Method and device for detecting rare cells and application of method and device

Abstract

A method of detecting rare cells in a biological sample, comprising: providing single cells from a biological sample; providing magnetic beads; enclosing the plurality of single cells and the magnetic beads in a single droplet; based on the single cell nucleic acid sequence captured by the first capture sequence in the single droplet, constructing a DNA library, and carrying out sequencing and data analysis; and screening specific marker sequence information of a rare cell from the single cell nucleic acid sequence, and determining whether the rare cell exists in the biological sample.

Description

Method, device and application for detecting rare cells Technical Field

[0001] The present application relates to the field of cell detection technology, and in particular to a method, device and application thereof for detecting rare cells. Background Art

[0002] Rare cells refer to a type of cell with low abundance in biological fluids. These cells play an important role in growth, development, and disease progression. The isolation and detection of rare cells are of great significance in clinical research and disease monitoring. Rare cells are present in very small numbers in body fluids, such as 10 8 -10 9 of blood cells or every 10 6 -10 7 There is only one circulating tumor cell (CTC) among all normal white blood cells. Currently, the isolation and detection of these rare cells remain challenging. Relevant technologies for the isolation and detection of these rare cells include magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), and microfluidics.

[0003] Single-cell sequencing technology based on microfluidic systems is currently widely used. Compared with other technologies, the consumption of microscale droplet reagents in microfluidics is extremely small, and each droplet with stable morphology can be regarded as an independent microreactor, thus reducing cross-contamination in the experiment and facilitating the manipulation of the droplet; the larger specific surface area of ​​the droplet can also accelerate various reaction rates and heat transfer rates, so microfluidic technology is an ideal choice for single-cell research. However, the detection cost of traditional single-cell sequencing methods based on microfluidic systems is relatively expensive, and its cost increases linearly with the number of cells processed. In addition, traditional droplet microfluidics methods require the cell density to be adjusted to a very low level to avoid the occurrence of twins or multiples of cells in the droplets (i.e., more than one cell is encapsulated in a droplet). Such an experimental strategy requires higher reagent and labor costs when using this technology to detect rare cells in samples, and the probability of capturing rare cells is also extremely low.

[0004] Therefore, it is urgent to establish a high-throughput detection technology for rare cells with high sensitivity, strong specificity and low detection cost.

[0005] Summary of the Invention

[0006] The present application solves at least one of the problems of the prior art from the following aspects.

[0007] To this end, embodiments of the present application provide a method, device, and application thereof for detecting rare cells.

[0008] The first aspect of the present application provides a method for detecting rare cells in a biological sample, comprising: providing a single cell from a biological sample; providing magnetic beads, wherein the magnetic beads are connected to multiple oligonucleotide sequences, each oligonucleotide sequence comprising a barcode tag sequence, a unique molecular identifier sequence (UMI), and a first capture sequence; enclosing multiple single cells and the magnetic beads in a single droplet; constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet and performing sequencing and data analysis; and screening the single-cell nucleic acid sequence for specific marker sequence information of rare cells, and determining whether the rare cells are present in the biological sample.

[0009] In some embodiments, the biological sample is from a mammal, optionally the biological sample is from a pregnant female or a subject having or suspected of having a disease.

[0010] In some embodiments, the biological sample is at least one of peripheral blood, urine, saliva, or tissue.

[0011] In some embodiments, the rare cells include embryonic cells, stem cells, progenitor cells, cancer stem cells, and / or tumor cells.

[0012] In some embodiments, the rare cells include circulating tumor cells and / or fetal free cells, optionally, the fetal free cells are fetal nucleated red blood cells.

[0013] In some embodiments, 100,000 to 10 million, preferably 100,000 to 1 million, oligonucleotide sequences are linked to a single magnetic bead. Optionally, based on the same single magnetic bead, the barcode tag sequences in the multiple oligonucleotide sequences are the same, and based on different single magnetic beads, the barcode tag sequences are different from each other. Optionally, based on the same single magnetic bead, the unique molecular identifier sequences (UMIs) in the multiple oligonucleotide sequences are different from each other.

[0014] In some embodiments, the first capture sequence is selected from at least one of poly(dT) and a DNA capture sequence, preferably the first capture sequence is poly(dT), optionally, the first capture sequence is located at the 3' end of the oligonucleotide sequence on the magnetic bead, optionally, each of the oligonucleotide sequences further includes a linker sequence and / or a second capture sequence.

[0015] In some embodiments, constructing a DNA library based on the single-cell nucleic acid sequences captured by the first capture sequence in the single droplet includes: lysing the multiple single cells to obtain single-cell nucleic acid sequences, wherein the single-cell nucleic acid sequences are captured by the first capture sequence; destroying the droplet to release magnetic beads connected to the oligonucleotide sequence and the single-cell nucleic acid sequence; and constructing a DNA library based on the single-cell nucleic acid sequences.

[0016] In some embodiments, the single-cell nucleic acid sequence is an mRNA sequence, wherein constructing a DNA library based on the single-cell nucleic acid sequence further comprises: reverse transcribing the mRNA sequence into a DNA sequence and then constructing the DNA library.

[0017] In some embodiments, the single droplet contains 1-10, preferably 5-7, single cells.

[0018] In some embodiments, each droplet contains 1-10 magnetic beads, preferably 1 magnetic bead.

[0019] In some embodiments, the single droplet further comprises an Index carrier, wherein the Index carrier is connected to a plurality of Index sequences, and the Index sequence recognizes the second capture sequence in the oligonucleotide sequence of the magnetic bead in the single droplet and is captured by the second capture sequence.

[0020] In some embodiments, at least 100,000 Index sequences are connected to the Index vector.

[0021] In some embodiments, the droplet contains 5-15, preferably 8-10 Index carriers.

[0022] In some embodiments, determining whether the rare cell is present in the biological sample comprises:

[0023] The method further includes calculating, based on a single droplet, a ratio between the number of unique molecular identifier sequences (UMIs) corresponding to a single-cell nucleic acid sequence aligned to a specific marker sequence of the rare cell and the total number of all unique molecular identifier sequences (UMIs); comparing the ratio with a set ratio threshold; and determining the presence of the rare cell in the biological sample when the positive ratio is greater than the set ratio threshold.

[0024] In some embodiments, the set ratio threshold is 1-10%, preferably 1%-5%.

[0025] The second aspect of the present application provides an apparatus for detecting rare cells in a biological sample, comprising: a droplet sealing device for sealing a plurality of single cells and magnetic beads in a single droplet, wherein the plurality of single cells are from the biological sample, and the magnetic beads are connected to a plurality of oligonucleotide sequences, each oligonucleotide sequence comprising a barcode tag sequence, a unique molecular identifier sequence UMI, and a first capture sequence; a DNA library construction device connected to the droplet sealing device for constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet; a sequencing device connected to the DNA library construction device for sequencing the DNA library; and an analysis device connected to the sequencing device for data analysis, and screening specific marker sequence information of rare cells from the single-cell nucleic acid sequence, and determining whether the rare cells are present in the biological sample.

[0026] In some embodiments, the biological sample is from a mammal, optionally the biological sample is from a pregnant female or a subject having or suspected of having a disease.

[0027] In some embodiments, the biological sample is at least one of peripheral blood, urine, saliva, or tissue.

[0028] In some embodiments, the rare cells include embryonic cells, stem cells, progenitor cells, cancer stem cells, and / or tumor cells.

[0029] In some embodiments, the rare cells include circulating tumor cells and / or fetal free cells, optionally, the fetal free cells are fetal nucleated red blood cells.

[0030] In some embodiments, 100,000 to 10 million, preferably 100,000 to 1 million, oligonucleotide sequences are linked to a single magnetic bead. Optionally, based on the same single magnetic bead, the barcode tag sequences in the multiple oligonucleotide sequences are the same, and based on different single magnetic beads, the barcode tag sequences are different from each other. Optionally, based on the same single magnetic bead, the unique molecular identifier sequences (UMIs) in the multiple oligonucleotide sequences are different from each other.

[0031] In some embodiments, the first capture sequence is selected from at least one of poly(dT) and a DNA capture sequence, preferably the first capture sequence is poly(dT), optionally, the first capture sequence is located at the 3' end of the oligonucleotide sequence on the magnetic bead, optionally, each of the oligonucleotide sequences further includes a linker sequence and / or a second capture sequence.

[0032] In some embodiments, the DNA library construction device is further used to: lyse the multiple single cells to obtain single-cell nucleic acid sequences, wherein the single-cell nucleic acid sequences are captured by the first capture sequence; destroy the droplets to release magnetic beads connected to the oligonucleotide sequences and the single-cell nucleic acid sequences; and construct a DNA library based on the single-cell nucleic acid sequences.

[0033] In some embodiments, the single-cell nucleic acid sequence is an mRNA sequence, wherein the DNA library construction device is further used to: reverse transcribe the mRNA sequence into a DNA sequence and then construct the DNA library.

[0034] In some embodiments, the single droplet contains 1-10, preferably 5-7, single cells.

[0035] In some embodiments, each droplet contains 1-10 magnetic beads, preferably 1 magnetic bead.

[0036] In some embodiments, the single droplet further comprises an Index carrier, wherein the Index carrier is connected to a plurality of Index sequences, and the Index sequence recognizes the second capture sequence in the oligonucleotide sequence of the magnetic bead in the single droplet and is captured by the second capture sequence.

[0037] In some embodiments, at least 100,000 Index sequences are connected to the Index vector.

[0038] In some embodiments, the droplet contains 5-15, preferably 8-10 Index carriers.

[0039] In some embodiments, the analysis device is further configured to: calculate, based on a single droplet, a ratio between the number of unique molecular identifier sequences (UMIs) corresponding to a single-cell nucleic acid sequence mapped to a specific marker sequence of the rare cell and the total number of all unique molecular identifier sequences (UMIs); compare the ratio with a set ratio threshold; and determine the presence of the rare cell in the biological sample when the positive ratio is greater than the set ratio threshold.

[0040] In some embodiments, the set ratio threshold is 1-10%, preferably 1%-5%.

[0041] The third aspect embodiment of the present application proposes a method for diagnosing cancer, comprising: determining the presence of circulating tumor cells in a biological sample from a subject based on the method for detecting rare cells in a biological sample described in any embodiment of the first aspect embodiment of the present application; analyzing the number of circulating tumor cells, the expression of disease-related genes, and / or the regulation of open chromatin regions; and identifying whether the subject has a risk of suffering from a related disease, the development of the disease, and / or the prognosis of the disease based on the number of circulating tumor cells, the expression of disease-related genes, and / or the regulation of open chromatin regions.

