CN109996877A - A kind of gene label, kit and its application for nucleic acid samples mark - Google Patents
A kind of gene label, kit and its application for nucleic acid samples mark Download PDFInfo
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Abstract
This application discloses a kind of gene label, kit and its applications for nucleic acid samples mark.The application for nucleic acid samples mark gene label, for a segment length be greater than 130bp nucleic acid, nucleic acid sequence 3 ' end have polyA tail, and in nucleic acid sequence random site inserted at least one section of index sequence.
Description
This application involves nucleic acid samples process fields, more particularly to a kind of gene label, kit and its application for nucleic acid samples mark.
Life science has been widely applied in high throughput sequencing technologies, its basic process is that nucleic acid substances are extracted from biological tissue, DNA or RNA, these nucleic acid substances are then changed specific sequential structure by Protocols in Molecular Biology, that is sequencing library, this process are known as building library.After sequencing library passes through quality inspection, high-flux sequence is carried out.
As sequencing price is more and more cheaper, gene sequencing technological applications are more and more wider, so that more people participate in sequencing research field, while the sample number for building library is also just more and more, so that the complexity for building library process becomes larger, the probability that personnel's operating mistake occurs is higher and higher.The problems such as anti-sample tune, cross contamination are especially easy to appear during building library.
Two samples for usually building library simultaneously can pass through information analysis techniques if it is different plant species in sequencing data, and sequencing data is compared with reference to gene, compares similarity, by comparison rate, carries out analysis identification.But if two samples are same species, using the method for reference data matching identification with regard to unworkable.And usually get together be all belong to a batch of sample same species probability it is relatively high；Therefore, the correctness for being difficult to ensure two samples is compared by reference to gene.
It, can be in the connector company for building library process or PCR stage, by adding the sequence label of one section of 6-10bp in connector or primer, to distinguish each sample in sequencing for two samples of same species.But this method can only distinguish the sample after connector connects or after PCR, whether the sample before cannot identifying connector connection has tune anti-or cross contamination.
Furthermore it is also possible to design the multiple PCR primer of 21 high frequency SNP sites by 21 high frequency SNP sites for selecting sample, be then analyzed by mass spectrometry to PCR product, is compared with sequencing data, determine that sample is not adjusted instead.But this method temporarily can be only applied to DNA sequencing, and at high cost, individually do multiplex PCR and mass spectrum；More importantly sample of this method for some different parts from same tissue, such as cancer and cancer beside organism, it is difficult to differentiate between on genome SNP, therefore can not also play sample mark action.
Summary of the invention
The purpose of the application is to provide a kind of new gene label for nucleic acid samples mark, includes the gene
The application of the kit and gene label of label.
To achieve the goals above, the application uses following technical scheme:
The one side of the application disclose it is a kind of for nucleic acid samples mark gene label, the gene label be a segment length be greater than 130bp nucleic acid, nucleic acid sequence 3 ' end have polyA tail, and in nucleic acid sequence random site inserted at least one section of index sequence.
It should be noted that, as long as segment is greater than the gene label that 130bp all can serve as the application from principle, no ceiling, because it is subsequent build library during there is DNA to interrupt step, long sequence can be also interrupted again, and distinguish the difference between sample, detect the gene label of wherein known array, whether it is interrupted as gene label, has no influence；Therefore, the gene label of the application is the nucleic acid that a segment length is greater than 130bp.
It should be noted that the gene label of the application, in use, adding it in sample DNA or RNA, eventually by the particular sequence or index sequence for detecting added gene label, so that it may know which sample is test object be；Wherein index sequence, that is, index sequence, the sequence can be using conventional index sequences in microarray dataset, and but, for different microarray datasets, index sequence is different, the application to specific index sequence without limitation；In addition, polyA tail is designed primarily directed to the banking process for needing polyA to capture in the gene label of the application.
It is understood that, the gene label of the application is for being identified to sample, to distinguish different samples, the nucleic acid sequence of certain gene label cannot be homologous with identified sample, that is, the nucleic acid of gene label and identified sample must be not no homologys, one section of random sequence can be usually used, and compared by blast, confirm its specificity.But, using the sequence in the mRNA molecule of ERCC in a kind of implementation of the application, ERCC cuts off and repairs complementary chiasma gene, it itself can be used as reference gene to be added in sequencing sample, therefore, in a kind of implementation of the application, one section of unduplicated sequence is had chosen respectively from 8 mRNA molecules of ERCC as gene label, it is combined by 8 gene labels that 8 groups of nucleic acid form, this will be described in detail in subsequent scheme.
