CN108300700A - A kind of preparation of gene sequencing calibration Reference Strains - Google Patents
A kind of preparation of gene sequencing calibration Reference Strains Download PDFInfo
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Abstract
A kind of gene sequencing for medical domain calibrates the preparation of Reference Strains, which is characterized in that the Genetic Mutant Cell with IgGFc receptors is connected as conjugate with the myeloma cell with IgGFab receptors by IgG；Or all have IgGFab receptors Genetic Mutant Cell respectively connected by IgG with myeloma cell after, conjugate is connected as by the SPA with IgGFc receptors again, followed by under the action of polyethylene glycol, or Genetic Mutant Cell and myeloma cell are under the directly effect of polyethylene glycol, it is fused to hybridoma, make mutator transplanting oncocyte and is infinitely expanded with oncocyte, identification through target Disease-causing gene and be made gene sequencing calibration Reference Strains, whole range quality control for gene sequencing, matter is commented, calibrate or substitute the comparison that existing gene comparison data library carries out sequencing data and its mutator, to improve existing gene order surveying method.
Description
Technical field
The present invention relates to the preparations that a kind of gene sequencing of medical domain calibrates Reference Strains, and it is prominent to be mainly used in DNA genes
Become the error correction of Sequence Detection.
Background technology
Gene sequencing (Gene Sequencing) or DNA sequencing (DNA Sequencing) refer to the specific DNA of analysis
The base sequence of segment.The appearance of gene sequencing technology has not only pushed the research of genomics, but also is the disease of complex disease
New approaches are also provided because learning research, while further promoting genetic test and being examined in pre-natal diagnosis, organ transplant distribution type, tumor cells
The application of disconnected and targeted therapy and drug individualized treatment etc..Gene sequencing technology has been developed to forth generation, wherein the
Two generations, the third generation and forth generation sequencing technologies are referred to as next-generation sequencing (NGS) technology.Gene sequencing principle is also correspondingly undergone
Sanger PCR sequencing PCRs are sequenced, single-molecule sequencing and nano-pore sequencing in synthesis.NGS technologies are because flux improves, cost drops
Low and sequencing cycle time advantage has been widely used in genomics, transcription group, metagenomics and apparent group etc.
Aspect.
The dideoxy chain termination (Sanger PCR sequencing PCRs) of the inventions of Sanger in 1977 is the goldstandard of sequencing technologies, is surveyed
For sequence length up to 1000bp, accuracy is almost 100%, but there are flux low, of high cost and the deficiency that time-consuming, is seriously affected
Its large-scale application produces second generation sequencing technologies thus.The central principle of second generation sequencing technologies is sequenced in synthesis,
Its basic step includes library preparation, the generation of monoclonal DNA clusters and sequencing reaction.Compared with first generation sequencing technologies, second
It is had the characteristics that for sequencing technologies：(1) high-throughput.Second generation sequencing technologies do not depend on traditional Capillary Electrophoresis, sequencing
Reaction carries out on chip, and millions of a points on chip can be sequenced simultaneously；(2) at low cost.Second generation sequencing technologies are per Mb alkali
Base cost ratio Sanger PCR sequencing PCRs reduce 96.0%-99.9%；(3) sensibility is high.Such as Roche454 microarray datasets " 1 segment
The design of=1 magnetic bead=1 reading length " can guarantee the detection to low abundance DNA information；(4) it is shorter to read length.It is not easy to follow-up number
Splicing when according to analysis；(5) PCR (polymerase chain Reaction, PCR) process may introduce partially
Partial and mispairing.Third generation sequencing technologies are to increase to read length on the basis of the second generation, reduce reagent cost, and accelerate operation speed
Degree.Its distinguishing feature is single-molecule sequencing, i.e., directly carries out being sequenced in synthesis without PCR, not only simplify sample treatment mistake
Journey avoids the mispairing that amplification may introduce, and not by guanine and cytimidine or the shadow of adenine and thymine content
It rings, therefore third generation sequencing technologies can directly be sequenced RNA and methylated DNA sequence.Forth generation sequencing technologies, that is, nanometer
Hole sequencing technologies, nano-pore sequencing can be used for mononucleotide, ssDNA, dsDNA and RNA sequencing, while nano-pore technology is additionally operable to
The qualitative and quantitative of DNA, Microrna, protein, anion, cation and organic molecule.Due to each base of DNA molecular
Size shape is different, DNA molecular under electrophoresis driving by the way that characteristic curent change can be caused when nanometer micropore built-up circuit, according to
This can determine the base type of DNA molecular and puts in order.But if DNA molecular by speed when some kinds of nano-pore too
Soon, it can be difficult to differentiate between base information and background noise, error rate is higher.