[0042] The fourth aspect embodiment of the present application proposes a non-invasive fetal diagnosis method, comprising: determining the presence of fetal free cells in a biological sample from a pregnant woman based on the method for detecting rare cells in a biological sample described in any embodiment of the first aspect embodiment of the present application; and analyzing the genomic or transcriptomic information of the fetal free cells to determine the developmental status of the fetus and / or whether it suffers from a genetic disease.

[0043] In the embodiments of the present application, the genetic disease is a chromosomal disease, a single-gene genetic disease or a multi-gene genetic disease.

[0044] The embodiments of the present application achieve the following beneficial effects:

[0045] The method for detecting rare cells based on the super-loading strategy proposed in this application uses the super-Poisson distribution of cells to increase the starting cell input for a single experiment to the million level, allowing multiple cells to be encapsulated in a droplet; once a rare cell is encapsulated in a droplet, the magnetic beads in the droplet can capture the characteristic cell markers of the rare cell; by setting a certain threshold, the magnetic beads with the characteristic markers of rare cells can be screened out, and the presence of rare cells in the droplet can be proved at the same time. This method can convert the probability of detecting rare cells from a large number of background cells (one in ten thousand or even one in a million) into the probability of detecting rare cells based on the magnetic beads in the target droplet (a few in one), thereby greatly improving the detection rate and accuracy of rare cells, while significantly reducing the cost of rare cell detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0047] FIG1 is a flow chart of a method for detecting rare cells according to an embodiment of the present application;

[0048] FIG2 is a schematic diagram of the structure of a magnetic bead according to an embodiment of the present application;

[0049] FIG3 is a droplet generating device according to an embodiment of the present application;

[0050] FIG4 is a partial schematic diagram of a droplet generating device according to an embodiment of the present application;

[0051] FIG5 is a schematic diagram of a C4V2 slide according to an embodiment of the present application;

[0052] FIG6 is a schematic diagram of the structure of a device for detecting rare cells according to an embodiment of the present application;

[0053] Figures 7A and 7B show the ratio of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells (NIH / 3T3) to background cells (HEK293T) is 1:100 according to Example 1 of the present application. The experiment was repeated in two groups, with Figure 7A showing repeat one and Figure 7B showing repeat two.

[0054] Figures 8A and 8B show the ratio of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells (NIH / 3T3) to background cells (HEK293T) is 1:1000 according to Example 1 of the present application. The experiment was repeated in two groups, Figure 8A shows repeat one and Figure 8B shows repeat two.

[0055] Figures 9A and 9B show the ratio of the number of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells (NIH / 3T3) to background cells (HEK293T) is 1:10,000 according to Example 1 of the present application. The experiment was repeated in two groups, with Figure 9A showing repeat one and Figure 9B showing repeat two.

[0056] FIG10 is a Seurat cluster diagram of PBMC (background cells) and MCF-7 (breast cancer cells) according to Example 2 of the present application;

[0057] FIG11 is a diagram showing the specific expression of four marker genes (FOLR1, KRT18, BASP1, and C4BPB) in MCF-7 cells according to Example 2 of the present application;

[0058] FIG12 is a schematic diagram of a method for detecting rare cells based on a super-loading strategy according to an embodiment of the present application. DETAILED DESCRIPTION

[0059] The embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present disclosure, but should not be understood as limiting the present disclosure.

[0060] The present disclosure is based on the inventors' discovery and understanding of the following facts and problems: rare cells are present in very small amounts in body fluids. In related technologies, the enrichment and detection process of rare cells is cumbersome, and there are many challenges in terms of throughput and cost. In the single-cell sequencing technology based on microfluidic systems that has been widely used in recent years, ideally, each droplet should contain a cell and a magnetic bead, so that a microfluidic chip can generate tens or even millions of independent single-cell reaction spaces. However, in actual applications, the number of effective droplets on a chip is far less than this order of magnitude. The reason is that in order to ensure that the vast majority of droplets do not contain more than one cell or magnetic bead, it is necessary to control the number of droplets generated to far exceed the amount of cells or magnetic beads input. This results in most droplets not containing cells and / or magnetic beads, that is, the cells or magnetic beads follow a Poisson distribution in the droplets.

[0061] Therefore, in the case that the reagent cost of single-cell microfluidics technology is already relatively expensive, such a Poisson distribution strategy requires the generation of a large number of droplets, and most of these droplets are empty droplets, that is, they do not encapsulate any cells or magnetic beads; at the same time, if double or multi-cell situations occur during the detection process, that is, a single droplet encapsulates more than one cell, it will affect the accuracy of subsequent experimental data. Therefore, during the experiment, it is necessary to control the amount of cell input within a certain value to avoid double or multi-cell problems. Such a strategy causes the reagent cost and labor cost to gradually increase as the number of test samples increases. In addition, since the number of rare cells is extremely small, and this technology is based on a large number of background cells, by analyzing all single cells (including a large number of background cells and a very small number of rare cells) to identify rare cells, the probability of capturing and detecting rare cells is also extremely low under the current magnetic bead encapsulation rate.

[0062] The method proposed in this application for detecting rare cells based on the super-loading strategy utilizes the super-Poisson distribution of cells to increase the starting cell input for a single experiment to the million level, allowing multiple cells / cells to be encapsulated in a droplet; once a rare cell is encapsulated in a droplet, the magnetic beads in the droplet can capture the characteristic cell markers of the rare cell; by setting a certain threshold, the magnetic beads that capture the characteristic markers of rare cells can be screened out, and the presence of rare cells in the droplet can be confirmed at the same time; background cell signals will be filtered and discarded. In some embodiments, the proposed method mainly includes the following steps:

[0063] 1. Providing a magnetic bead phase: A magnetic bead phase of a certain concentration is used, wherein each magnetic bead is attached to hundreds of thousands to tens of millions of oligonucleotide sequences. The oligonucleotide sequence comprises a barcode sequence and a first capture sequence. The barcode sequence is magnetic bead-specific, i.e., the barcode sequence attached to the same magnetic bead is the same, while the barcode sequences attached to different magnetic beads are different. The first capture sequence is capable of capturing nucleic acid sequences from multiple single cells in the droplet.

[0064] 2. Providing a cell phase: Millions of cells are prepared into a high-density cell phase, wherein the high-density cell phase includes background cells that account for the vast majority and a certain number of target cells mixed in a small proportion.

[0065] 3. Droplet generation: Based on droplet microfluidic single-cell technology, a high-density cell phase and a certain concentration of magnetic beads are combined to generate droplets on a microfluidic device. This process will allow one or more magnetic beads and one or more cells to be encapsulated in a droplet.

[0066] 4. Droplet-based cell post-processing: The droplets are used as independent reactors to lyse cells within the droplets and release nucleic acid sequences, which are captured by the first capture sequence on the magnetic beads. All collected droplets are then demulsified, and the nucleic acid sequences captured by the magnetic beads are subjected to cDNA amplification, fragmentation, library construction, and sequencing (see the examples for details).

[0067] 5. Target cell (rare cell) detection: Since the cells are overloaded in the droplets in this overloading strategy, that is, one magnetic bead corresponds to multiple cells, and since the number of rare cells is extremely low, most droplets only contain background cells, and a few droplets will encapsulate the target cells (rare cells). Therefore, in subsequent data processing, the sequenced data is screened using the target cell-specific marker, and the background cell signals are filtered and screened out. Magnetic beads that capture a certain proportion of rare cell signals will be considered positive magnetic beads, and at the same time prove that rare cells are detected in the droplets.

[0068] The above-mentioned method proposed in the examples of this application achieves the conversion of the probability of detecting rare cells from a large number of background cells in traditional detection technology (one in ten thousand or even one in a million) to the probability of detecting rare cells based on magnetic beads in target droplets (a few in one), thereby greatly improving the detection rate and accuracy of rare cells. At the same time, the above-mentioned method encapsulates multiple cells in a single droplet, avoiding the need to dilute cells to an extremely low density during cell phase preparation and eliminating the need to generate excessive droplets during the detection process, thereby significantly reducing the cost of rare cell detection.

[0069] In addition, this application uses a high-throughput microfluidic single-cell sequencing platform to detect rare cells, completing the capture and genomic analysis of rare cells in one step; and the platform is highly applicable and can be applied to various types of rare cells, and can analyze and identify rare cells based on the platform's data analysis process and personalized analysis.

[0070] The following describes the method and device for detecting rare cells in a biological sample proposed in the embodiments of the present application with reference to the accompanying drawings.

[0071] FIG1 is a flow chart of a method for detecting rare cells according to an embodiment of the present application. As shown in FIG1 , the method comprises the following steps:

[0072] Step S101: providing a single cell from a biological sample.

[0073] In the embodiments of the present application, rare cells refer to a type of cell with low abundance in biological fluids. In some embodiments, the types of rare cells include: embryonic cells, stem cells, progenitor cells, cancer stem cells and various types of tumor cells. Rare cells may be associated with pathological symptoms, or may be associated with conditions such as pregnancy. In some embodiments, rare cells include circulating tumor cells and fetal free cells, wherein fetal free cells may be fetal nucleated red blood cells. It is understandable that the methods and platforms proposed in the embodiments of the present application are universal for various types of rare cells, that is, the methods for detecting rare cells proposed in the embodiments of the present application can be used to identify any type of low-abundance cells that account for a small proportion in a population, and there is no limitation on the types of rare cells.

[0074] In the embodiments of the present application, a biological sample refers to a sample derived from an organism. In some embodiments, the biological sample is from a mammal. In some embodiments, the biological sample is from a pregnant woman or a subject suffering from or suspected of having a disease. It is understandable that the method for detecting rare cells proposed in the embodiments of the present application can detect target cells (i.e., rare cells) in a large number of background cells. Therefore, in the embodiments of the present application, the biological sample can be derived from the same subject or from different subjects, such as from multiple subjects with a certain type of tumor screening requirement; it can be derived from the same species or from different species, such as from mice and humans. There is no limitation on the source and composition of the biological sample.

[0075] In embodiments of the present application, the biological sample can be any biological fluid, tissue, or organ from which single cells can be extracted. In some embodiments, the biological sample is a body fluid, tissue, or organ from the same organism containing rare cells; in other embodiments, the biological sample is a body fluid, tissue, or organ from a different organism containing rare cells. In some embodiments, the biological sample is at least one of peripheral blood, urine, saliva, or tissue.

[0076] Step S102: providing magnetic beads, wherein a plurality of oligonucleotide sequences are connected to the magnetic beads, each oligonucleotide sequence comprising a barcode tag sequence, a unique molecular identifier sequence UMI, and a first capture sequence.

