
It states capture interval library and carries out high quality and efficient capture；
The capture interval library includes following region,
(a) all globin genes of alpha gene cluster and important regulating and controlling region, including the area HBA1, HBA2, HBZ, HBQ1, HS-40
Domain and 5 ' HVR, HVR, 3 ' HVR for the above full length gene segment and outward extend 70bp as target acquistion region；
(b) all globin genes of beta gene cluster and important regulating and controlling region, including HBB, HBD, HBG1, HBG2, HBE1, LCR
HS-1, LCR HS-2, LCR HS-3, LCR HS-4 and LCR HS-5 extend for the above full length gene segment and outward
70bp is as target acquistion region；
(c) using with having the deletion form alpha of record poor mutation breakpoint and deletion form beta poor mutation breakpoint is caught as target
Obtain region；
(d) using maximum range of loss involved in the deletion form alpha mutation recorded as target, within its scope, every
1kb choose 1 SNP site, selected SNP site in the MAF recorded in dbSNP between 0.3-0.5, meanwhile, selected SNP site
Place sequence is in the genome without other very high homology sequences, using selected SNP site as target acquistion region；
(e) using a series of SNP site on the poor modifier in ground as target acquistion region, wherein the poor modifier in ground includes
BCL11A, HBS1L, MYB, KLF1, BCL11A and MYB-HBS1L intergenic.
2. preparation method according to claim 1, it is characterised in that: the length of the capture probe is 50-120bp, is caught
The denaturation temperature for obtaining probe and hybridising region is 60-100 DEG C；Also, 0.6 is higher than for the G/C content of hybridising region or is lower than
0.3 capture probe, multiplier are greater than 2；Preferably, the length of the capture probe is 75bp, capture probe and hybridising region
Denaturation temperature is 80 DEG C.
3. the probe that preparation method according to claim 1 or 2 obtains.
4. genetic chip, reagent that probe according to claim 3 is used for thalassemia detection in Gene Mutation in preparation
Application in box or detection device.
5. a kind of genetic chip for thalassemia detection in Gene Mutation, it is characterised in that: fixed on the genetic chip
Probe described in having the right to require 3.
6. a kind of kit for thalassemia detection in Gene Mutation, it is characterised in that: contain in the kit and have the right
It is required that genetic chip described in probe described in 3 or claim 5.
7. a kind of detection method of thalassemia gene mutation, it is characterised in that: including using Tn5 transposase to sample to be tested
Genomic DNA carry out library construction, and using genetic chip pair described in probe as claimed in claim 3 or claim 5
Constructed library carries out thalassemia gene and its relevant modifications gene, controlling gene or region are enriched with, then, right
Enriched product carries out high-flux sequence.
8. detection method according to claim 7, it is characterised in that: carry out the connector of library construction using Tn5 transposase
Sequence includes Tn5MErev, Tn5ME-A and Tn5ME-B, and Tn5MErev is sequence shown in Seq ID No.1, Tn5ME-A Seq
Sequence shown in ID No.2, Tn5ME-B are sequence shown in Seq ID No.3.
9. detection method according to claim 7, it is characterised in that: the index sequence that high-flux sequence uses is N5 index
Sequence and N7 index sequence, N5 index sequence include sequence shown in Seq ID No.4 to Seq ID No.33, N7 index sequence packet
Include sequence shown in Seq ID No.34 to Seq ID No.63.
10. according to the described in any item detection methods of claim 7-9, it is characterised in that: further include using known Mediterranean
Anemia gene mutation is labeled all gene mutations detected；The gene mutation not marked to detecting carries out
Crowd's frequency annotation and function prediction.
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| CN113564248A (en) * | 2021-09-26 | 2021-10-29 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneously detecting multiple mutations of HBA1/2, HBB and HBD gene sites |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110184337A (en) * | 2019-05-16 | 2019-08-30 | 南京诺禾致源生物科技有限公司 | Probe compositions, the reagent comprising it, kit, detection method and application |
| CN110343756A (en) * | 2019-06-25 | 2019-10-18 | 广西识远医学检验实验室有限公司 | One group of probe and related kit and application for detecting thalassemia |
| CN110343756B (en) * | 2019-06-25 | 2023-02-24 | 广西识远医学检验实验室有限公司 | Group of probes for detecting thalassemia, related kit and application |
| CN110938685A (en) * | 2019-12-11 | 2020-03-31 | 福建福君基因生物科技有限公司 | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof |
| CN114645330A (en) * | 2020-12-21 | 2022-06-21 | 北京大学 | Preparation method and kit of pathogen macrotranscriptome sequencing library, and method and device for screening infection pathogens |
| CN113564248A (en) * | 2021-09-26 | 2021-10-29 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneously detecting multiple mutations of HBA1/2, HBB and HBD gene sites |
| CN115985399A (en) * | 2023-03-20 | 2023-04-18 | 广州迈景基因医学科技有限公司 | HRD panel site selection optimization method and system for high-throughput sequencing |
| CN115985399B (en) * | 2023-03-20 | 2023-07-04 | 广州迈景基因医学科技有限公司 | HRD panel site selection optimization method and system for high-throughput sequencing |
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