Preferably, gene label is the nucleic acid of a segment length 130bp~160bp.The nucleic acid that preferred gene label is a segment length 160bp.
It should be noted that, although front is it is stated that as long as the length of gene label is greater than 130bp；But for DNA synthetic technology and cost, it is both economical for selecting the short-movie section of 160bp, and technology is easy to accomplish.
Preferably, the length of index sequence is 6-10bp.
Preferably, uniformly inserted with 6 index sequences in nucleic acid sequence.
It should be noted that the nucleic acid sequence in gene label is uniformly inserted into multiple index sequences, it is the identity in order to further enhance gene label.
Preferably, the length of polyA tail is 24bp.
Preferably, nucleic acid is single stranded DNA, double-stranded DNA or RNA.
It should be noted that, if identified sample is RNA, the nucleic acid of gene label is RNA sequence as gene label；If identified sample is DNA, the nucleic acid of gene label is single stranded DNA or double chain DNA sequence.
Preferably, the nucleic acid of gene label sequence shown in Seq ID No.1 to Seq ID No.8 forms.
It should be noted that eight groups of nucleic acid shown in Seq ID No.1 to Seq ID No.8 design actually in a kind of implementation of the application in order to illustrate the gene label of the application；It is appreciated that this eight groups of nucleic acid can be completely applied to sequencing or other places, to be identified to sample；But the gene label of the application, it is not only limited in this eight groups of nucleic acid, can be other random sequences, quantity, the length of nucleic acid also can according to need the sample of mark and changes, and index sequence can also change according to different microarray datasets.
The another side of the application discloses application of the gene label of the application in nucleic acid sequencing.
It should be noted that, the gene label of the application is inherently directed to sample in nucleic acid sequencing process and is easy to adjust anti-or cross contamination and design, by the gene label for adding the application, in sequencing data analysis, it may determine that whether sample has tune anti-or cross contamination according to the sequence of gene label, to play mark action.
The application's discloses a kind of kit for nucleic acid samples mark, the gene label containing the application in the kit on one side again.
It should be noted that the gene label of the application can be added in sample to be tested as an independent sample of nucleic acid, mark action is played, therefore, the nucleic acid of gene label powder can be lyophilized into or be configured to high concentration nucleic acid solution, as kit, with convenient to use and transport；It can be used very advantageously in nucleic acid sequencing or other places for needing to be identified nucleic acid samples in this way.
Gene label based on the application, the application's discloses a kind of method for nucleic acid sequencing on one side again, the gene label including the application is added in original DNA or RNA sample, then carries out Jian Ku, the sequencing of upper machine again.
It should be noted that, the method for nucleic acid sequencing of the application, an actually concrete application of the gene label of the application, the gene label of the application is added i.e. in original DNA or RNA sample, to play the role of sample mark, avoid it is subsequent build library, occur in sequencing procedure to adjust the problems such as anti-, can also be whether to have cross contamination between sample survey.For example, if detecting identical gene label in two samples, then it represents that there are cross contamination during building library or sequencing, the sequencing result inaccuracy being achieved in that needs to be sequenced again the two samples.For another example, during building library or sequencing, the sample message recorded is not met with the gene label of detection, and it is another sample that the gene label detected is corresponding, it then indicates to have occurred tune instead, needs that the correction of corresponding sample message is come according to the gene label of detection.
It is appreciated that the gene label of the application, effect is just added in nucleic acid samples, plays mark action；Different samples, adds different gene labels, the sequence of these gene labels itself be it is known, therefore, pass through the nucleic acid sequence of detection gene label, so that it may judge which sample object is.Therefore, although the application gene label is studied for nucleic acid sequencing, still, is not only limited the use of in nucleic acid sequencing, and all places for needing to be identified nucleic acid samples can use the gene label of the application.Furthermore, it is contemplated that some special purposes, can also carry out a series of modification to the nucleic acid sequence of the gene label of the application, such as fluorescent decoration etc. is not specifically limited herein to enhance its recognition performance.