[0077] In the embodiments of the present application, multiple oligonucleotide sequences are attached to the magnetic beads. In some embodiments, 100,000 to 10 million oligonucleotide sequences are attached to a single magnetic bead. In some embodiments, 100,000 to 1 million oligonucleotide sequences are attached to a single magnetic bead. It is understood that the number of oligonucleotides attached to a single magnetic bead is sufficient to contain the nucleic acid sequences of all single cells encapsulated in a single droplet, and this application does not intend to limit the number of oligonucleotides.

[0078] Figure 2 is a schematic diagram of the magnetic bead structure according to an embodiment of the present application. As shown in Figure 2, in the embodiment of the present application, each oligonucleotide sequence attached to the magnetic bead comprises: a barcode tag sequence, a unique molecular identifier sequence (UMI), and a first capture sequence. The barcode tag sequences of multiple oligonucleotide sequences are identical for the same single magnetic bead; the barcode tag sequences for different single magnetic beads are different. In other words, the barcode tag sequence is bead-specific. In some embodiments, the barcode sequence length can be 8-16 bp. In some embodiments, the barcode sequence length can be 10-20 bp.

[0079] In embodiments of the present application, the first capture sequence is used to capture nucleic acid sequences released after cell lysis in a single droplet, i.e., the first capture sequence has a structure that is reverse complementary to the nucleic acid sequence released by the cells. In some embodiments, the first capture sequence is selected from at least one of poly(dT), a DNA capture sequence, or a capture sequence with a specific function, wherein poly(dT) is used to capture mRNA sequences, DNA capture sequences are used to capture DNA sequences, and capture sequences with specific functions are used to capture sequences with special recognition functions designed according to needs. In some embodiments, sequences with specific functions can be designed based on experimental design and objectives. Sequences with specific functions can include sequences conjugated to oligos on capture antibodies, sequences conjugated to oligos on capture index carriers, and so on. In some embodiments, the first capture sequence is poly(dT). In some embodiments, the first capture sequence is located at the 3' end of the oligonucleotide sequence on the magnetic bead. In some embodiments, the first capture sequence can be 10-40 bp in length. In some embodiments, the first capture sequence can be 10-20 bp in length.

[0080] In an embodiment of the present application, a unique molecular identifier (UMI) is used to mark the nucleic acid sequence captured by the first capture sequence, wherein the unique molecular identifier sequences UMIs in the multiple oligonucleotide sequences are different from each other based on the same single magnetic bead. In other words, the UMI has unique specificity, that is, the UMI carried by each captured single-cell nucleic acid sequence is different. In some embodiments, the original nucleic acid template is marked by the UMI, so that during subsequent data analysis, repeated amplification sequences with the same UMI sequence can be removed according to the UMI sequence (i.e., UMI correction), thereby reducing the bias and amplification errors caused by subsequent library amplification. In an embodiment of the present application, the length of the UMI can be 5-20bp. In some embodiments, the length of the UMI can be 10-20bp. In some embodiments, the length of the UMI can be 10bp.

[0081] In the embodiments of the present application, the oligonucleotide sequence may further comprise a linker sequence, which may be used for primer binding in subsequent amplification, library construction, and sequencing.

[0082] Step S103: enclosing the plurality of single cells and the magnetic beads in a single droplet.

[0083] In an embodiment of the present application, a microfluidic device or other device that can realize droplet generation is used to perform droplet sealing. Figures 3 and 4 are respectively a droplet generating device according to an embodiment of the present application and a partial schematic diagram of the droplet generating device, and Figure 5 is a schematic diagram of the C4V2 slide used for droplet generation. In an embodiment of the present application, single cells derived from biological samples are prepared as a high-density cell phase, and the initial input amount thereof is in the millions or even higher; the initial input amount of magnetic beads can also be from hundreds of thousands to millions. As shown in Figure 4, in an embodiment of the present application, the high-density cell phase and the magnetic bead phase are encapsulated by using an oil phase to achieve the encapsulation of a single / multiple magnetic beads and a single / multiple cells in a single droplet. In an embodiment of the present application, it is preferred to encapsulate a single magnetic bead and multiple cells in a single droplet.

[0084] In an embodiment of the present application, a single droplet may contain 1-10 single cells. In some embodiments, a single droplet may contain 1-5 single cells. In some embodiments, a single droplet may contain 5-7 single cells. It is understandable that in an embodiment of the present application, by increasing the initial input amount of single cells, the need to dilute the cells to an extremely low density during the cell phase preparation process is avoided. At the same time, the input amount of magnetic beads can also be moderately increased. For example, in some embodiments, the input amount of magnetic beads is increased to 1 million, which can maintain the magnetic bead encapsulation rate above 95%, avoid the generation of excessive empty droplets during the droplet generation process, and maximize the capture rate of rare cells. This strategy significantly reduces the detection cost of rare cells and improves the detection rate of rare cells.

[0085] In embodiments of the present application, each droplet contains 1-10 magnetic beads. In some embodiments, each droplet contains 1, 2, 3, or 4 magnetic beads. In some embodiments, each droplet contains 5-8 magnetic beads. In some embodiments, each droplet contains 1 magnetic bead.

[0086] It is understandable that in Example 1 of the present application, since the initial input amount of cells is in the millions or even higher (high-density cell phase), and the initial input amount of magnetic beads is about hundreds of thousands (standard density magnetic bead phase), the two orders of magnitude are very different. Therefore, when the droplet is closed by the microfluidic device, in most cases, multiple cells and only one / two magnetic beads will be wrapped in a single droplet. In this case, the barcode in the tens of millions of independent and identical oligonucleotides connected to the magnetic beads will mark the nucleic acid sequences of multiple single cells wrapped in this droplet, so that the nucleic acid sequences of multiple single cells wrapped in the same droplet all carry the same barcode. In this way, during subsequent data analysis, by detecting the characteristic markers (markers) of rare cells (i.e., target cells), positive magnetic beads with captured rare cell nucleic acid sequences can be determined, thereby increasing the detection rate of rare cells in a large number of background cells from one in ten thousand or even one in a million to a fraction of the detection rate of rare cell markers on magnetic beads in droplets, thereby improving detection sensitivity and significantly reducing detection costs.

[0087] In addition, in Example 2 of the present application, in the process of droplet encapsulation, due to the increase in the input amount of magnetic beads, a single droplet will be encapsulated with multiple magnetic beads and multiple cells. It is understandable that in this case, the different barcodes in the oligonucleotide sequences connected to the multiple magnetic beads will mark the nucleic acid sequences of all single cells encapsulated in this droplet, and the positive magnetic beads will still be determined by detecting the characteristic markers (markers) of rare cells (i.e., target cells), and at the same time, rare cells will be determined in multiple cells corresponding to these magnetic beads. Therefore, even when multiple magnetic beads are encapsulated in the same droplet, the above-mentioned rare cell detection can still be achieved, and the above-mentioned high detection rate and high sensitivity of one fraction can be achieved.

[0088] Step S104: constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet and performing sequencing and data analysis.

[0089] In some embodiments, step S104 includes: lysing multiple single cells to obtain single-cell nucleic acid sequences, wherein the single-cell nucleic acid sequences are captured by a first capture sequence; destroying droplets to release magnetic beads connected to the oligonucleotide sequences and the single-cell nucleic acid sequences; and constructing a DNA library based on the single-cell nucleic acid sequences.

[0090] In an embodiment of the present application, after the magnetic beads and multiple single cells are enclosed in a single droplet, the lysis solution in the droplet will lyse the multiple single cells to obtain single-cell nucleic acid sequences. These released nucleic acid sequences will then be captured by the first capture sequence in the oligonucleotide sequence attached to the magnetic beads to connect the nucleic acid sequence to the magnetic beads. This process occurs independently in the reactor formed by a single droplet. It can be understood that this capture step causes each nucleic acid sequence attached to the magnetic beads to be connected to the oligonucleotide sequence carried on the magnetic beads, including barcodes and UMIs, etc., wherein the nucleic acid sequences attached to the same magnetic bead have the same barcode and different UMIs; the nucleic acid sequences attached to different magnetic beads have different barcodes and different UMIs, thereby achieving magnetic bead labeling of the nucleic acid sequence and labeling of the original template.

[0091] In some embodiments, after capturing the nucleic acid sequence, all droplets are collected and a breakage buffer is used to break the oil-in-water structure of the droplets to release the magnetic beads attached with the oligonucleotide sequence and the single-cell nucleic acid sequence. It is understood that this breakage step can be performed on individual droplets, or all droplets can be collected and then treated together. As long as the oil phase used to form the droplet envelope is destroyed, the treatment process is not limited herein. In some embodiments, the breakage buffer is an emulsifier, etc.

[0092] In some embodiments, a DNA library is constructed based on the single-cell nucleic acid sequence using a standard library construction process. In some embodiments, the standard library construction procedure can be the BGI standard library construction procedure (see Example 1 below for details). In other embodiments, the standard library construction procedure can also be a standard library construction procedure adapted for sequencing platforms such as Illumina, Roche, and SOLiD that has been implemented in the relevant art, without limitation.

[0093] In some embodiments, before constructing a DNA library based on the single-cell nucleic acid sequence using a standard library construction procedure, the method further includes: using the released magnetic beads that captured the single-cell nucleic acid sequence as a template to amplify the single-cell nucleic acid sequence and the associated oligonucleotide sequences (including barcodes, UMIs, etc.). In some embodiments, the single-cell nucleic acid sequence is an mRNA sequence. Furthermore, if the captured single-cell nucleic acid sequence is mRNA, the mRNA needs to be reverse transcribed after the capture step, and then amplified based on the synthesized cDNA and the associated oligonucleotide sequences (including barcodes, UMIs, etc.).

[0094] Step S105: Screening the specific marker sequence information of rare cells from the single-cell nucleic acid sequence, and determining whether the rare cells exist in the biological sample.

[0095] In this embodiment of the present application, step S105 includes: calculating, based on a single droplet, a ratio between the number of unique molecular identifier sequences (UMIs) corresponding to a single-cell nucleic acid sequence mapped to a specific marker sequence of the rare cell and the total number of all unique molecular identifier sequences (UMIs); comparing the ratio with a set ratio threshold; and determining the presence of the rare cell in the biological sample when the positive ratio is greater than the set ratio threshold.

[0096] In the embodiment of the present application, the DNA library constructed in step S104 is sequenced on a machine to obtain sequencing data. Sequencing can be performed on various sequencing platforms of the related art. In the embodiment of the present application, the sequencing platform is the MGI platform.