As other experiments, the experimental error of gene sequencing is unavoidable.In gene sequencing, DNA changes through sequencing
For initial data, initial data, which is changed into through the processing such as screening and splice, can do the specific data of genetic analysis, and then and base
Because database compares, mutator is found out, and analyze the pathogenic of mutator.It is carried in the sample collection of gene sequencing, DNA
It takes, any link will produce in PCR amplification, microarray dataset, sequencing reagent, sequencing depth, data analysis and database comparison
Experimental error, especially in terms of data analysis, there are many high throughput DNA sequencing data sequence matching process at present, however, what
All high-throughput DNA sequencing data all can not be mapped back gene by kind method by the matching with reference gene group sequence
Group, always have part sequencing data because can not with reference gene group sequences match and map back it and cause to analyze in the source on DNA
Error.
2015, FDA set up FDA genetic tests calibration item (precision FDA), from global more than 500 machine
2400 multidigit experts of structure take part in the formulation of " calibration " standard, repeatability, accuracy and the non-Tongfang detected with test cdna
The consistency of method testing result.It is found by " FDA calibrations " project testing, it is same with the operating software " reading " of gene sequencing company
The DNA sequencing of a cdna sample is as a result, almost 50% ocr software can not repeat same result.FDA thinks, precisely cures
Core is to read the accuracy of gene code.For gene code, the especially reading quality of full genome coding, only
It establishes more massive database to be used as with reference to data, be possible in establishment and test cdna ocr software, by a large amount of
" trial and error " find out Bugs, it is final to realize really precisely genetic test " reading ".
J etc. reports [J Clin Oncol.2016 Dec 1；34(34)：4071-4078], 26% is examined
The gene mutation of survey obtains different decipherings, and 11% detection is mutated to obtain antipodal deciphering.Genetic test result at present
More difficult to recognize each other, identical sample can obtain different as a result, the reason is that the sequencing approach of each mechanism is not united through different institutions detection
One.If with the comparison data library of the same microarray dataset, the same data analysis as, including the same sample collection,
The same DNA extractions, it is the same build the sequencing depth of library, the same PCR amplification as, be just easy to get consistent result.
In particular, if gene sequencing calibration Reference Strains can be prepared, and surveyed under the same conditions with tested sample with calibrating Reference Strains
Sequence carries out whole range quality control, calibration and comparison, makes it easier to obtain consistent result.
But there is presently no this gene sequencing to calibrate Reference Strains.
Invention content
In order to prepare a kind of gene sequencing calibration Reference Strains, and to calibrate Reference Strains whole range quality control, calibration and compare each machine
The DNA sequencing of the similar tested sample of structure, present inventors have proposed the present invention.
The invention aims to provide a kind of gene sequencing to calibrate Reference Strains and preparation method thereof；Another object is that carry
It comments, calibrate and data comparison method for a kind of Quality Control of gene sequencing based on calibration Reference Strains, matter.
The object of the present invention is achieved like this：Genetic Mutant Cell is with myeloma cell in specific antibody or poly- second two
Hybridoma is fused under the action of alcohol, mutator is implanted oncocyte and is expanded with the unlimited amplification of oncocyte, into
And the calibration Reference Strains of known mutations gene are made, comment, calibrate or substitute existing base for the whole range quality control of gene sequencing, matter
Because mutation database carries out the comparison of sequencing data and its mutator.