Due to using the technology described above, the beneficial effects of the present application are as follows:
The problems such as gene label for nucleic acid samples mark of the application, can very easily be added in nucleic acid samples, by detecting the particular sequence of gene label, effectively distinguish different nucleic acid samples, cross contamination anti-so as to avoid sample tune；The gene label of the application is applied to nucleic acid sequencing, can preferably ensure sequencing quality, is avoided because adjusting anti-or cross-contamination effects sequencing result.
Fig. 1 is the base distribution figure of sequencing result in the embodiment of the present application.
With the raising of nucleic acid sequencing efficiency, sequencing cost is accordingly reduced, and more and more researchs are related to that link is sequenced.This makes the processing of sample, and the sample size for especially building library is more and more huger, in this process, is easy to appear that sample tune is anti-or cross contamination.It is unavailable that cross contamination directly results in sequencing result, if the problem of not finding cross contamination, the nucleic acid sequencing of mistake can be provided for researcher as a result, influencing follow-up study.And in all correct situation of other programs and process, it is anti-in case of sample tune, then it is to be more difficult the problem of being found, leads to the nucleic acid sequencing for being supplied to researcher in this way as a result, being entirely different species, or is runed counter to expected result.Therefore, the application has studied a kind of gene label for nucleic acid samples mark, in use, directly the gene label is added in the nucleic acid samples at the beginning of original or processing, records gene label added by good each nucleic acid samples；By it is subsequent build library and sequencing after, according to gene label detected in sequencing result, then it can accurately know which nucleic acid samples the sequencing result belongs to, effectively avoid the anti-problem of sample tune, cross contamination can also be intuitively judged whether there is very much, to ensure sequencing quality.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment is only further described the application, should not be construed as the limitation to the application.
Embodiment
One, gene label design and synthesis
This example selects exogenous RNA with reference to group (External RNA Controls consortium, ERCC 8 mRNA molecules), intercept 124 bases at its 3 ' end, it can guarantee that the sequence of each molecule will not repeat in this way, wherein, it all include the polyA tail of a 24bp in 3 ' 124 bases in end of 8 mRNA molecules.Select 8 index sequences, 8 index sequences are inserted into respectively in the segment of 8 mRNA molecules interception, each mRNA molecule interception segment adds a kind of index sequence, also, is uniformly repeatedly inserted into the gene label that 6 index sequences obtain this example in each mRNA molecule interception segment.8 gene label P1-P8 of this example, nucleic acid sequence is respectively as shown in Seq ID No.1 to Seq ID No.8.
P1 (Seq ID No.1):
P2 (Seq ID No.2):
P3 (Seq ID No.3):
P4 (Seq ID No.4):
P5 (Seq ID No.5):
P6 (Seq ID No.6):
P7 (Seq ID No.7):
P8 (Seq ID No.8):
Underscore thickened portion, that is, index sequence in sequence shown in Seq ID No.1 to Seq ID No.8.8 gene labels of this example are synthesized by the Hong Kong Thermofisher branch company, and it is spare to be then diluted to 15nM with water dissolution.
Two, library is built
This example refers to RNA with the general mankind, and the RNA standard items of (Universal Human Reference RNA, abridge UHRR) carry out building library and sequencing test.The gene label P1 that 4 μ L concentration are 15nM is added in the RNA standard items (brand: Aglient, article No.: 740000-Universal Human Reference RNA) of 200ng, is subsequently used for subsequent building library and sequencing.
This example carries out building library using kit TruSeq_RNA_SamplePrep_v2kit (article No.: RS-122-2001/RS-122-2002), builds the specific steps in library with reference to kit specification TruSeq_RNA_SamplePrep_v2_Guide_15026495_A (second edition sheet).Building library, detailed process is as follows, and building library process below is calculated according to a reacting dose.
(1) it the purifying of mRNA and interrupts
This example is purified using RNA standard items of mRNA purifying magnetic bead (mRNA Purification Beads) to addition gene label, specifically, the RNA Purification Beads of 50 μ L is added into the RNA standard items of addition gene label, then 1min is placed in 65 DEG C of 5min, on ice, is stored at room temperature 5min
It places it in and is stored at room temperature 5min on magnetic frame again, remove supernatant, retain magnetic bead；The magnetic bead cleaning buffer solution (Bead Washing Buffer) of 150 μ L is added thereto, cleaning is primary, likewise, removing cleaning solution after being stored at room temperature 5min on magnetic frame；The dissolution buffer (Elution Buffer) of 50 μ L is added thereto, then 80 DEG C of processing 2min place 1min, then place it in and be stored at room temperature 5min on magnetic frame on ice, take supernatant；It carries out a magnetic bead absorption, washing and elution again to supernatant, that is, obtains the RNA sample of purifying.