[0097] In the embodiment of the present application, for sequencing data, the original offline data is first formatted using scRNA_parse software, and barcode correction and filtering are performed according to the whitelist; then, STAR is used to align the filtered sequences to the reference genome / transcriptome of background cells and target cells (rare cells); PISA software is used to process and annotate the files, and the ratio of the UMI annotated to the target cell reference genome / transcriptome in a magnetic bead to all UMIs on the magnetic bead is calculated based on the statistically obtained barcode and UMI data. The corresponding ratio threshold is set according to the data accumulation. When the ratio exceeds the ratio threshold, it is considered that the target cell is detected, and the magnetic bead can be determined to be a positive magnetic bead. It is understandable that for the above-mentioned quality control, filtering, alignment and annotation steps, any scripts and software that can be implemented in the relevant technology can be used, and there is no limitation here.

[0098] In an embodiment of the present application, the ratio threshold may be set to 1%-10%. In some embodiments, the ratio threshold may be set to 1%-5%. In some embodiments, the ratio threshold may be set to 5%-10%.

[0099] In the examples of this application, bead-specific barcodes are used to identify the bead source of sequencing data, and unique molecular identifiers (UMIs) are used to label the original nucleic acid sequence for subsequent correction, thereby minimizing bias and errors introduced during library construction and amplification.

[0100] The method for detecting rare cells proposed in this embodiment transforms the probability of detecting rare cells from a large number of background cells in traditional detection techniques (one in ten thousand or even one in a million) into the probability of detecting rare cells based on magnetic beads within the target droplet (a fraction of a percent), thereby significantly improving the detection rate and accuracy of rare cells. Furthermore, the method encapsulates multiple cells within a single droplet, avoiding the need to dilute cells to an extremely low density during cell phase preparation. Furthermore, by increasing the amount of magnetic beads input, the magnetic bead encapsulation rate within the droplet is greatly improved, allowing for the capture of a larger number of cells during the droplet generation process. This allows for a high cell input in a single test, significantly reducing the cost of rare cell detection.

[0101] It is understood that the high detection rate (a fraction of a percent) achieved by the detection method proposed in the above embodiment is based on the magnetic beads in the target droplet. Specifically, by calculating the ratio of UMIs carrying rare cell-specific nucleic acid sequences on the same magnetic bead based on the barcode, positive magnetic beads are obtained, confirming the detection of rare cells. In this regard, another embodiment of the present application, based on the above-mentioned magnetic bead-based rare cell detection method, further proposes a method for detecting rare cells by further increasing the amount of magnetic beads used.

[0102] In the method for detecting rare cells proposed in Example 2 of the present application, the oligonucleotide sequence connected to the magnetic bead further includes a second capture sequence for additionally capturing non-single-cell nucleic acid sequences. In this embodiment, the second capture sequence is used to capture the Index sequence to achieve the labeling of the droplet source of the magnetic beads. Specifically, by adding an Index carrier to the cell phase, the single droplet generated in the droplet generation (closure) step further includes an Index carrier. In some embodiments, the Index carrier is a solid magnetic bead. Multiple Index sequences are connected to the Index carrier, and the Index sequence also includes a barcode sequence, a UMI sequence, and a capture sequence (collectively referred to as an Index sequence), and each Index carrier has a unique barcode sequence. After the Index carrier is encapsulated into a droplet, the Index sequence breaks off from the Index carrier and captures the second capture sequence in the oligonucleotide sequence of the magnetic bead in the same droplet, thereby complementarily pairing with the second capture sequence on the magnetic bead. This establishes a connection between the Index sequence and the barcode on the magnetic bead, thereby labeling the magnetic beads in a single droplet. That is, when two or more magnetic beads are present in a single droplet, the Index carrier encapsulated in this droplet releases the Index sequence, which binds to the second capture sequence and simultaneously labels the barcode on the magnetic bead. In subsequent analysis, the similarity of the Index sequences in the same droplet can be used to distinguish and identify the magnetic beads and cells from the same droplet, thereby tracing the droplet origin of the cells.

[0103] In some embodiments, each Index vector is connected to hundreds of thousands of Index sequences. In some embodiments, each Index vector is connected to at least 100,000 Index sequences. In some embodiments, the Index sequence length can be 30-90 bp.

[0104] In some embodiments, a single droplet contains 5-15 Index vectors. In some embodiments, a single droplet contains 8-10 Index vectors.

[0105] According to the droplet-based detection method proposed in Example 2 of the present application, the labeling and tracing of magnetic beads and cells from the same droplet are achieved. It also achieves the above-mentioned conversion of the probability of detecting rare cells from a large number of background cells in traditional detection technology (one in ten thousand or even one in a million) into the probability of detecting rare cells based on magnetic beads in the target droplet (a few percent), thereby greatly improving the detection rate and accuracy of rare cells. At the same time, this method further increases the probability of rare cell capture by increasing the amount of magnetic beads input and the encapsulation rate of magnetic beads in the droplet. At the same time, a high amount of cell input is achieved in a single test, thereby significantly reducing the cost of rare cell detection.

[0106] Furthermore, an embodiment of the present application also provides a device for detecting rare cells.

[0107] FIG6 is a schematic diagram of the structure of a device for detecting rare cells according to an embodiment of the present application. As shown in FIG6 , the device 100 for detecting rare cells includes: a droplet sealing device 110 , a DNA library construction device 120 , a sequencing device 130 , and an analysis device 140 .

[0108] The droplet sealing device 110 is used to seal multiple single cells and magnetic beads in a single droplet, wherein the multiple single cells are from a biological sample, and the magnetic beads are connected to multiple oligonucleotide sequences, each of which includes a barcode tag sequence, a unique molecular identifier sequence UMI, and a first capture sequence.

[0109] The DNA library construction device 120 is connected to the droplet sealing device and is used to construct a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in a single droplet.

[0110] The sequencing device 130 is connected to the DNA library construction device and is used to sequence the DNA library.

[0111] The analysis device 140 is connected to the sequencing device for data analysis, screening specific marker sequence information of rare cells from single-cell nucleic acid sequences, and determining whether rare cells exist in the biological sample.

[0112] It should be noted that the device for detecting rare cells proposed in this embodiment executes the above-mentioned method for detecting rare cells and corresponds one-to-one with the above-mentioned method. The above explanation of the embodiment of this method is also applicable to the device 100 for detecting rare cells in this embodiment and will not be repeated here.

[0113] The device for detecting rare cells proposed in the embodiments of the present disclosure achieves the conversion of the probability of detecting rare cells from a large number of background cells in traditional detection technology (one in ten thousand or even one in a million) to the probability of detecting rare cells based on magnetic beads in target droplets (a few percent), thereby greatly improving the detection rate and accuracy of rare cells. At the same time, the above method encapsulates multiple cells in a single droplet. At the same time, this method further increases the probability of rare cell capture by increasing the amount of magnetic beads input and the encapsulation rate of magnetic beads in the droplet. A single test achieves a high input of cells / magnetic beads, significantly reducing the cost of rare cell detection.

[0114] Furthermore, the disclosed embodiments also provide a method for diagnosing cancer, comprising determining the presence of circulating tumor cells in a biological sample from a subject based on the method for detecting rare cells described in any of the above embodiments;

[0115] Analyze the number of circulating tumor cells, expression of disease-associated genes, and / or regulation of accessible chromatin regions; and

[0116] Based on the number of circulating tumor cells and the expression of disease-related genes and / or the regulation of chromatin open regions, it is identified whether the subject has a risk of developing a related disease, the development of the disease and / or the prognosis of the disease.

[0117] The method for diagnosing cancer proposed in the embodiment of the present application determines the presence of circulating tumor cells in a biological sample from a subject by using the method for detecting rare cells based on any of the above embodiments. It is understandable that the method for detecting rare cells proposed in the embodiment of the present application can achieve the separation of single cell data in the droplet, that is, if the droplet is wrapped in a rare cell, then the magnetic beads in the droplet can capture the transcriptome or genomic information of these rare cells. The expression profile of such rare cells is often quite different from that of background cells, such as in the expression of certain disease-related genes, the regulation of chromatin open regions, etc. Therefore, this information can be used as an important basis for identifying whether a subject has a certain disease risk, disease development and prognosis process, and fetal-related genetic diseases. Therefore, the method for detecting rare cells in the embodiment of the present application not only involves the use of specific markers to screen the presence and quantity of rare cells, but also can use the sequenced rare cell information for subsequent more in-depth analysis, thereby achieving efficient and accurate diagnosis of cancer.

[0118] Furthermore, the embodiments of the present disclosure also provide a non-invasive fetal diagnostic method, comprising: the method for detecting rare cells described in any of the above embodiments, determining the presence of fetal free cells in a biological sample from a pregnant woman; and analyzing the genomic or transcriptomic information of the fetal free cells to determine the developmental status of the fetus and / or whether it suffers from a genetic disease.

[0119] In some embodiments, the genetic disease can be a chromosomal disease, a single gene disease, or a multi-gene disease.

[0120] The non-invasive fetal diagnostic method proposed in the embodiments of the present application determines fetal free cells in a biological sample from a pregnant woman based on the method for detecting rare cells in the embodiments of the present application, and further conducts in-depth analysis of the genomic information or transcriptome information of the fetal free cells, thereby obtaining relevant information such as the developmental status of the fetus and whether it suffers from genetic diseases, thereby achieving efficient acquisition and accurate analysis of fetal information under non-invasive conditions.

[0121] Figure 12 is a schematic diagram of a method for detecting rare cells based on an overload (super-loading) strategy according to an embodiment of the present application. As shown in Figure 12, part a represents that when traditional detection does not carry out droplet microfluidic cell super-loading, rare cell signal intensity is extremely weak, and the event rate in background cells is 1 / 10,000 or even 1 / 1,000,000 (as shown in part e). Figure b partially shows a microfluidic droplet generating device applicable to the method for detecting rare cells based on a super-loading strategy proposed in an embodiment of the present application, in which cells are super-loaded so that magnetic beads and multiple cells are wrapped in a single droplet. Figure c partially represents the method for detecting rare cells proposed in an embodiment of the present application, after forming closed droplets, these droplets wrapped with a large number of cell colonies in the sample can be divided into two categories, i.e., droplets with target cells (i.e., rare cells) and droplets without target cells, only droplets with target cell features are screened out and analyzed by genome / transcript features, and target cells can be detected. Each individual droplet is regarded as a system, as shown in part d in Figure 12, the detection probability of target cells will be much greater than the detection probability of target cells in a million populations.

[0122] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0123] Example 1

[0124] In this example, human embryonic kidney cells HEK293T were used as background cells, and mouse embryonic fibroblasts 3T3 were used as target cells (i.e., rare cells) as experimental materials. A method and platform for detecting rare cells based on a super-loading strategy was used. Rare cells were detected at a ratio of target cells to background cells of 1:100, 1:1000, and 1:10000, respectively. Two replicates were performed for each group. The specific cell numbers and experimental groups are shown in Table 1.

