Background technique
The abrupt climatic change of gene transcripts refers to produced by the gene order of RNA expressed by experimentally identification individualThe technology in mutational site.
The detection of mutation can be carried out at the genomic level, can also be carried out in transcript profile level.If using baseIt is that sample carries out Testing and appraisal to the series jump of individual because organizing, can be extracted by DNA, target sequence is expanded using special primerIncreasing carries out generation sequencing identification mutation again.The building of two generation sequencing libraries can also be carried out with the DNA that extracting obtains, and library is carried outAfter sequencing, the information that resulting sequencing data obtains genes of individuals mutation is analyzed.The flux of former technique is low, but compare throughJi, speed is fast, still identifies the conventional means of series jump so far.The advantages of second method is that data volume is big, and flux is high, is led toSeries jump information in most gene group can be obtained by crossing once sequencing, but a disadvantage is that the period is long, complicated degree of analysis is high,It spends high.And it may not be needed there are many information.Very good solution portion is sequenced by the capture that second scheme derivesDivide problem, the relatively heavy sequencing in measurement region is small very much, and the data volume of required measurement is adjusted according to the size of target, does not needSurvey a large amount of data.But because or using two generations sequencing scheme, in some long segments with part repeat sequenceThe sequencing of column can then encounter obstacle in post analysis.
In addition, carrying out sequencing to the RNA especially mRNA of individual is also to find a kind of means in expressing gene mutational site,If researcher is concerned with the mutation of some gene or certain several gene, can by expand target gene cDNA sequence,Sequencing is carried out again, to obtain relevant information.It is interested if it is being mutated to unknown mRNA sequence, then just with individualMRNA be template build library carry out the sequencing of two generations, by sequencing data carry out analysis obtain sequence abrupt information.
Although above experimental program can make researcher reach research purpose, both methods is all built uponA kind of identification of means on large organization foundation, some researchs need to confirm that the particular sequence mutation of some gene occurs at someIn cell, this just needs to identify the sequence information in each cell, to obtain the sequence information in individual cells.Such asCell in tumor sample is there are high heterogeneity, and that there may be gene is a variety of for multiple cells of a tumor sampleMutant form, and different mutant forms may will affect the morbid state of individual.At this moment if each cell can be found outIn sequence variations the case where can provide more accurately information for the diagnosing and treating of disease.In addition, gene editing meansIt is widely used in scientific research now, but after once being edited to gene, to by the gene of editor's cellThe confirmation in series jump site is also necessary.And the unicellular sequencing technologies occurred in recent years provide for the solution of this problemApproach, the research that the especially nearly 2 years some unicellular instruments occurred composes researcher to monocell expressing, which becomes, to be showedIt is real.But because current single cell analysis instrument is to carry out sequencing using two generation sequencing approaches mostly, sequencing length byTo limitation, it can not splice after some long segment sequences, the abrupt information of transcript is caused to cannot get, therefore in identification overall lengthTranscript mutation aspect is subject to certain restrictions, some research work are unable to complete.
For the sequencing confirmation for solving the mutation of unicellular transcript, in view of the foregoing drawbacks, we single cell analysis method andLong segment sequencing combines, and according to the detection target sequence design primer of target gene and expands target sequence again to sequence progress threeFor long segment be sequenced, it is intended to high throughput solve the problems, such as overall length transcript it is unicellular in gene mutation.
Specific embodiment
1 preferred embodiment of the invention introduced below keeps its technology contents more clear and is easy to understand.The present invention canTo be emerged from by many various forms of embodiments, protection scope of the present invention is not limited only to the implementation mentioned in textExample.
Whether the nucleotide A for detecting 2103 positions in unicellular middle STAT3 gene mRNA sequence sports G:
1, it is carried out using single cell suspension of 10 × Genomics Chromium Controller to mutational cell line singleCell marking and cDNA synthesis, and cDNA expansion is carried out according to the experimental program of 10 × Genomics Chromium ControllerIncrease, purify and carry out quality inspection with Bioanalyzer 2100.
2, the cDNA sample of quality inspection qualification is divided into two parts, and portion is for conventional 10 × Genomics ChromiumController builds library.Another is used for template amplification target fragment.
3, primer sequence used in target fragment: F:5 '-AATGATACGGCGACCACCGAG-3 ', R:5 is expanded '-CATGGGGGAGGTAGCGCACTCCGA-3 ', PCR ingredient: 5X PrimeSTAR GXL buffer, 10 μ L；CDNA, 10 μ L；DNTP Mix (2.5mM each), 4 μ L；Primer F, 1 μ L；Primer R, 1 μ L；DdH2O, 23 μ L；PrimeSTAR GXLDNA Polymerase (1.25U/ μ L), 1 μ L.It after mixture prepares, is expanded using PCR instrument, PCR condition is as follows: 98 DEG C,30 seconds；98 DEG C, 10 seconds；65 DEG C, 15 seconds；68 DEG C, 2 minutes, 28 circulations；68 DEG C, 5 minutes, EP (end of program).
4, the purifying resulting product of PCR amplification is followed these steps:
1 × magnetic beads for purifying purifies for the first time.
1)PB magnetic beads magnetic bead is placed at room temperature in advance, and oscillation mixes.
2) magnetic bead is added to sample tube according to 1 × sample volume.
3) in vortex oscillator, 2000rpm, 10min are mixed, are gently got rid of.