More specific says, the Genetic Mutant Cell with IgGFc receptors and myeloma cell's quilt with IgGFab receptors
IgG is connected as the conjugate of Genetic Mutant Cell-IgG- myeloma cells；Or all have the Genetic Mutant Cell of IgGFab receptors
After respectively being connected by IgG with myeloma cell, then it is connected as by the staphylococcal protein A (SPA) with IgGFc receptors
The conjugate of Genetic Mutant Cell-IgG-SPA-IgG- myeloma cells, followed by under the action of polyethylene glycol or gene mutation
With myeloma cell under the directly effect of polyethylene glycol, Genetic Mutant Cell merges cell with myeloma cell, is made
Hybridoma makes mutator transplant oncocyte, and then gene sequencing calibration Reference Strains are made.
The present invention is bridged according to the surface receptor of Genetic Mutant Cell and myeloma cell using specific antibody or antibody
Effect, so that cell is easy to after birth contact and special fusion under the action of antibody, increase effective integration rate, it is invalid to reduce
Fusion, Genetic Mutant Cell and the fusion method of myeloma cell are designed with this, and full mutator group is implanted into oncocyte for the first time
And expanded by means of the amplification of oncocyte, the monoclonal hybridoma strain of genomic abnormality is then screened and prepares, with high-resolution
Gene sequencing calibration is made with reference to thin in chromosome karyotype analysis, fluorescence in situ hybridization, PCR or sequence verification targeted mutagenesis gene
Born of the same parents, for the whole range quality control of gene sequencing, matter comment, calibrate or substitute existing gene mutation database carry out sequencing data and its
The comparison of mutator to significantly improve the accuracy of sequencing, and reaches the more preferable of different institutions testing result and recognizes each other.
Specific implementation mode
Fig. 1 is that the IgG of the present invention is helped and melted schematic diagram.
Fig. 2 is that the SPA collaboration double antibodies of the present invention help and melt schematic diagram.
Fig. 3 is the cell fusion real scene shooting figure of the present invention
If Fig. 1,1 indicates the myeloma cell with IgGFab receptors, 2 indicate monoclonal antibody (IgG), and 3 indicate have
Another end Fab of the Genetic Mutant Cell of IgGFc receptors, monoclonal antibody (IgG) may also be combined with a myeloma cell, lead to
It crosses monoclonal antibody cell to be fused links together, is conducive to merge under the action of PEG.
If Fig. 2,1 indicates the staphylococcal protein A (SPA) with IgGFc receptors, 2 indicate that number is 6 and 7
The monoclonal antibody of cell to be fused, 3 indicate the monoclonal antibody for the cell to be fused that number is 4 and 5, in monoclonal antibody
[2] after the cell combination to be fused that the cell combination to be fused for being 6 and 7 with number, monoclonal antibody [3] are 4 and 5 with number,
Connection through SPA again, the cell of number 4,5,6 and 7 are conducive to merge under the action of PEG.
Such as Fig. 3, under the helping and melt of monoclonal antibody and/or SPA, screens 2 weeks through HAT, shot under inverted microscope
(40X), obtains hybridoma cell clone.
With reference to Fig. 1, Fig. 2 and Fig. 3, embodiment of the present invention is described in detail.
1, cell to be fused is acquired
(1) Genetic Mutant Cell：Base including monogenic disease, polygenic disease, chromosomal disorder, mitochondriopathy or body cell disease
Because lymphocyte, monocyte, amniocyte and the villi tissue cell of mutation such as lacking, repeating；Gene mutation includes normal
Mutation, the unknown mutation of the cause of disease and target disease cause mutation.The present invention acquires the lymphocyte of people β-patients with thalassemia.
(2) myeloma cell：Mouse SP2/0 oncocytes or people H929 myeloma cell, the present invention are thin using mouse SP2/0 tumors
Born of the same parents.
2, experiment reagent
(1) pre-confluent agent：The CD138 of supplying and myeloma cell's equivalence is mono- in the DMEM culture mediums of 10% fetal calf serum
It is anti-that (myeloma cell: monoclonal antibody=1 CD138: 1), or being incorporated other corresponding antibodies, CD138 monoclonal antibodies or other antibody can commodity purchases
.
(2) staphylococcal protein A (SPA).
