阅读说明：本技术 一种高通量的单细胞转录组测序方法和试剂盒 (High-throughput single-cell transcriptome sequencing method and kit ) 是由 赵星 金皓玄 李罗权 赵小莹 冯太青 齐晓娟 周清 李贵波 李计广 王磊 李阳 于 2018-07-05 设计创作，主要内容包括：本申请公开了一种高通量的单细胞转录组测序方法和试剂盒。本申请的高通量单细胞转录组测序方法,包括采用液滴生成系统将单细胞与带有标签的微珠包裹在一个液滴中,并在液滴中进行逆转录。本申请的方法,通量可达9000个细胞,与10×genomics相当,液滴破乳之后微珠相互污染小,提高了有效数据占比。本申请的方法在液滴中进行逆转录,所需试剂少,成本低。本申请的优选方案中,逆转录采用SMART模板转换技术,其产物可以直接用于后续的Tn5文库构建以及BGISeq-500平台测序,无需进行文库转化；避免了多次扩增引入偏差,实现了液滴微流控平台与BGISeq-500测序平台连用,简化并方便了大规模单细胞测序。(The application discloses a high-throughput single-cell transcriptome sequencing method and a kit. The high-throughput single-cell transcriptome sequencing method comprises the steps of wrapping single cells and microbeads with labels in a droplet by adopting a droplet generation system, and carrying out reverse transcription in the droplet. According to the method, the flux can reach 9000 cells, which is equivalent to 10 × genomics, the mutual pollution of the microbeads is small after the liquid drops are demulsified, and the effective data ratio is improved. The method carries out reverse transcription in the liquid drop, and has the advantages of less required reagent and low cost. In a preferred scheme of the application, the reverse transcription adopts a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; avoids the deviation caused by multiple amplifications, realizes the connection of the droplet microfluidic platform and the BGISeq-500 sequencing platform, simplifies and facilitates the large-scale single-cell sequencing.)

1. A high throughput single cell transcriptome sequencing method, characterized in that: comprises the steps of wrapping single cells and microbeads with labels in one liquid drop by using a liquid drop generating system, and carrying out reverse transcription in the liquid drop to obtain first strand cDNA.

2. The single cell transcriptome sequencing method of claim 1, characterized in that: the reverse transcription employs SMART template switching technology to introduce bases at the 5 'end of reverse transcribed first strand cDNA of mRNA to distinguish from 3' end sequences.

3. The single cell transcriptome sequencing method of claim 2, characterized in that: carrying out SMART PCR pre-amplification on the SMART template conversion product by adopting SMART PCRPRIMER, and obtaining a 3' end marker sequence by amplification; wherein SMARTPCR PRIMER is a specific primer at the 3' end.

4. The single cell transcriptome sequencing method of claim 3, wherein: the SMART PCR PRIMER is a sequence shown in SEQ ID NO. 1;

SEQ ID NO.1：5’-AAGCAGTGGTATCAACGCAGAGT-3’。

5. the single cell transcriptome sequencing method of claim 3, wherein: the method also comprises the steps of carrying out PCR amplification enrichment on the 3 'end marker sequence by adopting a specific primer group aiming at the 3' end marker sequence, then carrying out fragment selection on a PCR amplification enrichment product, cyclizing the selected fragment, and directly sequencing by adopting a BGISeq-500 platform.

6. The single cell transcriptome sequencing method of claim 5, wherein: the upstream primer of the specific primer group is a sequence shown by SEQ ID NO.2, the downstream primer is a sequence shown by SEQ ID NO.3, and the 5' end of the upstream primer has phosphorylation modification;

SEQ ID NO.2：5’-GAACGACATGGCTACGATCCGACTTAAGCAGTGGTATCAACGCAGAGTAC-3’

SEQ ID NO.3：5’-TGTGAGCCAAGGAGTTGTTGTCTTCGTCTCGTGGGCTCGG-3’

the Splint oligo adopted for fragment cyclization is a sequence shown in SEQ ID NO.4,

SEQ ID NO.4：5’-GCCATGTCGTTCTGTGAGCCAAGG-3’

wherein, the last two bases of the upstream primer of the sequence shown in SEQ ID NO.2 are modified by sulfo.

7. The single cell transcriptome sequencing method of any one of claims 1 to 6, wherein: in the Reverse transcription, 1mL of reaction solution comprises 10% Triton-X10. mu.L, 0.1M DTT 125. mu. L, RnaseOUT 75. mu.L, 10Mm eachDNTPS 50. mu.L, 5 XT buffer 400. mu.L, Super Script II Reverse Transcriptase 75. mu.L, Template Switch Oligo 20. mu. L, Rnase-free H₂O265. mu.L; the reaction conditions of reverse transcription are 30min at 37 ℃, 60min at 65 ℃ and standby at 4 ℃.