RNA sample is after purification, using dissolution, primer, fragmentation mixture, referred to as interrupt mixture (Elute, Prime, Fragment Mix), that is Fragment Mix, sample is interrupted, specifically, be added 19.5 μ L in RNA sample after purification interrupts mixture (Fragment Mix), in 94 DEG C of processing 8min, that is, obtain the RNA solution interrupted.
(2) cDNA synthesis and purifying
CDNA synthesis includes that the synthesis of mono- chain of cDNA and bis- chain of cDNA synthesize, specific as follows:
The reaction system of one chain synthesis are as follows: the 17 μ L of RNA solution interrupted adds the reverse transcriptase (SuperScript II) of 1 μ L and a chain synthesis reaction mixture (First Strand Master Mix) of 7 μ L, reacted after mixing.Reaction condition are as follows: 25 DEG C of 10min, 42 DEG C of 50min, 70 DEG C of 15min, it is standby at 4 DEG C after the reaction was completed.
The reaction system of two chains synthesis are as follows: 25 μ L of a chain product adds the two chain synthesis reaction mixtures (Second Strand Master Mix) of 25 μ L, reacted after mixing.Reaction condition are as follows: at 16 DEG C, 350rpm interrupted oscillating 15s, static 2min, so reaction 1h.
Two chain product purifications: 1.8 volume of purifying magnetic bead (Ampure XP Beads 1.8x) of 90 μ L is added into the two chain products of 50 μ L, it mixes, it is placed at room temperature for 5min, magnetic frame is stored at room temperature 5min, abandon supernatant, then 80% ethyl alcohol that 200 μ L are added thereto is washed twice, and buffer (Resuspension Buffer) is resuspended after drying and recycles 60 μ L.
(3) end is repaired
The system that end is repaired are as follows: reaction mixture (End Repair Mix) is repaired in the end that 40 μ L are added into 60 μ L, bis- chain purified product, in 30 DEG C of reaction 30min.After the reaction was completed, using purifying 1.6 volume of magnetic bead (Ampure XP Beads 1.6x) purified product, recycling obtains 17.5 ends μ L and repairs product, and specific purification process refers to two chain product purifications.
(4) add polyA tail
Add the system of polyA tail are as follows: the end that 12.5 μ L are added into 17.5 ends μ L reparation product adds A reaction mixture (A-Tailing Mix), in 37 DEG C of reaction 30min.Obtain about 30 μ L adds polyA end reaction product.
(5) adjunction head
The system of adjunction head are as follows: add to 30 μ L and connection reaction mixture is added in polyA end reaction product
(Ligation Mix) 2.5 μ L, buffer (Resuspension Buffer) 3 μ L, 2 μ L of connector-label (Adaptor Index), after mixing, 30 DEG C of reaction 10min are resuspended.Then connection stopping of reaction buffer (Stop Ligation Buffer) 5 μ L are added thereto, reaction was completed.
(6) Sample Purification on Single
Adjunction head product is using purifying 1 times of volume of magnetic bead (Ampure XP Beads 1x) purifying, purifying is resuspended buffer (Resuspension Buffer) and recycles 50 μ L for the first time, it is purified to carrying out second to a purified product, buffer (Resuspension Buffer) recycling is resuspended and obtains 20 μ L products.The detailed process purified twice refers to two chain product purifications.
(7) PCR amplification and purifying
PCR amplification is carried out to the adjunction head product of purifying, reaction system are as follows: PCR primer mixture (PCR Primer Cocktail) 5 μ L are added into 20 μ L purified products, PCR reaction mixture (PCR Master Mix) 25 μ L, are reacted after mixing.
Reaction condition is 98 DEG C of 30s, is recycled subsequently into 12: 98 DEG C of 30s, 60 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 5min after circulation terminates, then 10 DEG C it is standby.
Pcr amplification product is resuspended buffer (Resuspension Buffer) recycling and obtains 30 μ L products, the detailed process of purifying refers to two chain product purifications using purifying 1 times of volume of magnetic bead (Ampure XP Beads 1x) purifying.