[0125] Table 1

[0126] Group 293T number 3T3 number 1, 21000000100003, 4100000010005, 61000000100

[0127] 1. Cell Suspension Preparation

[0128] 1.1 Prepare cell suspensions for 293T cell lines and 3T3 cell lines respectively, wash the cells 1-2 times with PBS (containing 0.04% BSA) and filter through a 40 μm cell sieve.

[0129] 1.2 Use a cell counting chamber or a cell counter to detect the concentration of cells or cell nuclei.

[0130] 1.3 Based on the concentration of the 293T cell suspension, calculate the required volume of cell suspension, assuming 1.1 million 293T cells per group. Centrifuge the cell suspension containing 1.1 million 293T cells at 300g, 25°C, for 5 minutes to collect the cell pellet, and add 22.5μl of Cell Resuspension Buffer to resuspend the cells or nuclei.

[0131] 1.4 Adjust the 3T3 cell concentration to 1000 cells / μl, 100 cells / μl, and 10 cells / μl, respectively. Then, mix 11 μl of each of the three concentration gradients of 3T3 cell suspension with 22.5 μl of the 293T cell suspension prepared in step 1.3 above. Set up two replicates for each of the three concentration gradients (see Table 1).

[0132] 1.5 Add 33μl Cell Additive, 5.5μl RNase Inhibitor and 38μl Index carrier to each group. The total volume of each cell suspension system is 110μl.

[0133] 2. Magnetic Bead Preparation

[0134] 2.1 Vortex and mix Single Cell Barcoded Beads-V2. Pipette 100 μl of Single Cell Barcoded Beads-V2 into a PCR tube for each group. Place the PCR tube on a magnetic rack and let it stand for 2 minutes. Remove the supernatant.

[0135] 2.2 Remove the PCR tube from the magnetic stand and add 99 μl of Lysis Buffer-V2 to the tube to suspend the magnetic beads.

[0136] 2.3 Before loading the tube, add 11 μl of CR reagent to the magnetic bead phase, pipette to mix, and then place on ice until loading.

[0137] 3. Droplet Generation

[0138] 3.1 Use a C4V2 slide and place it in the slide slot area of ​​the droplet generating device C4V2 as shown in Figure 3.

[0139] 3.2 Add 100 μl of Droplet Generation Oil to the C4V2 collection tube, tighten the tube cap, and place the collection tube vertically on the fixed rack.

[0140] 3.3 Insert the A end of the first connecting tube on the collection tube cover (this tube contacts the bottom of the collection tube, as shown in Figure 4) into the Outlet hole of the slide.

[0141] 3.4 Adjust the position of the C4V2 syringe piston and place it in the initial position on the C4V2 base. Use a needle to connect the syringe to the B end of the second connecting tube on the collection tube cap (as shown in Figure 3, this tube does not touch the bottom of the collection tube).

[0142] 3.5 Mix the cell suspension prepared in step 1.5 by pipetting and add 100 μL of cell buffer to the cells well of the chip.

[0143] 3.6 Mix the magnetic bead suspension prepared in step 2.3 by pipetting, and add 100 μl of the magnetic bead suspension to the beads well of the chip.

[0144] 3.7 Quickly add 450 μL of Droplet Generation Oil to the Oil well of the chip.

[0145] 3.8 Quickly pull the syringe piston to the 20mL mark and secure the syringe to the fixed bracket.

[0146] 3.9 Collect the droplets. This process lasts for about 10 minutes. When the liquid in either the cell well or the beads well is about to be loaded, immediately loosen the cap of the collection tube, pull out the connecting tube from the chip outlet hole, and stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube.

[0147] 3.10 Place the collection tube vertically and let it stand at room temperature for 20 minutes before immediately proceeding with the subsequent demulsification operation.

[0148] 4. Demulsification

[0149] 4.1 Preparation of 6×SSC (approximately 50 mL per sample): Add 35 mL of Molecular Grade Water to 15 mL of 20×SSC and vortex to mix.

[0150] 4.2 Breakage Buffer Preparation (approximately 10 mL per sample): Add 400 μl of Additive B to 20 mL of 6× SSC.

[0151] 4.3 Connect the C4 V2 filter to the vacuum pump and start the vacuum pump.

[0152] 4.4 Add approximately 20 mL of 6×SSC to pre-rinse the C4 V2 filter.

[0153] 4.5 When there is no liquid left on the filter membrane, pour all the liquid in the collection tube evenly onto the filter membrane surface, and wash the collection tube twice with 2 mL of 6×SSC, and pour the washing solution into the filtration device.

[0154] 4.6 Mix the Breakage Buffer by inverting vigorously and add it slowly to the filter in batches.

[0155] 4.7 When there is no liquid residue on the filter membrane, add 30 mL of 6×SSC to wash the magnetic beads.

[0156] 4.8 When there is no liquid residue on the filter membrane, turn off the vacuum pump and disconnect the vacuum pump from the filter.

[0157] 4.9 Transfer the filter to a clean bench and add 700 μl Collection Buffer to suspend the magnetic beads.

[0158] 4.10 Transfer the collected solution containing magnetic beads to a 1.5 mL low-binding centrifuge tube.

[0159] 4.11 Resuspend the remaining magnetic beads in 700 μl Collection Buffer.

[0160] 4.12 Transfer the collected solution containing magnetic beads to the same 1.5 mL centrifuge tube as above.

[0161] 4.13 Place the centrifuge tube on a magnetic rack, let it stand for 3 minutes, and slowly remove the supernatant.

[0162] 4.14 Add 1 ml of 6×SSC, let it stand for 2 minutes, and slowly remove the supernatant.

[0163] 4.14 Add 200 μl of 6× SSC to the 1.5 ml tube and mix thoroughly by pipetting. Transfer the recovered magnetic bead suspension to a 0.2 ml PCR tube and place it on a magnetic stand to allow it to stand. Aspirate the supernatant.

[0164] 4.15 Add another 200 μl of 6×SSC to the above 1.5 ml tube, pipette to mix again, and transfer the magnetic bead suspension to the same PCR tube containing magnetic beads in step 4.14.

[0165] 4.16 Place the 0.2 mL PCR tube on a magnetic rack, let it stand for 2 minutes, and slowly remove the supernatant.

[0166] 4.17 Keep the PCR tube on the magnetic rack, add 200 μL 6×SSC, let it stand, and prepare the reverse transcription reaction system.

[0167] 5. Reverse Transcription Reaction

[0168] 5.1 Prepare the reverse transcription reaction system on ice for each PCR tube according to the recipe shown in Table 2 below.

[0169] Table 2

[0170]

[0171] 5.2 Slowly remove the 6×SSC supernatant from step 4.17 and add 100 μl of reverse transcription reaction system to suspend the magnetic beads.

[0172] 5.3 Incubate on a PCR instrument according to the reaction conditions shown in Table 3.

[0173] Table 3

[0174]

[0175] 5.4 After the reaction is completed, place the PCR tube on a magnetic rack and let it stand for 2 minutes before removing the supernatant.

[0176] 5.5 Remove the PCR tubes from the magnetic stand and add 200 μl Wash Buffer E (TE-SDS) to each tube to terminate the reaction.

[0177] 5.6 Place the PCR tube on a magnetic rack, let it stand for 2 minutes, and remove the supernatant.

[0178] 5.7 Keep the PCR tubes on the magnetic rack, add 200 μl Wash Buffer F (TE-TW) to each tube, let it stand for 2 minutes, and remove the supernatant.

[0179] 5.8 Repeat step 5.7 again.

[0180] 6. Second-chain synthesis

[0181] 6.1 Place the PCR tube in 5.8 on a magnetic rack, let it stand for 2 minutes, and remove the supernatant.

[0182] 6.2 Add 200 μl of diluted Solution S1 to each tube, remove the PCR tube from the magnetic stand, and gently flick to mix the magnetic beads.

[0183] 6.3 After mixing, incubate at room temperature for 5 minutes.

[0184] 6.4 Centrifuge briefly, place the PCR tube on a magnetic rack, let it stand for 2 minutes, and then remove the supernatant.

[0185] 6.5 Keep the centrifuge tubes on the magnetic rack, add 200 μl Solution S2 to each tube, let it stand for 2 minutes, and remove the supernatant.

[0186] 6.6 Keep the centrifuge tubes on the magnetic rack, add 200 μL Wash Buffer F (TE-TW) to each tube, let it stand, and prepare the next reaction system.

[0187] 6.7 Prepare the second-strand synthesis reaction system on ice according to the recipe in Table 4.

[0188] Table 4

[0189]

[0190] 6.8 Slowly remove the supernatant from the PCR tubes in step 6.6, add 100 μl of the second-strand synthesis reaction system to each tube, vortex to mix, and briefly centrifuge.

[0191] 6.9 Incubate on a PCR instrument according to the reaction conditions shown in Table 5.

[0192] Table 5

[0193]

[0194] 6.10 After the reaction is completed, centrifuge briefly, place the PCR tube on a magnetic rack, let it stand for 2 minutes, and remove the supernatant.

[0195] 6.11 Remove the PCR tubes from the magnetic rack, add 200 μl Wash Buffer E (TE-SDS) to each tube, vortex to mix, and terminate the reaction.

[0196] 6.12 Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand for 2 minutes and remove the supernatant.

[0197] 6.13 Keep the centrifuge tubes on the magnetic rack, add 200 μl Wash Buffer F (TE-TW) to each tube, let it stand for 2 minutes, and remove the supernatant.

[0198] 6.14 Remove the PCR tubes from the magnetic stand, add 200 μl Wash Buffer F (TE-TW) to each tube, and mix thoroughly by pipetting. Divide the magnetic bead suspension evenly into four portions and transfer them to new PCR tubes, with approximately 50 μl per tube.

[0199] 6.15 Add 200 μl of Wash Buffer F (TE-TW) to the original PCR tube and mix thoroughly by pipetting. Divide the magnetic bead suspension into four equal portions and transfer them to the four PCR tubes loaded with magnetic beads in step 6.14, with approximately 50 μl in each tube.

[0200] 6.16 Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand and prepare the next reaction system.

[0201] 7. cDNA Amplification

[0202] 7.1 Prepare the cDNA amplification reaction system on ice according to the recipe in Table 6.

[0203] Table 6

[0204]

[0205] 7.2 Slowly remove the supernatant from each PCR tube in step 6.16, add 100 μl of the prepared cDNA amplification reaction system to each tube, vortex to mix, and centrifuge briefly.

[0206] 7.3 Incubate on a PCR instrument according to the reaction conditions shown in Table 7.