(3) other reagents：DMEM culture mediums, HAT Selective agar mediums are purchased from Sigma companies, and fetal calf serum (FBS) is purchased from
Gibco companies；DMSO (-- methyl sulfoxide) it is domestic analytical reagents.
3, cell fusion method
(1) preparation of myeloma cell：Fusion the last week takes out the myeloma cell (SP2/0) frozen out of liquid nitrogen container,
Or human myeloma cell H929, it is immediately placed in hot water defrosting.Appropriate complete culture solution is added after thawing, 1000r/m centrifuges 3min；
It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solutions, sets C02 incubator cultures, once passed within 3-4 days
In generation, expands culture, merges and adjusts cell state in first 24 hours, ensures that cellular morphology is good, it is vigorous to grow before fusion.Fusion
When SP2/0 is blown down from culture bottle, be transferred in centrifuge tube, 1000r/m centrifuges 5-10min, repeated washing cell 2 times, gently
Beat mixing, take a small amount of suspension to count, adjust density, make density when fusion be 80% or so (using it is most be Sp2/0
Cell strain, the cell strain growth and fusion efficiencies are good, itself does not secrete any heavy chain immunoglobulin or light chain, and cell is most
Seedling height scale is 9 × 105/ml, and the doubling time is usually 10~15h；It is same with consideration selection in the relevant practical application of human body
Source cell strain, the NCI-H929 human myeloma cells strain introduced to ATCC cell banks such as the Shanghai bio tech ltd Fu Xiang).
(2) preparation of Genetic Mutant Cell to be fused：Lymphocyte, monocyte, amniocyte, villi tissue cell with
DMEM culture solutions (basal medium) adjust total cell number to 1 × 108~2 × 108, expect that blue dyeing, phase contrast microscope are examined with platform
It looks into, viable count should be higher than that 80% as qualification.
(3) pre-confluent is handled：1. for the Genetic Mutant Cell (B cell) of the receptor containing IgGFc, make in pre-confluent processing
CD138 monoclonal antibodies, with 1: 1 allotment, make the CD138 receptors of myeloma cell just be combined by CD138 monoclonal antibodies, lead to myeloma cell
The IgGFc section target capture Genetic Mutant Cells of CD138 monoclonal antibodies are crossed, it is thin to form myeloma cell-CD138 monoclonal antibodies-gene mutation
The conjugate of born of the same parents.2. for containing IgGFab receptors Genetic Mutant Cell and myeloma cell, make respectively to be connected by IgGFab
After connecing, then it is connected as by the SPA with IgGFc receptors the conjugate of Genetic Mutant Cell-IgG-SPA-IgG- myeloma cells.
3. for the Genetic Mutant Cell of no IgGFc receptors and IgGFab receptors, pre-confluent processing is not done.3. pre-confluent processing side
Method：By myeloma cell and CD138 monoclonal antibodies by etc. potency be formulated in DMEM, 37 DEG C 2 hours, 1000r/min centrifuges 5min, discards
Supernatant gently beats tube bottom to cell grainless and precipitates, 5-10 times of bone-marrow-derived lymphocyte is added and is mixed evenly, 1000r/min from
Heart 5min, discards supernatant, and gently beats tube bottom to cell grainless and precipitates.
(4) cell fusion：Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, aseptically by preheating after taking-up
The 30%PEG3000 of 1000 μ L is added drop-wise to along tube wall in fusion pipe in 60s, while gently rotating centrifugal pipe, later will preheating
25mL basal mediums be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal during addition
Pipe, is then allowed to stand in 37 DEG C of water-bath 10min, and (remove combine antibody) is diffused antibody 5 minutes in 56 DEG C of water-baths of transposition, immediately with
1500r/m centrifuges 5min, discards supernatant, and 50mL HAT culture mediums are added, is inoculated into 96 well culture plates, is placed in after appropriate mixing
37 DEG C, cultivate in 5%C02 incubators.
4, the screening of fused cell (hybridoma cell strain)
96 orifice plate cell growth status are observed, being only effective integration cell after 7-10 days can grow, and discard HAT trainings at this time
Base is supported, the complete medium containing HT is replaced, continues to cultivate.
