A single-cell sequencing method for barcode droplets

Abstract

The present invention belongs to the technical field of single-cell sequencing methods, and particularly relates to a single-cell sequencing method for barcode droplets, which is a single-cell sequencing based on barcode droplets of nano- and micron-sizes for single cells and subcellular structures, and is suitable for full-length mRNA sequencing and random primer-based mRNA sequencing.

Description

Technical Field

[0001] The present invention belongs to the technical field of single-cell sequencing methods, and particularly relates to a single-cell sequencing method for barcode droplets. Background Art

[0002] The 10X technology is based on the principle of droplet capture. By encapsulating a single magnetic bead and one cell in a droplet, a reaction occurs between the magnetic bead and the cell: after the cell lyses, mRNA is released, and the primer tail on the magnetic bead can specifically bind to the mRNA, thus achieving the purpose of capture. At the same time, through the cell barcode sequence and UMI sequence carried by the magnetic bead primer itself, different transcripts of different cells can be labeled and distinguished. Because the primer fragment on the magnetic bead carries mRNA sequence information, through reverse transcription or replication amplification on this basis, all transcriptional information will carry the cell barcode and UMI.

[0003] The BD technology is based on the principle of microwell capture. In a microwell, one cell matches with a magnetic bead labeled with a barcode; the cell lyses, and the mRNA is captured by the barcode sequence on the magnetic bead; then the magnetic bead that has captured the mRNA is recovered, and cDNA synthesis, library construction, and sequencing are completed; finally, through data analysis, the mRNA sequences of different cells are distinguished according to the barcode sequence.

[0004] The 10X technology adopts the principle of droplet capture, which can integrate single-cell sorting, amplification, and library construction. The capture efficiency of single cells can be as high as 65%, and the probability of capturing multiple cells in a single droplet is extremely low. However, using 10x sequencing can only obtain the transcriptional information at the 3' end, and the information at the 5' end is lost; 10X sequencing has very high requirements for the quality of the sample. For a single sample, the starting amount of cells should reach 10 5 -10 6 cells, and the number of live cells needs to be above 80%.

[0005] The BD technology is based on the principle of microwell capture. In a microwell, each transcript in a single cell is specifically labeled with a molecular tag using a molecular label, which can achieve absolute quantification of the gene expression profile at the single-cell level. At the same time, each cell will also be labeled with a specific cell tag, making high-throughput parallel library construction possible. BD sequencing has its own unique single-cell separation technology, and 100 - 10,000 single-cell libraries can be prepared in a single experiment. Users can customize primers according to their needs, concentrating the detection range on target genes, which is convenient for significantly reducing the subsequent sequencing cost. The BD technology can achieve mixed capture of multiple samples, and the number of wells in the microwell plate can be as high as 220,000; it has an imaging system and can observe cell-related information. However, compared with 10X, the BD technology has disadvantages such as a long library construction cycle, high cost, and low throughput.

[0006] The 10X and BD technologies are not applicable to single-cell sequencing of micropore plate chips for smaller microdroplets such as platelets, exosomes, bacteria, viruses, or mycoplasmas.

[0007] Currently, the main consumables for single-cell sequencing are 10X and BD. However, both are based on the barcode-encoded magnetic bead system. The biggest drawback of magnetic beads is that the number of barcodes bound to a single magnetic bead is limited. To achieve an effective sequencing depth, their volume must be at least 30 μm (the barcode upper limit is 10 6 times / magnetic bead). When performing single-cell sequencing of smaller cells and organelles such as platelets, exosomes, bacteria, viruses, mycoplasmas, and mitochondria on a micropore plate chip, the corresponding magnetic beads and micropores will be smaller. However, the number of barcodes that can be coupled to microspheres of this volume cannot support the sequencing depth required. Therefore, it is necessary to develop microdroplets with a smaller volume but capable of loading enough barcodes for single-cell sequencing.

[0008] Therefore, it is particularly important to develop a method for single-cell sequencing applicable to smaller microdroplets. Summary of the Invention

[0009] In view of the above problems, the purpose of the present invention is to provide a method for single-cell sequencing of barcode microdroplets.