[0207] Table 7

[0208]

[0209] 7.4 Mix the 4 tubes of PCR products in each group into a 1.5 mL tube, with a total volume of approximately 400 μl.

[0210] 7.5 Pipette 266 μl (0.6×) Cleanup Beads A-v2 into the mixed PCR product and gently pipette to mix thoroughly, then incubate at room temperature for 10 min.

[0211] 7.6 Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand for 5 minutes until the liquid becomes clear.

[0212] 7.7 Aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube and label it as oligo product.

[0213] 7.8 Keep the centrifuge tube in step 7.6 on the magnetic rack, add 1000 μl 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0214] 7.9 Repeat the previous step.

[0215] 7.10 Keep the centrifuge tube on the magnetic rack, open the tube cover, and dry the magnetic beads at room temperature.

[0216] 7.11 Remove the centrifuge tube from the magnetic stand, add 32 μl of Nuclease-Free Water to elute the magnetic beads, and gently pipette to mix thoroughly. Then incubate at room temperature for 5 minutes.

[0217] 7.12 Briefly centrifuge the tube and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer the supernatant (30 μl) to a new 1.5 mL centrifuge tube and label it as cDNA product.

[0218] 7.13 Take 1 μl of cDNA product and use the Qubit dsDNA HS Kit to determine the concentration. Take another 1 μl of cDNA product and use the Agilent DNA analysis kit to determine the fragment distribution.

[0219] 7.14 Add 266 μl (0.8×) Cleanup Beads A-v2 to the oligo product retained in step 7.7 and gently pipette to mix thoroughly. Then incubate at room temperature for 10 min.

[0220] 7.15 Keep the centrifuge tube on the magnetic rack, add 1000 μl 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0221] 7.16 Repeat the previous step.

[0222] 7.17 Keep the centrifuge tube on the magnetic rack, open the tube cap, and dry the magnetic beads at room temperature.

[0223] 7.18 Remove the centrifuge tube from the magnetic stand, add 32 μl of Nuclease-Free Water for elution, and gently pipette to mix thoroughly. Then incubate at room temperature for 5 minutes.

[0224] 7.19 Briefly centrifuge the tube and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer the supernatant (30 μl) to a new 1.5 mL centrifuge tube and label it as the oligo product.

[0225] 7.20 Take 1 μl of the Oligo product and use the Qubit dsDNA HS Kit to detect the concentration.

[0226] Quality control (QC) standards: cDNA concentration greater than 5 ng / μl, and the main peak of fragment distribution is between 900-1500 bp.

[0227] 8. Oligo product secondary amplification

[0228] 8.1 Take a new 0.2mL PCR tube and take 8μL of the oligo product from step 7.19 for secondary amplification. The reaction system is shown in Table 8:

[0229] Table 8

[0230]

[0231] 8.2 Incubate on a PCR instrument according to the reaction conditions in Table 9.

[0232] Table 9

[0233]

[0234] 8.3 Pipette 35 μl of Cleanup Beads A-v2 into the PCR product and gently pipette to mix thoroughly. Then incubate at room temperature for 5 minutes.

[0235] 8.4 Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear. Aspirate the supernatant and transfer it to a new PCR tube.

[0236] 8.5 Pipette 35 μl of Cleanup Beads A-v2 into the supernatant of the PCR tube in step 8.4, pipette gently to mix thoroughly, and then incubate at room temperature for 5 minutes.

[0237] 8.6 Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid becomes clear and discard the supernatant.

[0238] 8.7 Keep the centrifuge tube on the magnetic stand, add 200 μl 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0239] 8.8Repeat the previous step.

[0240] 8.9 Keep the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature for 1 minute.

[0241] 8.10 Remove the centrifuge tube from the magnetic stand, add 32 μl of Nuclease-Free Water, and gently pipette until completely mixed.

[0242] 8.11 Incubate at room temperature for 5 minutes.

[0243] 8.12 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer 30 μl of the supernatant to a new centrifuge tube.

[0244] 8.13 Take 1 μl of the purified product and use the Qubit dsDNA HS Kit to determine the concentration.

[0245] Quality control (QC) standard: oligo concentration greater than 5 ng / μl.

[0246] 9. Library Construction

[0247] 9.1 Interruption

[0248] 9.1.1 Remove the Fragmentation Enzyme-v2, gently invert and mix 10 times, centrifuge briefly, and place on ice until ready to use.

[0249] 9.1.2 Prepare the disruption reaction solution on ice according to Table 10.

[0250] Table 10

[0251]

[0252] 9.1.3 Depending on the concentration of the cDNA product, take 200 to 300 ng of the cDNA product purified in 7.12 and place it in a new 0.2 mL PCR tube. Use Nuclease-Free Water to make up the volume to 16 μl.

[0253] 9.1.4 Add 4 μl of the prepared disruption reaction solution to the PCR tube in 9.1.3, vortex, and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0254] 9.1.5 Set the reaction conditions according to Table 11. When the temperature of the PCR instrument drops to 4°C, immediately place the PCR tube in the PCR instrument and perform the reaction at 37°C.

[0255] Table 11

[0256]

[0257] 9.1.6 After the reaction is completed, immediately remove the PCR tube and place it on ice. Add 30 μl of Stop Solution-v2 to the PCR tube and vortex to mix to terminate the reaction.

[0258] 9.1.7 Briefly centrifuge to collect the reaction solution at the bottom of the tube and place the PCR tube on ice.

[0259] 9.2 Magnetic bead double selection of interruption products

[0260] 9.2.1 Add 30 μl of Cleanup Beads A to the PCR product, pipette gently to mix thoroughly, and incubate at room temperature for 5 min.

[0261] 9.2.2 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3 to 5 minutes until the liquid becomes clear. Transfer the supernatant to a new PCR tube.

[0262] 9.2.3 Add 20 μl of Cleanup Beads A to the supernatant in the previous PCR tube, pipette gently to mix thoroughly, and incubate at room temperature for 5 minutes.

[0263] 9.2.4 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3 to 5 minutes until the liquid becomes clear. Remove the supernatant.

[0264] 9.2.5 Keep the centrifuge tube fixed on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0265] 9.2.6 Repeat the previous step.

[0266] 9.2.7 Keep the centrifuge tube fixed on the magnetic rack, open the tube cover, and dry the magnetic beads at room temperature.

[0267] 9.2.8 Remove the centrifuge tube from the magnetic stand, add 42 μl ddH2O to elute the DNA, and incubate at room temperature for 5 minutes.

[0268] 9.2.9 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3 to 5 minutes until the liquid becomes clear. Transfer 40 μl of the supernatant to a new 0.2 ml PCR tube.

[0269] 9.3 End Repair

[0270] 9.3.1 Prepare the end-repair reaction solution on ice according to Table 12.

[0271] Table 12

[0272]

[0273] 9.3.2 Pipette 10 μl of the prepared end-repair reaction solution into the new PCR tube prepared in step 9.2.9. Vortex and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0274] 9.3.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 13.

[0275] Table 13

[0276]

[0277] 9.3.4 After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube and place the PCR tube on ice.

[0278] 9.4 Connector Connection

[0279] 9.4.1 Prepare the adapter ligation reaction solution on ice according to Table 14.

[0280] Table 14

[0281]

[0282] 9.4.2 Pipette 30 μl of the prepared adapter ligation reaction solution into the PCR tube prepared in step 9.3.4. Vortex to mix thoroughly and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0283] 9.4.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 15.

[0284] Table 15

[0285]

[0286] 9.4.4 After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0287] 9.4.5 Add 20 μl of TE Buffer to the PCR tube to make the total volume 100 μl.

[0288] 9.5 Purification of Ligation Product

[0289] 9.5.1 Pipette 50 μl of Cleanup Beads A-v2 into the adapter-ligated product from step 9.4.5, gently pipette to mix thoroughly, and incubate at room temperature for 10 min.

[0290] 9.5.2 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid becomes clear. Remove the supernatant.

[0291] 9.5.3 Keep the centrifuge tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0292] 9.5.4 Repeat the previous step.

[0293] 9.5.5 Keep the centrifuge tube on the magnetic rack, open the tube cap, and dry the magnetic beads at room temperature.

[0294] 9.5.6 Remove the centrifuge tube from the magnetic stand, add 48 μl of Nuclease-Free Water to elute the DNA, and incubate at room temperature for 5 minutes.

[0295] 9.5.7 Briefly centrifuge, place the tube on a magnetic rack, and let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer 46 μl of the supernatant to a new 0.2 mL PCR tube.

[0296] 9.6 PCR Amplification

[0297] 9.6.1 Add 4 μl of iDrop Index Primer to the PCR tube in step 9.5.7.

[0298] 9.6.2 Add 50 μl of AMP Ready Mix-v2 to the PCR tube, vortex and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0299] 9.6.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 16.

[0300] Table 16

[0301]

[0302] 9.7 Screening of PCR Amplification Product Fragments

[0303] 9.7.1 Pipette 60 μl of Cleanup Beads A-v2 into the PCR product, mix thoroughly, and incubate at room temperature for 10 minutes.

[0304] 9.7.2 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid becomes clear. Aspirate the supernatant and transfer it to a new PCR tube.

[0305] 9.7.3 Pipette 20 μl of Cleanup Beads A-v2 into the transferred supernatant, pipette to mix thoroughly, and incubate at room temperature for 5 minutes.

[0306] 9.7.4 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid becomes clear. Remove the supernatant.

[0307] 9.7.5 Keep the centrifuge tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 seconds, and remove the supernatant.

[0308] 9.7.6 Repeat the previous step.

[0309] 9.7.7 Keep the centrifuge tube on the magnetic rack, open the tube cap, and let it stand at room temperature for 1 minute.

[0310] 9.7.8 Remove the centrifuge tube from the magnetic stand, add 42 μL of TE Buffer, pipette until completely mixed, and incubate at room temperature for 5 minutes.

[0311] 9.7.9 Briefly centrifuge, place the tube on a magnetic rack, and let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer 40 μl of the supernatant to a new 0.2 mL PCR tube.

[0312] 9.7.10 Take 1 μl of the fragment screening product and use the Qubit dsDNA HS Kit to detect the concentration. Take another 1 μl of the fragment screening product and use the Agilent DNA analysis kit to detect the fragment distribution.

[0313] Quality control (QC) standards: product concentration greater than 3 ng / μl, and the main peak of fragment distribution is between 300 and 500 bp.

[0314] 9.8 Denaturation

[0315] 9.8.1 Transfer 0.75 to 1.5 pmol of PCR product (300-400 ng for cDNA, 300-400 ng for oligo) to a new 0.2 mL PCR tube and add TE Buffer to a total volume of 45 μl.