8. The single cell transcriptome sequencing method of claim 7, wherein: the Template SwitchOligo has a sequence shown in SEQ ID NO.5,

SEQ ID NO.5：5’-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3’

in the Template Switch Oligo of the sequence shown in SEQ ID NO.5, the penultimate bases 2 and 3 are riboguanoside modified, and the last base is LNA modified.

9. The single cell transcriptome sequencing method of any one of claims 1 to 6, wherein: in the microbeads with the labels, the labels are primer sequences with poly T random sequences, the primer sequences are sequences shown in SEQ ID NO.6, and the 5 'end and the 3' end of the primer sequences are respectively provided with poly T tails; two sections of barcode are arranged between the 3 'end of the primer sequence and the poly T tail of the 3' end, the first section of barcode is cell barcode and is used for marking single cells, and the second section of barcode is used for marking transcripts;

SEQ ID NO.6：5’-AAGCAGTGGTATCAACGCAGAGTAC-3’；

preferably, the tag sequence is a sequence shown in SEQ ID NO.7,

SEQ ID NO.7：5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’

in the sequence shown in SEQ ID NO.7, the first 12 Ns are the random sequence of the first segment of barcode, and the last 8 Ns are the random sequence of the second segment of barcode.

10. A kit for high throughput single cell transcriptome sequencing, characterized in that: comprises a SMART conversion template, a cDNA 3' end marking primer, a PCR amplification enrichment primer group, a Splint oligo and a micro-bead with a label;

the SMART conversion template is a sequence shown in SEQ ID NO.5 and is used for introducing base at the 5 'end of the first strand cDNA of the transcript of mRNA so as to be distinguished from a 3' end sequence;

the cDNA3 'end marker primer is a sequence shown in SEQ ID NO.1 and is used for obtaining a 3' end marker sequence in amplification;

the upstream primer of the PCR amplification enrichment primer group is a sequence shown by SEQ ID NO.2, and the downstream primer is a sequence shown by SEQ ID NO.3, and is used for carrying out PCR amplification enrichment on the 3' end marker sequence;

the Splint olig is a sequence shown as SEQ ID NO.4 and is used for cyclizing fragmented nucleic acid;

in the microbead with the tag, the tag sequence is shown in SEQ ID NO.7, wherein the first 12N are random sequences of a first section of barcode, and the last 8N are random sequences of a second section of barcode.

Technical Field

The application relates to the field of single cell sequencing, in particular to a high-throughput single cell transcriptome sequencing method and a kit.

Background

The disease occurrence mechanism and their detection and prevention are mainly analyzed by detecting the variation of cellular genome and abnormal expression of transcriptome. The single cell sequencing technology can reveal the complex heterogeneity of cells in tissues, and provide accurate information for diagnosis and treatment of diseases. Due to the problems of processing flux, cost and the like of the single cell sequencing technology, a lot of large-scale research work cannot be carried out, and the problems can be well solved by combining the microfluidic technology and the single cell sequencing technology; meanwhile, the single-molecule sequencing technology can avoid errors caused by nucleic acid amplification, and has great application prospect and requirements in basic scientific research and clinical medical research.

The instrument throughput has been increased to a point where hundreds of single cells can be analyzed at one time with a fully automated system of C1 single cells, introduced by Fludigm corporation in 2014. The advent of this system has been the focus of attention of the world-wide group of single-cell research topics. However, the Fluidigm C1 system, which uses micro valves to distinguish single cells, can only do 938 cells at most due to the limited chambers on the chip and low throughput, and is costly.

The Wafergen system uses a micropore chip and a mechanical arm to realize the separation of single cells, the number of micropores is 72, and compared with the Fluidigm C1 system, the Wafergen system flux can reach 1000-; however, the flux is still low, and the use requirement of large-scale research work cannot be well met; in addition, the wafer system has a large reaction system, so that a large amount of reagents are consumed, the cost is high, and the sample loss amount is large.