4) sample tube is placed on magnetic frame, sucks supernatant.
5) add 75% ethyl alcohol, remove supernatant, be repeated 2 times.
6) remove residual liquid.
7) plus 100 μ L EB elute magnetic bead and sample, and 2000rpm in vortex oscillator, 10min are mixed.
8) sample tube is placed on magnetic frame, draws supernatant, obtains product DNA.
1 × magnetic beads for purifying, second of purifying.
1)PB magnetic beads magnetic bead is placed at room temperature in advance, and oscillation mixes.
2) magnetic bead is added to sample tube according to 1 × sample volume.
3) in vortex oscillator, 2000rpm, 10min are mixed, are gently got rid of.
4) sample tube is placed on magnetic frame, sucks supernatant.
5) add 75% ethyl alcohol, remove supernatant, be repeated 2 times.
6) remove residual liquid.
7) plus 21 μ L Elution Buffer elute magnetic bead and sample, and 2000rpm in vortex oscillator, 10min are mixed.
8) sample tube is placed on magnetic frame, draws supernatant, be purpose product DNA.
5, long segment sequencing library constructs:
1) end-filling
The DNA expanded, 1.5 μ g；DNA repairs buffer, 5 μ L；NAD+, 0.5 μ L；ATP high, 5 μ L；DNTP, 0.5 μL, DNA damage repair mixed liquor, 2 μ L, H2O, supplement reaction system to 50 μ L of total volume；It plays even, gently gets rid of, be centrifuged；In PCR instrumentOperation reaction, 37 DEG C of 20min, 4 DEG C of 2min.
2) end is repaired
The DNA sample of end-filling, 50 μ L；End repair 2.5 μ L of mixed liquor, 52.5 μ L of total volume, mix parent, it is of short duration fromThe heart runs on PR instrument and reacts, and 25 DEG C, 5 minutes.
3) DNA is purified
A, PB magnetic bead is pre-equilibrated using room temperature.
B, the PB magnetic bead for taking 1 × sample volume, is loaded onto sample cell, and finger flicks, and gently gets rid of.
C, it is placed on oscillator, 2000rpm, 10min are gently got rid of.
D, sample cell is placed on magnetic frame, until supernatant is limpid, discards supernatant.
E, it with fresh 75% ethanol washing 2 times, gently gets rid of, discards residual liquid.
F, dry 1min, sample-adding 30ul Elution Buffer elution, 2000rpm, 10min.
G, sample tube is placed on magnetic frame, draws supernatant, collect End-Repaired DNA.
4) blunt end cloning sequence measuring joints
End DNA plerosis and the 30 μ L of sample purified；Blunt-ended adaptor, 2 μ L；After mixing, template is added and prepares 4 μ of buffer2 μ L of L, ATP low is mixed, of short duration centrifugation；1 μ L of ligase is added, water is added to be supplemented to 40 μ L, of short duration rear centrifugation is mixed, in PCR15 minutes are kept the temperature for 25 DEG C on instrument, and 65 degrees Celsius keep the temperature 10 minutes, and 4 DEG C keep the temperature 2 minutes
5) DNA fragmentation without connecting top connection is removed
Connect 40 μ L, EXO III of sample, 1 μ L, EXO VII 1 the μ L, 42 μ L of total volume, 37 after mixing of sequence measuring jointsDEG C heat preservation 60 minutes.
6) purified templates
It purifies for the first time:
A, PB magnetic bead shifts to an earlier date equilibrium at room temperature.
B, the PB magnetic bead for taking 1* sample volume respectively, is loaded onto sample cell, and finger flicks, and gently gets rid of.
C, it is placed on oscillator, 2000rpm, 3-4 turns, and 10min is gently got rid of.
D, sample cell is placed on magnetic frame, until supernatant is limpid, discards supernatant.
E, it with fresh 75% ethanol washing 2 times, gently gets rid of, discards residual liquid.
F, dry 1min, sample-adding 50ul Elution Buffer elution.
Second of purifying:
A, PB magnetic bead shifts to an earlier date equilibrium at room temperature.
B, the PB magnetic bead for taking 1* sample volume is loaded onto the 1st 50ul DNA after purification, goes to 1.5ml sample cell,Finger is light
Bullet is gently got rid of.
C, it is placed on oscillator, 2000rpm, 3-4 turns, and 10min is gently got rid of.
D, sample cell is placed on magnetic frame, until supernatant is limpid, discards supernatant.
E, it with fresh 75% ethanol washing 2 times, gently gets rid of, discards residual liquid.
F, dry 1min, sample-adding 15ul Elution Buffer elution, 2000rpm, 3-4 turn, and 1min collects DNA, i.e.,For final library, -20 DEG C
It saves.
7) library quality inspection
Qubit and 2100 quality inspections.
Machine is sequenced on 6
Library is sequenced using PacBio sequenator.Data analysis is carried out to sequencing result, identifies mutational site.Since the barcode of 10X marks each cell, the STAT3 in which cell can be obtained according to this mark informationThe nucleotide of 2301 positions changed.Unicellular transcript profile literature data judgement further according to 10XGenomics is eachThe variation of stat3 gene order in cell reaches the specifying information of stat3 gene mutation in identification individual cells and is owningMutant proportion in cell.
In addition, the sequencing of the overall length transcript of TCR and BCR can also be carried out with this programme.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without woundThe property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the artPass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's ideaScheme, all should be within the scope of protection determined by the claims.