Three, nucleic acid sequencing and interpretation of result
This example is sequenced using the Hiseq4000 microarray dataset of Illumina company, and PE100 is sequenced, and 1G is surveyed in library.
(1) by sequencing result compared with the sequence of P1, choose the sequence containing gene label.
(2) sequencing result and ERCC reference data are compared.
The results show that can significantly detect the nucleic acid sequence of gene label P1 in sequencing result, the sequencing result of RNA standard items is also identical as its sequence, is consistent with expection.
Also, it is lower that P1 sequence accounts for sample data ratio, and only 0.0033%, data volume will not be wasted, sample will not be influenced and use ERCC internal reference.
In addition, the base distribution figure directly exported from microarray dataset, as shown in Figure 1, because the segment that this example is added is single, due to the sequencing principle limitation of Illumina, if the identical segment of a certain sequence equal length, ratio is excessively high to will lead to base fluctuation, and it was verified that the gene label for the single segment that this example is added does not result in the fluctuation of base fierceness, therefore it will not influence sequencing.Analysis reason thinks, may be this example be added gene label account for sequencing sample nucleic ratio it is small, in addition the reverse transcription reaction stage, initiation site is different, make same segment, after ultimately forming library, fragment length has changed, sequencing time series synchronism has been disturbed, so not will lead to base fluctuation.
Therefore, generally speaking, the gene label of design is added in this example in sample to be tested, can effectively distinguish nucleic acid samples, and will not influence sequencing result.
In addition, this example has also carried out identical test respectively for seven gene labels of P2 to P8 of this example design, as a result suitable with P1, the gene label of display this example design can be effectively used for distinguishing nucleic acid samples, have no effect on being normally carried out for sequencing.
In addition, eight gene labels of this example design, other than it can be identified by not homotactic gene label to nucleic acid samples, it can also distinguish, i.e., nucleic acid samples are identified according to the concentration of the gene label added in different nucleic acid samples according to the same gene label for adding various concentration in different nucleic acid samples.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that the specific implementation of the application is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, without departing from the concept of this application, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection scope of the application.
Claims (10)
- It is a kind of for nucleic acid samples mark gene label, it is characterised in that: the gene label be a segment length be greater than 130bp nucleic acid, nucleic acid sequence 3 ' end have polyA tail, and in nucleic acid sequence random site inserted at least one section of index sequence.
- Gene label according to claim 1, it is characterised in that: the gene label is the nucleic acid that a segment length is 130bp~160bp.
- Gene label according to claim 1, it is characterised in that: the length of the index sequence is 5~10bp.
- Gene label according to claim 1, it is characterised in that: uniformly inserted with 1~15 index sequence in the nucleic acid sequence.
- Gene label according to claim 1, it is characterised in that: the length of the polyA tail is 15~70bp.
- Gene label according to claim 1, it is characterised in that: the nucleic acid is single stranded DNA, double-stranded DNA or RNA.
- Gene label according to claim 1-6, it is characterised in that: the nucleic acid of gene label sequence shown in Seq ID No.1 to Seq ID No.8 forms.
- Application of the gene label according to claim 1-7 in nucleic acid sequencing.
- A kind of kit for nucleic acid samples mark, it is characterised in that: contain the described in any item gene labels of claim 1-7 in the kit.
- A kind of method for nucleic acid sequencing, it is characterised in that: including the described in any item gene labels of claim 1-7 are added in original DNA or RNA sample, then carry out Jian Ku, the sequencing of upper machine again.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2016/110457 WO2018107481A1 (en) | 2016-12-16 | 2016-12-16 | Gene tag for nucleic acid sample identification, kit, and application thereof |
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| CN109996877A true CN109996877A (en) | 2019-07-09 |
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| CN110656157B (en) * | 2019-10-16 | 2023-09-08 | 重庆市人口和计划生育科学技术研究院 | Quality control product for tracing high-throughput sequencing sample and design and use method thereof |
| CN111304309A (en) * | 2020-03-06 | 2020-06-19 | 上海韦翰斯生物医药科技有限公司 | Detection method for sequencing platform tag sequence pollution |
| CN112251501B (en) * | 2020-10-28 | 2023-07-25 | 深圳人体密码基因科技有限公司 | Internal reference gene set, screening method thereof, universal primer group, kit, reaction system and application |
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