[0010] The technical content of the present invention is as follows:

[0011] The present invention provides a method for single-cell sequencing of barcode microdroplets, including the following steps:

[0012] 1) Preparation and characteristics of barcode microdroplets

[0013] 1.1) Establishment of barcode microdroplets

[0014] The barcode microdroplets are divided into multiple large droplet libraries for library construction. The microdroplets are separated using a microfluidic chip system, including barcode library 1, barcode library 2, barcode library 3,... and so on;

[0015] The barcode library 1 includes 100 subdivisions, namely barcode1-1, barcode1-2,..., barcode1-100);

[0016] The barcode1-1 prepares 10 n droplets (n > 1), and each droplet contains no less than 10 6Root barcode1-1. Similarly, prepare 100 droplet libraries (barcode1-1 - barcode1-100) of subdivided barcodes "1-n", and mix them into droplet library barcode library 1, which contains 10 n+2 droplets;

[0017] Similarly, prepare other barcode "N-n" droplet libraries N, such as barcode library 2, barcode library 3, etc.;

[0018] The number of barcode microdroplet libraries depends on the storage capacity required for detection, but is greater than 2. The principle is that the combination of the storage capacities of all libraries meets the number of sequencing cells. For example, if the number of sequencing cells is 10 5 , then barcode1 library × barcode2 library × barcode "n" library ≥ 10 5 ;

[0019] For example, if the conventional minimum detection requirement is 10 6 , then barcode library 1 × barcode library 2 × barcode library 3 × barcode library n ≥ 10 6 , so if there are only 2 libraries, then the storage capacity of each microdroplet library is not less than 10 3 , that is, from barcode1-1 to barcode1-1000 and from barcode2-1 to barcode2-1000.

[0020] 1.2) When preparing and separating microdroplets, take 1 droplet from the mixed library 1 and several subsequent libraries respectively. Mix the multiple taken droplets with the amplification mix to form an amplified droplet, and amplify according to Figure 1 ;

[0021] After amplification, demulsify the product, collect the cDNA and construct a library, and perform sequencing and analysis;

[0022] 2) Construct a library and sequence the full-length mRNA and primer mRNA;

[0023] The library construction and sequencing of the full-length mRNA described in step 2) include the following steps:

[0024] Use Barcodes containing multiple segments to label cells, and a UMI linked to the Barcode to label transcripts;

[0025] The Barcode is composed of 2 to 6 segments of sequences, preferably 2 - 3 segments, which are the upstream Barcode, the middle Barcode, and the downstream Barcode respectively;

[0026] The 3'-end of the upstream Barcode is composed of 3 to 5 Gs for RACE reverse transcription, and the 5'-end is an adapter sequence for connecting the sequencing adapter;

[0027] The sequence of the upstream Barcode is: 5'-adapter+jjjjjj+GGG-3';

[0028] Taking 3 Barcodes as an example, the 3'-end of the middle Barcode is a polyT sequence for the reverse transcription of mRNA, and the 5'-end is a linker;

[0029] The sequence of the middle Barcode is: 3'-polyT+jjjjjj+linker-5';

[0030] The 3'-end of the downstream Barcode is an anti-linker, which is complementary to the linker of the previous middle Barcode. The middle is a cell label sequence, and the 5'-end is a UMI sequence;

[0031] The sequence of the downstream Barcode is: 5'-nnnnnnnnnnnnnn+jjjjjj+anti-linker-3'.

[0032] In the above sequences, j is the cell label nucleic acid sequence cell label, and n is the UMI;

[0033] The UMI is composed of 4 to 10 randomly arranged nucleotides. After the reverse transcription of mRNA, it enters the library, and each mRNA randomly attaches a UMI. Therefore, different UMIs can be counted, and finally the counting of mRNA can be achieved;

[0034] The Barcode is composed of multiple random bases, and its combination mode is 4 n , and its sequence length (i.e., n) is determined according to the sequencing depth. Multiple Barcodes are combined to encode a cell. Taking 3 Barcodes as an example, the combination number is 4 n ×4 n ×4 n (Barcode1×Barcode2×Barcode3), and its combination number is generally not less than 10 6 to meet the minimum single-cell sequencing depth requirement;

[0035] 3) Use the transposase method or the random primer method to amplify the cDNA to obtain a double-stranded DNA library, and use the Illumina high-throughput sequencing system for sequencing.

[0036] The beneficial effects of the present invention are as follows:

[0037] The single-cell sequencing method of barcode droplets of the present invention is based on barcode droplets of nano- and micron-sizes for single-cell sequencing of single cells and subcellular structures, and is suitable for full-length mRNA sequencing and random primer-based mRNA sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the schematic diagram of the mRNA capture principle described in the present invention;

[0039] Figure 2 It is the schematic diagram of the establishment of the barcode droplet library of the present invention;

[0040] Figure 3 It is the schematic diagram of the acquisition of single-cell amplified droplets of the present invention;

[0041] Figure 4 It is the schematic diagram of the basic principle of library construction by the transposase method in Example 1;

[0042] Figure 5 It is the schematic diagram of the principle of library construction by the random primer method in Example 2;

[0043] Figure 6 It is the schematic diagram of the double-barcode library construction mode in Example 2;

[0044] Figure 7 It is the schematic diagram of the mixing of the two libraries in Example 2;

[0045] Figure 8 It is the schematic diagram of the mixing of the library and cells in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be further described in detail below through specific implementation cases and the description of the drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the appended claims of this application.