5, the screening (subclone) of monoclonal hybridoma strain
When fused cell clonal growth area reaches 1/10 cell hole, culture supernatant is gone, selects hybridoma life
The good culture hole of long status, label cell growth position, size under microscope, will be thin in the position of mark using sterile pipette tips
Born of the same parents clone be drawn to it is new have in the culture hole of complete medium, then successively doubling dilution to below count holes, make below each
Cell number in hole is 0-1, and 37 DEG C, the interior culture of 5%C02 incubators about 1 week, microscopically observation cell growth status waits for
When the cell clone in (inverse) hole is covered with to 1/10 or more hole floor space below, the cell in (inverse) back hole is selected to go again
Monoclonal screens, and prepares monoclonal hybridoma strain.
6, the verification of monoclonal hybridoma strain target Disease-causing gene
(1) karyotype is verified：The karyotype of comparative analysis SP2/0 myeloma cell and hybridoma, meter
50 mitotic figures of number are observed chromosome number purpose in hybridoma and are changed.There is part human chromosome not have after cell fusion
It is individually present, is merged with the chromosome of rat bone marrow tumour cell.
(2) target Disease-causing gene is verified：Using fluorescence in situ hybridization (FISH) technology and/or round pcr (QF-PCR/RT-
PCR/ quantitative fluorescent PCRs/real-time PCR) and/or sequencing technologies (generation sequencing/Hiseq sequencings/target area sequencing/depth survey
Sequence/GC corrections and one two-way segment of information comparison judgement CNV/ sliding windows analysis sequencing data/binary segmentation adjust threshold value point
Analyse sequencing data) verification target Disease-causing gene, it confirms contained target Disease-causing gene and classifies.
7, it the preparation of calibration Reference Strains and packing and freezes
By DNA extraction kit specification, monoclonal hybridoma strain DNA is extracted, measures target Disease-causing gene DNA's
Concentration is scaled comparable cell quantity by DNA dosages as defined in gene sequencing kit, according to target Disease-causing gene DNA concentration
It prepared respectively with the relationship of cell content and the type of target Disease-causing gene, dispense monoclonal hybridoma strain, as base
Because of the calibration Reference Strains of sequencing, set frozen in -196 DEG C of liquid nitrogen it is spare.
8, the STABILITY MONITORING of Reference Strains is calibrated
(1) periodic detection of target Disease-causing gene：3 calibration Reference Strains frozen were taken every 1 month, by above-mentioned 6th step
The detections such as PCR or the gene sequencing of target Disease-causing gene are carried out, the stability of target Disease-causing gene is verified.
(2) the regular culture of Reference Strains is calibrated：3 calibration Reference Strains frozen were taken every 1 month, carried out recovery culture,
The survival rate and secondary culture situation of cell are observed, PCR or survey that above-mentioned 6th step carries out target Disease-causing gene are pressed after passing 5-10 generations
Sequence etc., verifies the stability of target Disease-causing gene, and stabilization person continues to freeze.
9, the application of Reference Strains is calibrated
(1) carry out the Internal Quality Control of gene sequencing：1. judging the accuracy of sequencing result：2 plants of calibration Reference Strains of selection, with
The tested sample for doubting the Disease-causing gene containing same target is sequenced under the same conditions, if the known target contained by calibration Reference Strains
Disease-causing gene is all detected, and the target Disease-causing gene not contained is not detected, and illustrates that sequencing system is in Quality Control in control shape
State, the similar target Disease-causing gene sequencing result with batch is reliable, can send out report：2. drawing the Internal Quality Control of gene sequencing
Figure：Using the target Disease-causing gene of Reference Strains as positive control, the mutation of non-targeted Disease-causing gene is as negative control, by every batch of
The secondary positive and negative control testing result are recorded on intuitive Internal Quality Control figure, to monitor the stability of indoor detection method
And whether controlling.
(2) microarray dataset is calibrated：According to the Internal Quality Control of calibration Reference Strains as a result, if it find that sequencing system is not at
Quality Control should then search reason out of control, including calibrate or replace sequenator and sequencing reagent, use Data Analysis Software instead in control state
With mutator comparison data library and its comparison method.