Compared with a Fludigm C1 system and a Wafergen system, the 10 Xgenomics and Dolomite flux based on the droplet microfluidic principle is higher, microbeads with labels and single cells are wrapped in one droplet by utilizing a microfluidic chip, the single cells are separated and marked, and tens of thousands of cells can be analyzed simultaneously. However, when the Dolomite system breaks emulsion, the no-load primers on the microbeads are easy to combine with free mRNA, so that the cross contamination is large; the formed library cannot be directly cyclized and cannot be directly connected with a BGISeq-500 platform; additional library transformations are required, introducing data bias, and reducing the effective utilization rate of data. The 10 Xgenomics has high requirements on samples and high cost; the formed library can not be directly cyclized, and can not be directly connected with a BGISeq-500 platform; likewise, additional library transformations are required, introducing data bias, reducing the effective use of data.

In addition, there are several single cell separation techniques, such as oral pipettes, flow cytometry, etc. However, the mouth pipette is micro-operated, the method has low flux, long time consumption and complicated process. The flow cytometer has the advantages of large sample demand, accurate control, damage to cells and high requirement on subsequent library construction.

In general, the existing single cell isolation or single cell transcriptome sequencing technology is limited by the problems of flux, cost and the like, and a lot of large-scale research work cannot be carried out. On the one hand, low throughput is more difficult to achieve in detail analysis of cellular heterogeneity and different cell subsets. On the other hand, the increase of the double-packet rate caused by the increase of the flux becomes a bottleneck of the current high-flux platform.

Disclosure of Invention

It is an object of the present application to provide a novel high throughput single cell transcriptome sequencing method and kit.

The application specifically adopts the following technical scheme:

the first aspect of the application discloses a high-throughput single-cell transcriptome sequencing method, which comprises the steps of wrapping single cells and microbeads with labels in one droplet by using a droplet generation system, and carrying out reverse transcription in the droplet to obtain first-strand cDNA.

It should be noted that, the single-cell transcriptome sequencing method of the present application, which adopts a droplet technology to separate single cells, has a throughput of about 9000 cells in a single experiment, which is equivalent to the highest throughput of 10 × genomics in the current market. The method realizes and provides reverse transcription in the liquid drop through research, and has the advantages that firstly, mutual pollution of microbeads after the liquid drop demulsification is effectively reduced, and the ratio of effective data is improved; secondly, reverse transcription is carried out in the liquid drops, and the required reagent is relatively less, so that the cost is low; lays a foundation for large-scale single cell research work.

Preferably, reverse transcription employs SMART template switching technology to introduce bases at the 5 'end of the first strand cDNA of the transcript of mRNA to distinguish from 3' end sequences.

The single-cell transcriptome sequencing method obtains the first-strand cDNA by a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; the method not only avoids the deviation caused by multiple amplifications in the library transformation process, but also realizes the combination of the droplet microfluidic platform and the BGISeq-500 sequencing platform, greatly simplifies and facilitates large-scale single cell sequencing.

Preferably, the single-cell transcriptome sequencing method further comprises performing SMARTPCR pre-amplification on the SMART template conversion product by using SMART PCR PRIMER, and obtaining a 3' end marker sequence by amplification; wherein SMARTPCRPRIMER is a specific primer at the 3' end.

It should be noted that the purpose of SMARTPCR pre-amplification is to amplify a target product at the 3 'end, so as to facilitate specific amplification of a labeled 3' sequence in the subsequent library construction. In one implementation of the present application, a specific tag, i.e., the sequence shown in SEQ ID NO.5, is added to the 3' end at the same time as the reverse transcription of the switch template.

Preferably, SMARTPCRPRIMER is the sequence shown in SEQ ID NO. 1;

SEQ ID NO.1：5’-AAGCAGTGGTATCAACGCAGAGT-3’。

it should be noted that SMARTPCRPRIMER of the sequence shown in SEQ ID NO.1 is only a specific implementation manner in the examples of the present application, and it is understood that other 3' end specific primers can be used under the basic concept of the present application, and are not limited herein.

Preferably, the single-cell transcriptome sequencing method further comprises the steps of performing PCR amplification enrichment on the 3 'end marker sequence by using a specific primer group aiming at the 3' end marker sequence, then performing fragment selection on the PCR amplification enrichment product, and directly sequencing by using a BGISeq-500 platform after cyclizing the selected fragment.

It should be noted that PCR amplification enrichment is a step in the construction of Tn5 library, and after PCR amplification enrichment and fragment selection, a more accurate sequencing library can be obtained, thereby improving sequencing efficiency and quality.