[0316] 9.8.2 Add 5 μl Split Oligo-v6 to the PCR tube, vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0317] 9.8.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 17.

[0318] Table 17

[0319]

[0320] 9.8.4 After the reaction is completed, immediately place the PCR tube on ice and let it stand for 5 minutes.

[0321] 9.9 Single-chain cyclization

[0322] 9.9.1 Prepare the single-stranded circularization reaction solution on ice according to Table 18.

[0323] Table 18

[0324]

[0325] 9.9.2 Pipette 10 μl of the prepared single-stranded circularization reaction solution into the PCR tube prepared in step 9.8.4. Vortex to mix thoroughly, then briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0326] 9.9.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 19.

[0327] Table 19

[0328]

[0329] 9.9.4 After the reaction is completed, centrifuge the PCR tube briefly and place it on ice before proceeding to the next step.

[0330] 9.10 Enzymatic Digestion

[0331] 9.10.1 Prepare the enzyme digestion reaction solution on ice according to the recipe in Table 20.

[0332] Table 20

[0333]

[0334] 9.10.2 Pipette 4 μl of the prepared enzyme digestion reaction solution into the PCR tube prepared in step 9.9.4, shake to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0335] 9.10.3 Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 21.

[0336] Table 21

[0337]

[0338] 9.10.4 After the reaction is completed, immediately add 3 μl of Stop solution-v2, vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube.

[0339] 9.11 Purification of Enzymatic Digestion Products

[0340] 9.11.1 Pipette 90 μl of Cleanup Beads B into the digestion product from step 9.10.4, gently pipette to mix thoroughly, and incubate at room temperature for 10 min.

[0341] 9.11.2 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid becomes clear. Discard the supernatant.

[0342] 9.11.3 Keep the centrifuge tube on the magnetic rack and add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds and remove the supernatant.

[0343] 9.11.4 Repeat the previous step.

[0344] 9.11.5 Keep the centrifuge tube on the magnetic rack, open the tube cover, and dry the magnetic beads at room temperature.

[0345] 9.11.6 Remove the centrifuge tube from the magnetic stand, add 32 μl of TE Buffer, pipette gently until completely mixed, and incubate at room temperature for 10 minutes.

[0346] 9.11.7 Briefly centrifuge, place the centrifuge tube on a magnetic rack, and let it stand for 2 to 5 minutes until the liquid becomes clear. Transfer 30 μl of the supernatant to a new 1.5 mL centrifuge tube.

[0347] 9.11.8 Take 1 μl of the enzyme digestion product and use ssDNA Assay Kit was used to detect the concentration.

[0348] Quality control (QC) standard: concentration greater than 0.5 ng / μl.

[0349] 10. Sequencing

[0350] The qualified libraries were subjected to second-generation double-end sequencing, with left and right read lengths (read1 and read2) of 150 bp each.

[0351] 11. Data Processing

[0352] The data obtained in 11.1 is a double-ended Fastq compressed file. Read1 retains 30bp of tag sequence information, specifically a 20bp barcode (10bp barcode1 + 10bp barcode2) + 10bp UMI. Read2 retains cDNA sequence information, specifically a 100bp cDNA fragment. scRNA_parse software is used to perform barcode correction (matching to a barcode whitelist), quality filtering (filtering sequences with an average quality score below 4), and formatting (adding the corrected barcode and UMI to the read2 Fastq file ID line).

[0353] 11.2 Alignment to the Reference Genome

[0354] 11.2.1 Align the processed single-end fastq files to the human-mouse hybrid reference genome using STAR (2.7.9a) to obtain a sam file.

[0355] 11.2.2 Use PISA (v0.7) to convert the sam file to a bam file, and transfer the bead barcode and UMI sequence in the read ID to the tags of the bam file, denoted as CB and UR respectively.

[0356] 11.2.3 Use PISA to annotate the bam file based on the human-mouse hybrid reference genome and add species tags.

[0357] 11.2.4 Use PISA to correct the UMI of the annotated bam file (Hamming distance is 1, corrected to a more abundant UMI).

[0358] 11.3 Count the number of UMIs and genes corresponding to each bead barcode in human and mouse based on the bam file.

[0359] 11.4 Use barcodeRanks to perform a sudden drop intercept based on the statistical file in 11.3 and select valid cells.

[0360] 11.5 Recalculate the number of UMIs and genes corresponding to the human and mouse bead barcodes based on the obtained valid cells.

[0361] 11.6 If the number of mouse UMIs in a cell accounts for greater than 99% of the total number of human and mouse UMIs, the cell is considered a rare cell (signal); otherwise, the cell is considered a background cell (background). A scatter plot is drawn using the R ggplot2 package (see Figures 7 to 9).

[0362] Figures 7A and 7B show the ratio of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells to background cells is 1:100, where Figure 7A is for repeat one and Figure 7B is for repeat two.

[0363] Figures 8A and 8B show the ratio of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells to background cells is 1:1000, where Figure 8A is for repeat one and Figure 8B is for repeat two.

[0364] Figures 9A and 9B show the ratio of mouse transcripts captured by each magnetic bead to the total number of captured transcripts when the ratio of target cells to background cells is 1:10,000, where Figure 9A is for repeat one and Figure 9B is for repeat two.

[0365] It can be seen that Figures 7 to 9 specifically show the distribution of magnetic beads that capture mouse transcripts under conditions of different mixing ratios of human and mouse cells when the ratio threshold is set to 5%. The vertical axis of the scatter plot is the ratio of the number of mouse transcripts captured by each magnetic bead to the total number of captured transcripts, and the horizontal axis is the magnetic bead serial number. When the ratio of the number of mouse transcripts captured by the magnetic beads is greater than 5%, it is considered that the magnetic beads have captured mouse cells. The hollow diamond patterns in the scatter plot represent target cells (NIH / 3T3), and the solid circular patterns represent background cells (HEK293T). The upper right corner of the scatter plot shows the number of rare cells and background cells. It can be observed from the scatter plots shown in Figures 7 to 9 above that the highest sensitivity of this round of testing reached 1 / 10000, that is, it can effectively detect 1 target cell among 10,000 background cells, proving that the rare cell detection method described in this embodiment has ultra-high sensitivity.

[0366] Example 2

[0367] In this example, female peripheral blood mononuclear cells (PBMCs) were used as background cells, and the human breast cancer cell line MCF-7 was used as the target cells (i.e., rare cells) as the experimental material. A method and platform based on a super-loading strategy was used to detect rare cells. The main variable in this test was the amount of magnetic beads input. The ratio of target cells to background cells was 1:10,000. Two replicates were performed for each group. The specific amount of magnetic beads input, number of cells, and experimental groups are shown in Table 22.

[0368] Table 22

[0369] Group PBM CCF-7 magnetic beads input amount 1, 250W5035W3, 450W5070W5, 650W50105W7, 850W035W9, 1002.2W35W

[0370] Experimental process reference example 1

[0371] Data processing:

[0372] 1. The resulting data are compressed cDNA and oligo double-end Fastq files. The cDNA read1 retains a 30bp tag sequence, specifically a 20bp barcode (10bp barcode1 + 10bp barcode2) + 10bp UMI; the read2 retains the cDNA sequence information, specifically a 100bp cDNA fragment; the oligo library read1 retains a 20bp barcode, specifically a 10bp barcode1 + 10bp barcode2; the read2 retains a 30bp UMI and the oligo bead-specific barcode information (i.e., the barcode information on the index carrier), specifically a 10bp UMI + 10bp barcode1 + 10bp barcode2. The raw Fastq data are barcode-corrected (matching the barcode whitelist), quality-filtered (filtering sequences with an average quality value below 4), and formatted (adding the corrected barcode and UMI to the read2fastq file ID line).

[0373] 2. Align to the reference genome

[0374] 2.1 Align the processed single-end fastq file to the reference genome using STAR (2.7.9a) to obtain a sam file.

[0375] 2.2 Use PISA (v0.7) to convert the sam file into a bam file, and transfer the cell barcode and UMI sequence in the reads ID to the tags of the bam file, recorded as CB and UR respectively.

[0376] 2.3 Use PISA to correct the UMI of the annotated bam file (Hamming distance is 1, corrected to a UMI with higher abundance).

[0377] 2.4 Count the number of UMIs and genes corresponding to each cell barcode based on the bam file.

[0378] 3. Beads merging

[0379] 3.1 Use barcodeRanks to perform a sudden drop interception based on the statistical file in 2.4 and select valid beads.

[0380] 3.2 Calculate the cosine value between beads according to the oligo sequence, filter out background beads with a cosine value greater than that of valid beads, and merge the filtered beads as cells.

[0381] 3.3 Count the cell and gene values ​​and generate a matrix.

[0382] 4. Cell Quality Control and Clustering

[0383] 4.1 Cell-gene matrix quality control filtering: filtering conditions included a mitochondrial ratio greater than 5%, a gene number less than 200 and greater than the upper quartile, and the use of DoubletFinder (version 2.0.3) to remove doublets based on a doublet rate of 5%.

[0384] 4.2 Perform normalization (NormalizeData) and standardization (ScaleData).

[0385] 4.3 Perform PCA dimensionality reduction, and then select the first 50 dimensions for clustering and UMAP dimensionality reduction.

[0386] 5. Marker gene screening

[0387] 5.1 Figure 10 shows a Seurat clustering diagram of PBMC (background cells) and MCF-7 (breast cancer cells) according to this example. As shown in Figure 10 , when Seurat objects are constructed for PBMC (background cells) and MCF-7 (breast cancer cells), MCF-7 cells can be clustered independently and do not overlap with PBMC cells.

[0388] Figure 11 is a graph showing the specific expression of four marker genes (FOLR1, KRT18, BASP1, and C4BPB) in MCF-7 cells according to Example 2 of the present application. In this example, four marker genes were selected based on existing PBMC and MCF data. Figure 11 shows the expression levels of the four genes in MCF-7 cells. It can be seen that FOLR1, KRT18, BASP1, and C4BPB are specifically highly expressed in MCF cells. Therefore, these four genes can be used as marker genes for MCF-7 cells.

[0389] 5.2 The number of test samples was counted based on the four selected marker genes, and the intersection of the four genes was taken as the predicted MCF-7 cell number of the sample (see Table 23).