[0047] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0048] Example 1

[0049] A single-cell sequencing method of barcode droplets

[0050] 1) Preparation and characteristics of barcode droplets

[0051] 1.1) Establishment of barcode droplets

[0052] The barcode droplets are divided into multiple large droplet libraries for library construction. A microfluidic chip system is used to separate the droplets, including barcode library 1, barcode library 2, barcode library 3, and so on;

[0053] The said barcode library 1 includes 100 subdivisions (barcode1-1, barcode1-2,...., barcode1-100);

[0054] 10 droplets are prepared from the said barcode1-1, 4 and each droplet contains no less than 10 6 barcode1-1. Similarly, a droplet library of 100 subdivisions of barcode1 (barcode1-barcode100) is prepared, and they are mixed into droplet library barcode library 1, which contains 10 6 droplets;

[0055] Similarly, other barcode droplet libraries are prepared, such as barcode library 2, barcode library 3, etc., as Figure 2 shown;

[0056] 1.2) When preparing and separating the droplets, 1 droplet is taken from each of the mixed libraries 1, 2, and 3 to form an amplified droplet, as Figure 3 shown, and then the amplified droplet is amplified according to Figure 1 ;

[0057] Finally, ethanol is used to demulsify and precipitate the DNA of the product. After washing, the cDNA is collected and library construction is carried out for sequencing and analysis;

[0058] 2) Library construction and sequencing are carried out on the full-length mRNA;

[0059] Barcodes containing multiple fragments are used to label cells, and a UMI linked to the Barcode is used to label transcripts;

[0060] The said Barcode is composed of a 3-segment sequence, namely the upstream Barcode, the middle Barcode, and the downstream Barcode;

[0061] The 3' end of the upstream Barcode is 3 Gs, which are used for RACE reverse transcription, and the 5' end is an adapter sequence, which is used to connect the sequencing adapter;

[0062] The sequence of the upstream Barcode is: 5'-adapter+jjjjjj+GGG-3';

[0063] The 3'-end of the middle Barcode is a polyT sequence for the reverse transcription of mRNA, and the 5'-end is a linker;

[0064] The sequence of the middle Barcode is: 3'-polyT + jjjjjj + linker-5';

[0065] The 3'-end of the downstream Barcode is an anti-linker, which is complementary to the linker of the previous middle Barcode. The middle is the cell label sequence, and the 5'-end is the UMI sequence;

[0066] The sequence of the downstream Barcode is: 5'-nnnnnnnnnnnnnn + jjjjjj + anti-linker-3'.

[0067] In the above sequences, j is the cell label and n is the UMI;

[0068] The UMI is composed of 4 to 10 randomly arranged nucleotides. After the reverse transcription of mRNA, it enters the library, and each mRNA randomly attaches a UMI. Therefore, different UMIs can be counted, and finally the counting of mRNA can be achieved;

[0069] The Barcode is composed of multiple random bases, and its combination mode is 4 n , and the length of its sequence (i.e., n) is determined according to the sequencing depth. Multiple Barcodes are combined to code a cell, and the combination number is 4 n ×4 n ×4 n (Barcode1 × Barcode2 × Barcode3), and its combination number is generally not less than 10 6 to meet the minimum single-cell sequencing depth requirement;

[0070] 3) Use the transposase method to amplify cDNA to obtain a double-stranded DNA library, as Figure 4 shown, and use the Illumina high-throughput sequencing system for sequencing.

[0071] Example 2

[0072] A single-cell sequencing method for barcode droplets ( Figures 6 - 8 )

[0073] 1) Preparation and characteristics of barcode droplets

[0074] 1.1) Establish barcode droplets

[0075] The barcode droplets are divided into multiple large droplet libraries for library construction, and a microfluidic chip system is used to separate the droplets, including barcode library 1 and barcode library 2;

[0076] The said barcode library 1 includes 100 subdivisions (barcode1-1, barcode1-2,...., barcode1-1000);

[0077] 10 droplets (n≥3) are prepared from the said barcode1-1, and each droplet contains no less than 10 n barcode1-1. Similarly, a droplet library of 1000 subdivisions of barcode1 (barcode1-barcode1000) is prepared, and they are mixed into droplet library barcode library 1, which contains 10 6 droplets; n+3 n+3

[0078] Similarly, a barcode2 droplet library is prepared, as Figure 2 shown;

[0079] 1.2) When preparing and separating the droplets, 1 droplet is taken from each of the mixed libraries 1 and 2 to form an amplified droplet, and amplification is carried out according to Figure 1 ;

[0080] Finally, the product is demulsified, the cDNA is collected and library construction is carried out, and sequencing and analysis are carried out;

[0081] 2) Library construction and sequencing are carried out on the full-length mRNA and primer mRNA;

[0082] The library construction and sequencing of the full-length mRNA in step 2) include the following steps:

[0083] Barcodes containing multiple fragments are used to label cells, and a UMI linked to the Barcode is used to label transcripts;

[0084] The said Barcode has 2 segments, namely the upstream Barcode (Barcode1) and the downstream Barcode (Barcode2);

[0085] The 3' end of the upstream Barcode is 3 Gs, which are used for RACE reverse transcription, and the 5' end is an adapter sequence, which is used to connect the sequencing adapter;

[0086] The sequence of the upstream Barcode is: 5'-adapter+jjjjjj+GGG-3’;

[0087] The downstream Barcode 3' end is a polyA sequence for mRNA capture and reverse transcription. The middle is the celllabel sequence, and the 5' end is the UMI sequence;

[0088] The sequence of the downstream Barcode is: 5'-nnnnnnnnnnnnnn+jjjjjj+AAAAAAA-3’.

[0089] In the said sequence, j is the cell lable and n is the UMI;

[0090] The UMI is composed of 4 to 10 randomly arranged nucleotides. After mRNA reverse transcription, it enters the library, and each mRNA randomly attaches a UMI. Therefore, different UMIs can be counted, and finally the counting of mRNA is achieved;

[0091] The Barcode is composed of multiple random bases, and its combination mode is 4 n , and its sequence length (i.e., n) is determined according to the sequencing depth. The combination of multiple Barcodes encodes a cell, and the combination number is 4 n ×4 n (Barcode1×Barcode2), and its combination number is generally not less than 10 6 to meet the minimum single-cell sequencing depth requirement;

[0092] 3) Use the random primer method to amplify cDNA to obtain a double-stranded DNA library, as Figure 5 shown, and use the Illumina high-throughput sequencing system for sequencing.

Claims

1. A single-cell sequencing method for barcode droplets, characterized in that It includes the following steps: 1) Preparation and characteristics of barcode droplets 1.1) Establishment of barcode droplets The barcode droplets are divided into multiple large droplet libraries for library construction. The microfluidic chip system is used to separate the droplets, including barcode library 1, barcode library 2, barcode library 3, …, and so on; The barcode library 1 includes 100 subdivisions, namely barcode1-1, barcode1-2,...., barcode1-100; 10 droplets are prepared from the said barcode1-1, where n > 1, and each droplet contains no less than 10 n barcode1-1s. Similarly, 100 droplet libraries barcode1-1-barcode1-100 of the subdivided barcode "1-n" are prepared, and they are mixed into a droplet library barcode library 1, which contains 10 6 droplets; n+2 ​ Similarly, other barcode "N-n" droplet libraries N are prepared; 1.2) When preparing and separating the droplets, 1 droplet is taken from the mixing library 1 and the subsequent n libraries respectively, n≥1. The multiple taken droplets are mixed with the amplification mix to form an amplification droplet; After amplification, finally, the product is demulsified, the cDNA is collected and library construction is carried out, and sequencing and analysis are performed; 2) Library construction and sequencing of full-length mRNA and primer mRNA: Barcodes containing multiple fragments are used to label cells, and a UMI linked to the Barcode is used to label transcripts; The Barcode consists of 3 sequences, namely the upstream Barcode, the middle Barcode, and the downstream Barcode; The 3' end of the upstream Barcode has 3-5 Gs for RACE reverse transcription, and the 5' end has an adapter sequence for connecting the sequencing adapter; The sequence of the upstream Barcode is: 5'-adapter+jjjjjj+GGG-3’; The j is the cell-labeling nucleic acid sequence cell lable; The 3' end of the middle Barcode has a polyT sequence for reverse transcription of mRNA, and the 5' end has a linker; The sequence of the middle Barcode is: 3'-polyT+jjjjjj+linker-5’; The j is cell lable; The 3' end of the downstream Barcode has an anti-linker, which is complementary to the linker of the previous middle Barcode. The middle is cell lable, and the 5' end has a UMI sequence; The sequence of the downstream Barcode is: 5'-nnnnnnnnnnnnnn++jjjjjj+anti-linker-3’; The n is UMI; The UMI consists of 4-10 randomly arranged nucleotides. After mRNA reverse transcription, it enters the library, and each mRNA is randomly linked to a UMI. Therefore, different UMIs can be counted, and finally, the counting of mRNA is realized; 3) Amplify the cDNA using the transposase method or the random primer method to obtain a double-stranded DNA library, and perform sequencing using the Illumina high-throughput sequencing system.

2. The single-cell sequencing method of barcode droplets according to claim 1, characterized in that Step 2) The Barcode is composed of multiple random bases, and its combination method is 4 n , and its sequence length n is determined according to the sequencing depth. Multiple Barcodes are combined to encode a cell, and the combination number is not less than 10 6 to meet the minimum single-cell sequencing depth requirement.

Images