Preferably, the specific primer group adopted in the PCR amplification enrichment has an upstream primer with a sequence shown by SEQ ID NO.2 and a downstream primer with a sequence shown by SEQ ID NO.3, and the 5' end of the upstream primer has phosphorylation modification;

SEQID NO.2：

5’-GAACGACATGGCTACGATCCGACTTAAGCAGTGGTATCAACGCAG AGTAC-3’

wherein, the last two bases of the upstream primer of the sequence shown in SEQ ID NO.2 are modified by sulfo;

SEQ ID NO.3：

5’-TGTGAGCCAAGGAGTTGTTGTCTTCGTCTCGTGGGCTCGG-3’；

the Splint oligo adopted for fragment cyclization is a sequence shown in SEQ ID NO.4,

SEQ ID NO.4：5’-GCCATGTCGTTCTGTGAGCCAAGG-3’。

it should be noted that the primers with sequences shown in SEQ ID nos. 2 and 3 and the Splint oligo with sequence shown in SEQ ID No.4 are also a specific implementation manner in the embodiment of the present application, and it is understood that other specific primer sets or Splint oligos can be adopted under the basic concept of the present application, and are not limited herein.

It should be noted that, in the downstream primer of the sequence shown in SEQ ID No.3, in order to distinguish samples from different sources, an index sequence of about 10bp may also be introduced into the primer sequence, for example, in an implementation manner of the present application, the downstream primer of the following sequence is specifically adopted:

5’-TGTGAGCCAAGGAGTTGTTGTCTTCNNNNNNNNNNGTCTCGTGG GCTCGG-3’。

preferably, in the reverse transcription, 1mL of the reverse transcription reaction solution comprises: 10 μ L of 10% Triton-X, 125 μ L, RnaseOUT 75 μ L of 0.1M DTT, 50 μ L of 10Mm each dNTPs, 400 μ L of 5 × RTbuffer, 75 μ L of Super Script IIReverse Transcriptase, 20 μ L, Rnase-free H of Template Switch Oligo₂O265. mu.L; the reaction conditions of reverse transcription are 30min at 37 ℃, 60min at 65 ℃ and standby at 4 ℃.

It should be noted that the single-cell transcriptome sequencing method of the present application provides, in particular, a reverse transcription reaction system suitable for the present application, which has not been performed in vitro or in droplets in the prior art.

Preferably, the Template Switch Oligo has the sequence shown in SEQ ID NO.5,

SEQ ID NO.5：5’-AAGCAGTGGTATCAACGCAGAGTGAATGGG-3’

in the Template Switch Oligo of the sequence shown in SEQ ID NO.5, the penultimate bases 2 and 3 are riboguanoside modified, and the last base is LNA modified.

It should be noted that a Template, namely a SMART conversion Template, and a Template, namely a Template of a SMART Switch shown in SEQ ID No.5, are also a specific implementation manner in the embodiments of the present application, and it is to be understood that, under the basic concept of the present application, other SMART conversion templates may also be used, and are not specifically limited herein.

Preferably, in the labeled microbeads, the label is a primer sequence with a poly T random sequence, the primer sequence is a sequence shown in SEQ ID NO.6, and the 5 'end and the 3' end of the primer sequence are respectively provided with a poly T tail; two sections of barcodes are arranged between the 3 'end and the polyT tail of the 3' end of the primer sequence, the first section of barcode is cellbarcode and is used for marking single cells, and the second section of barcode is used for marking transcripts;

SEQ ID NO.6：5’-AAGCAGTGGTATCAACGCAGAGTAC-3’。

it should be noted that, the primer sequence with poly T random sequence in the bead can hold mRNA released after cell lysis; wherein, the first section of barcode is cell barcode, is a random sequence, is generally represented by J12 and is used for marking single cells, and the cellbarcodes on the same microbead are the same; a second segment of barcode, also a random sequence, generally designated N8, also known as UMI, is used to label transcripts that differ in UMI on the same microbead.

Preferably, the tag sequence is a sequence shown in SEQ ID NO.7,

SEQ ID NO.7：

5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’

in the sequence shown in SEQ ID NO.7, the first 12 Ns are the random sequence of the first segment of barcode, and the last 8 Ns are the random sequence of the second segment of barcode.

It should be noted that the tag sequence of the sequence shown in SEQ ID No.7 is also a specific implementation manner in the embodiments of the present application, and it is understood that other tag sequences can also be used under the basic concept of the present application, and are not limited herein.