(3) verify data analysis software：Sequenator and sequencing reagent all in Quality Control in control under the premise of, available reference
Whether the sequencing data verify data analysis software of strain is accurate and reliable, analyzes its standard in the processing such as the screening and splicing of data
True property.
(4) gene database is verified：Exist all in Quality Control in sequenator, sequencing reagent and analysis software and its analysis method
Under the premise of control, the reliability in the sequencing data verification gene mutation comparison data library of available reference strain analyzes it in Reference Strains
Whether it is consistent with contained known mutations gene in the comparison and screening of targeted mutagenesis gene.
(5) sequencing result of mutator is proved：Reference Strains are sequenced with tested sample under the same conditions obtained
Sequencing data is compared with comparison data library simultaneously, if the mutator that is detected of Reference Strains and contained known mutations
Gene is consistent, then the mutator that tested sample identical with Reference Strains is detected should be judged as reliable result.
(6) the room interstitial for carrying out sequencing is commented：Calibration Reference Strains periodically can be issued to each unit attending to judge by quality management mechanism,
Carry out gene sequencing quality evaluation, according to the evaluation of result of return respectively participate in evaluation and electing laboratory gene sequencing accuracy and each room survey
The difference of sequence result, to find quality problems in time and to rectify and improve in time.
(7) sequencing result between room is recognized each other：It is commented according to room interstitial as a result, if it find that same reference strain is because being sequenced unit not
With and provide different as a result, quality is problematic between then illustrating room, reason should be searched, uniformly use reliable sequencing system instead, really
The accuracy and consistency for protecting constituent parts sequencing result, promote recognizing each other for each room result.
(8) it is the sequencing approach compared to create with Reference Strains：In conventional methods where, pass through sequencing data and gene database
Comparison obtain sequencing result, such as sequencing data and burst-normal database are compared, remove burst-normal gene, then with cause
Sick mutation database compares, and obtains similar pathogenic mutation gene.It is this to compare because tested ethnic group and testing conditions etc. are different, lack
Comparativity, creates the existing gene database of Reference Strains replacement and is compared, and has same detection with Reference Strains based on tested sample
The identical sequencing data of condition and both infer mutator having the same.
Claims (8)
1. a kind of preparation of gene sequencing calibration Reference Strains, which is characterized in that under the action of polyethylene glycol or specific antibody
Genetic Mutant Cell is merged to make mutator implantation oncocyte with myeloma cell and is expanded with oncocyte, is followed by screened
Monoclonal cell simultaneously identifies target Disease-causing gene, and calibration Reference Strains are made, are commented applied to the whole range quality control of gene sequencing, matter, school
Comparison that is accurate or substituting existing gene comparison data library progress sequencing data and its mutator.
2. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1, which is characterized in that the specificity is anti-
Body refers to CD138 monoclonal antibodies, IgG and SPA.
3. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1, which is characterized in that the gene mutation
Including the unknown mutation of burst-normal, the cause of disease and target disease cause mutation.
4. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1, which is characterized in that the myeloma is thin
Born of the same parents include mouse SP2/0 oncocytes or people's H929 oncocytes.
5. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1, which is characterized in that the calibration reference
Strain refers to the monoclonal hybridoma being mutated comprising known.
6. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1 and 5, which is characterized in that described known
Gene mutation refers to be identified through high resolution chromosome karyotyping, fluorescence in situ hybridization, PCR, genetic chip or sequencing technologies.
7. according to claim 1,5 and 6 a kind of gene sequencing calibration Reference Strains preparation, which is characterized in that it is described
Major gene is mutated the beautiful sick mutator of feeling the pulse with the finger-tip.
8. a kind of preparation of gene sequencing calibration Reference Strains according to claim 1 and 5, which is characterized in that the calibration
Reference Strains applied to gene sequencing refer to applied to carry out Internal Quality Control, calibration microarray dataset, verification sequencing data analysis software and
Gene database, evidence mutator sequencing result, development room interstitial are commented and recognize each other and create between the room of sequencing result to join
It is the improvement sequencing approach compared to examine strain.
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