[0390] Table 23

[0391]

[0392] Table 23 shows the results of predicting MCF-7 cells based on 4 marker genes (FOLR1, KRT18, BASP1 and C4BPB). As can be seen from Table 23, group 7, as a negative control, shows no rare cell detection in 50W background cells when MCF-7 cells (i.e., target cells or rare cells) are not put into the cells. Group 9 is used as a positive control. Under the input amount of 2.2W MCF-7 cells, the droplet microfluidic single cell library device captures 6659 mcf cells. These 6659 MCF-7 cells are screened separately by the above 4 marker genes, and the intersection (overlap) cells are taken, and a total of 6275 intersection MCF-7 cells are obtained, accounting for 94.23% of the total number of detected cells. It can be seen that these 4 marker genes can be effectively used as screening criteria for MCF-7 cells. At the same time, group 7 (negative control) does not detect rare cells, while group 9 (positive control) detects a large number of rare cells, proving that the method for detecting rare cells used in this embodiment has high accuracy.

[0393] In addition, according to the results shown in Table 23, when the magnetic bead input amount is 35W and the input ratio of MCF-7 cells is 0.01% (i.e., Group 1), the number of intersection cells is 1; when the magnetic bead input amount is 70W and the input ratio of MCF-7 cells is 0.01% (i.e., Group 3), the number of intersection cells is 2; when the magnetic bead input amount is 105W and the input ratio of MCF-7 cells is 0.01% (i.e., Group 5), the number of intersection cells is 3. It can be seen that when the input amount of MCF-7 cells in Groups 1, 3 and 5 is 50, based on 50W background cells, the method for detecting rare cells used in this embodiment can achieve effective detection of rare cells. At the same time, such experimental results also prove that the method for detecting rare cells used in this embodiment can effectively detect rare cells in the case of higher and lower magnetic bead input amounts, and the detection rate increases with the increase in the number of magnetic bead inputs. It can be seen that the method for detecting rare cells proposed in the embodiment of the present application can effectively detect rare cells with high accuracy.

[0394] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0395] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for detecting rare cells in a biological sample, comprising: Provide single cells from biological samples; Providing magnetic beads, wherein the magnetic beads are connected to multiple oligonucleotide sequences, each oligonucleotide sequence comprising a barcode tag sequence, a unique molecular identifier sequence (UMI), and a first capture sequence; enclosing a plurality of the single cells and the magnetic beads in a single droplet; constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet and performing sequencing and data analysis; and Specific marker sequence information of rare cells is screened from the single-cell nucleic acid sequence, and it is determined whether the rare cells exist in the biological sample.

2. The method of claim 1, wherein the biological sample is from a mammal, optionally from a pregnant woman or a subject suffering from or suspected of suffering from a disease.

3. The method according to claim 1 or 2, wherein the biological sample is at least one of peripheral blood, urine, saliva or tissue.

4. The method of any one of claims 1 to 3, wherein the rare cells comprise: Embryonic cells, stem cells, progenitor cells, cancer stem cells and / or tumor cells.

5. The method according to any one of claims 1 to 3, wherein the rare cells comprise circulating tumor cells and / or fetal free cells, optionally wherein the fetal free cells are fetal nucleated erythrocytes.

6. The method according to any one of claims 1 to 5, wherein 100,000 to 10 million, preferably 100,000 to 1 million, oligonucleotide sequences are attached to a single magnetic bead. Optionally, wherein the barcode tag sequences of the plurality of oligonucleotide sequences are identical based on the same single magnetic bead, and the barcode tag sequences are different from each other based on different single magnetic beads, Optionally, based on the same single magnetic bead, the unique molecular identifier sequences (UMIs) in the plurality of oligonucleotide sequences are different from each other.

7. The method according to any one of claims 1 to 6, wherein the first capture sequence is selected from at least one of poly(dT) and a DNA capture sequence, preferably the first capture sequence is poly(dT), Optionally, the first capture sequence is located at the 3' end of the oligonucleotide sequence on the magnetic bead, Optionally, each of the oligonucleotide sequences further comprises a linker sequence and / or a second capture sequence.

8. The method according to any one of claims 1 to 7, wherein constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet comprises: Lysing the plurality of single cells to obtain single-cell nucleic acid sequences, wherein the single-cell nucleic acid sequences are captured by the first capture sequence; destroying the droplets to release the magnetic beads connected to the oligonucleotide sequence and the single cell nucleic acid sequence; A DNA library is constructed based on the single cell nucleic acid sequence.

9. The method according to claim 8, wherein the single-cell nucleic acid sequence is an mRNA sequence, and wherein constructing a DNA library based on the single-cell nucleic acid sequence further comprises: After the mRNA sequence is reverse transcribed into a DNA sequence, the DNA library is constructed.

10. The method according to any one of claims 1 to 9, wherein the single droplet comprises 1-10, preferably 5-7, single cells.

11. The method according to any one of claims 1 to 10, wherein each droplet comprises 1-10 magnetic beads, preferably 1 magnetic bead.

12. The method according to any one of claims 1 to 11, wherein the single droplet further comprises an Index carrier, wherein a plurality of Index sequences are connected to the Index carrier, and the Index sequence recognizes the second capture sequence in the oligonucleotide sequence of the magnetic bead in the single droplet and is captured by the second capture sequence.

13. The method according to claim 12, wherein at least 100,000 Index sequences are connected to the Index vector.

14. The method according to claim 12 or 13, wherein the droplet contains 5-15, preferably 8-10 Index carriers.

15. The method of any one of claims 1 to 14, wherein determining the presence or absence of the rare cell in the biological sample comprises: Calculating, based on a single droplet, a ratio between the number of unique molecular identifier sequences (UMIs) corresponding to the single-cell nucleic acid sequence aligned to the specific marker sequence of the rare cell and the total number of all unique molecular identifier sequences (UMIs); comparing the ratio with a set ratio threshold; When the positive ratio is greater than the set ratio threshold, it is determined that the rare cells exist in the biological sample.

16. The method according to claim 4, wherein the set ratio threshold is 1-10%, preferably 1%-5%.

17. A device for detecting rare cells in a biological sample, comprising: A droplet sealing device, configured to seal multiple single cells and magnetic beads in a single droplet, wherein the multiple single cells are derived from the biological sample, and the magnetic beads are connected to multiple oligonucleotide sequences, each oligonucleotide sequence comprising a barcode tag sequence, a unique molecular identifier sequence (UMI), and a first capture sequence; a DNA library construction device, connected to the droplet sealing device, for constructing a DNA library based on the single-cell nucleic acid sequence captured by the first capture sequence in the single droplet; A sequencing device, connected to the DNA library construction device, for sequencing the DNA library; and An analysis device is connected to the sequencing device and is used for data analysis, screening specific marker sequence information of rare cells from the single-cell nucleic acid sequence, and determining whether the rare cells exist in the biological sample.

18. The device of claim 17, wherein the biological sample is from a mammal, optionally the biological sample is from a pregnant female or a subject suffering from or suspected of suffering from a disease.

19. The device according to claim 17 or 18, wherein the biological sample is at least one of peripheral blood, urine, saliva or tissue.

20. The device of any one of claims 17 to 19, wherein the rare cells comprise: Embryonic cells, stem cells, progenitor cells, cancer stem cells and / or tumor cells.

21. The device according to any one of claims 17 to 19, wherein the rare cells comprise circulating tumor cells and / or fetal free cells, optionally wherein the fetal free cells are fetal nucleated red blood cells.

22. The device according to any one of claims 17 to 21, wherein 100,000 to 10 million, preferably 100,000 to 1 million, oligonucleotide sequences are attached to a single magnetic bead. Optionally, wherein the barcode tag sequences of the plurality of oligonucleotide sequences are identical based on the same single magnetic bead, and the barcode tag sequences are different from each other based on different single magnetic beads, Optionally, based on the same single magnetic bead, the unique molecular identifier sequences (UMIs) in the plurality of oligonucleotide sequences are different from each other.

23. The device according to any one of claims 17 to 22, wherein the first capture sequence is selected from at least one of poly(dT) and a DNA capture sequence, preferably the first capture sequence is poly(dT), Optionally, the first capture sequence is located at the 3' end of the oligonucleotide sequence on the magnetic bead, Optionally, each of the oligonucleotide sequences further comprises a linker sequence and / or a second capture sequence.

24. The device according to any one of claims 17 to 23, wherein the DNA library construction device is further used for: Lysing the plurality of single cells to obtain single-cell nucleic acid sequences, wherein the single-cell nucleic acid sequences are captured by the first capture sequence; destroying the droplets to release the magnetic beads connected to the oligonucleotide sequence and the single cell nucleic acid sequence; A DNA library is constructed based on the single cell nucleic acid sequence.

25. The device according to claim 24, wherein the single-cell nucleic acid sequence is an mRNA sequence, and wherein the DNA library construction device is further used for: After the mRNA sequence is reverse transcribed into a DNA sequence, the DNA library is constructed.

26. The device according to any one of claims 17 to 25, wherein the single droplet comprises 1-10, preferably 5-7, single cells.

27. The device according to any one of claims 17 to 26, wherein each droplet comprises 1-10 magnetic beads, preferably 1 magnetic bead.

28. The device according to any one of claims 17 to 27, wherein the single droplet further comprises an Index carrier, wherein a plurality of Index sequences are connected to the Index carrier, and the Index sequence recognizes the second capture sequence in the oligonucleotide sequence of the magnetic bead in the single droplet and is captured by the second capture sequence. The device according to claim 28 , wherein at least 100,000 Index sequences are connected to the Index vector.

30. The device according to claim 28 or 29, wherein the droplet contains 5-15, preferably 8-10 Index carriers.

31. The device according to any one of claims 17 to 30, wherein the analysis device is further configured to: Calculate, based on a single droplet, a ratio between the number of unique molecular identifier sequences (UMIs) corresponding to the single-cell nucleic acid sequence aligned to the specific marker sequence of the rare cell and the total number of all unique molecular identifier sequences (UMIs); comparing the ratio with a set ratio threshold; When the positive ratio is greater than the set ratio threshold, it is determined that the rare cells exist in the biological sample.

32. The device according to claim 29, wherein the set ratio threshold is 1-10%, preferably 1%-5%.

33. A method for diagnosing cancer, comprising: Determining the presence of circulating tumor cells in a biological sample from a subject based on the method of any one of claims 1 to 16; Analyzing the number of circulating tumor cells, the expression of disease-related genes, and / or the regulation of chromatin open regions; and Based on the number of circulating tumor cells and the expression of disease-related genes and / or the regulation of chromatin open regions, it is identified whether the subject has a risk of developing a related disease, the development of the disease and / or the prognosis of the disease.

34. A method for non-invasive fetal diagnosis, comprising: Determining the presence of fetal free cells in a biological sample from a pregnant female based on the method of any one of claims 1 to 15; and The genomic or transcriptomic information of the fetal free cells is analyzed to determine the developmental status of the fetus and / or whether the fetus suffers from a genetic disease.

35. The method of claim 34, wherein the genetic disease is a chromosomal disease, a single gene genetic disease, or a multi-gene genetic disease.

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