The other side of the application discloses a kit for sequencing a high-throughput single-cell transcriptome, which comprises a SMART conversion template, a cDNA 3' end marker primer, a PCR amplification enrichment primer group, a Splint oligo and a microbead with a label; wherein the SMART conversion template is a sequence shown in SEQ ID NO.5 and is used for introducing base at the 5 'end of the first strand cDNA of the transcript of mRNA so as to be distinguished from a 3' end sequence; the cDNA3 'end marker primer is a sequence shown in SEQ ID NO.1 and is used for obtaining a 3' end marker sequence in amplification; the upstream primer of the PCR amplification enrichment primer group is a sequence shown by SEQ ID NO.2, and the downstream primer is a sequence shown by SEQ ID NO.3, and is used for carrying out PCR amplification enrichment on a 3' end marker sequence; splint oligo is a sequence shown in SEQ ID NO.4 and is used for circularizing fragmented nucleic acid; in the microbeads with the labels, the sequence of the labels is shown in SEQ ID NO.7, wherein the first 12N are random sequences of a first section of barcode, and the last 8N are random sequences of a second section of barcode.

It should be noted that the kit of the present application is actually the reagents used in the high-throughput sequencing method for single-cell transcriptome in the embodiment of the present application, and the reagents are combined into the kit, so that the high-throughput sequencing for single-cell transcriptome can be conveniently performed. It is to be understood that the kit may further include a reaction solution, an enzyme, and the like required in each reaction system for convenience of use, and is not particularly limited herein.

The beneficial effect of this application lies in:

according to the high-throughput single-cell transcriptome sequencing method, the single experiment flux can reach about 9000 cells, which is equivalent to the highest flux of 10 × genomics in the current market, and the microbeads subjected to liquid drop demulsification have small mutual pollution, so that the ratio of effective data is increased; in addition, the method carries out reverse transcription in the liquid drop, and has relatively less required reagent and low cost; lays a foundation for large-scale single cell research work. In a preferred scheme of the application, the reverse transcription adopts a SMART template conversion technology, and the product can be directly used for the subsequent Tn5 library construction and BGISeq-500 platform sequencing without library transformation; not only avoids the deviation caused by multiple amplifications, but also realizes the connection of the droplet microfluidic platform and the BGISeq-500 sequencing platform, greatly simplifies and facilitates large-scale single cell sequencing.

Drawings

FIG. 1 is an explanatory diagram of a case where a droplet generating system generates droplets in a chip in an embodiment of the present application;

FIG. 2 is a graph showing the observation results of 1 μ L of beads-coated droplets under a 4-fold mirror in the examples of the present application;

FIG. 3 is a graph showing the results of detecting a reverse transcription product produced in a droplet using 2100 in the example of the present application;

FIG. 4 shows the 2100 detection results of the fragment selection products of PCR amplification-enriched products after PCR amplification enrichment of the marker sequences at the 3' end by the specific primer set during the Tn5 library construction in the present example.

Detailed Description

High-throughput single-cell transcriptome sequencing is of great significance in basic scientific research and clinical medical research. Although high throughput single cell transcriptome sequencing protocols such as the C1 system, the Wafergen system, the 10 × genomics and Dolomite are available abroad, these protocols have some disadvantages; in addition, the high-throughput single cell sequencing technology is still blank in China at present. The application first establishes a sequencing platform of a high-throughput single-cell transcriptome. Combining droplet microfluidics and single cell RNA sequencing technology SMART-Seq, using labeled microbeads to capture mRNA, simultaneously performing reverse transcription experiments in droplets, performing high-throughput label separation and obtaining the 3 'ends of thousands of single cell mRNA, distinguishing the 3' end sequences and the 5 'end sequences of the mRNA by adding a plurality of basic groups on a TSO sequence, skillfully designing specific primers, and amplifying and enriching the products with the labeled 3' ends. The cost is reduced while the single cell flux is improved, the single experiment cell flux is 2000-9000, and the cost is reduced to 1-0.35 yuan per cell. Reverse transcription is carried out in liquid drops, so that the pollution rate is reduced and the operation is simplified; simplifies the library building process and realizes the direct use with a BGISeq-500 sequencer.

The cell flux is improved, the cost is reduced, the flux is equivalent to the highest 10 × genomics in the current market, about 9000 cells can be achieved in a single experiment, the reagent cost is one third of the reagent cost of 10 × genomics, and the labor cost is greatly reduced. Makes it possible to carry out single cell sequencing research on a large scale. Compared with the existing method, the method has the advantages that the reverse transcription is carried out in the liquid drop, the mutual pollution of the microbeads after the liquid drop demulsification is effectively reduced, and the ratio of effective data is improved. In addition, the application designs a library construction system and a sequencing scheme aiming at the BGISeq-500, compared with other commercial platforms, the library constructed by the application can be directly used for BGISeq-500 sequencing, and the data error caused by multiple PCR in library conversion is reduced.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.