Patent Application
20240191299
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence_Listing_76 PP-328958-WO, created Mar. 7, 2022, which is 4 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
The present disclosure relates generally to the field of molecular biology, specifically methods and compositions for sample indexing for high-throughput single-cell analysis.
Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) have become powerful techniques for studying heterogeneous transcription profiles in multicellular systems. With the advent of single-cell sequencing technologies, parallel transcriptional analysis of 103-105 cells or nuclei is now routine. This improves the prospects for screening of hundreds or even thousands of samples for high-throughput analysis of genetic, signal, and drug perturbations.
While next generation sequencing and library construction cost has been significantly reduced, routine analysis of thousands of cells is still expensive for individual laboratories. There are still a wide range of challenges, including reliable recognition of artifacts caused by cell multiplets or technology-dependent batch effects. It has been proven that in the integrated analysis of scRNA-seq experiments, technology and “batch” effects can mask biological signals, so experimental solutions are needed to alleviate these challenges.
Sample multiplexing methods such as CITE-seq, MULTI-seq, or CellTag Indexing addressed the issues by labeling cells with sample-specific barcodes before pooling and single cell separation. In these technologies, sample-specific barcodes just as transcripts are linked to cell barcodes during reverse transcription, such that cells are divided into sample groups by tracking cells with same sample-specific barcodes.
In a “Cell-Hashing” method, a known oligonucleotide sequence is conjugated with an antibody, which is then used to label a cell via an antigen-antibody specific reaction between the antibody and a cell surface protein. However, the price of antibodies is relatively expensive, which leads to higher costs. At the same time, the antigen-antibody reaction depends on the specific cell surface protein for labeling. For various types of clinical samples, it is impossible to label all types of cells, and thus causes certain cell types in the sample to be “lost.” Another method to label cells is called “Click-Tag,” in which methyltetrazine-modified DNA oligonucleotides are connected to cell proteins through an inverse-electron demand Diels-Alder (IEDDA) reaction with heterobifunctional, amine-reactive crosslinker NHS-trans-cyclooctene (NHS-TCO). However, this method is limited to labeling fixed cells, not living cells. It is known that cell fixation can cause cell damage, leading to changes in gene expression in cells.
There remains a need for efficient cell labeling and sample indexing methods for high-throughput analysis of various types of cells.
Provided herein include methods, reagents, compositions, systems and kits for cell labeling and high-throughput single-cell nucleic acid analysis.
Enclosed herein includes a method of indexing a plurality of samples. The method, in some embodiments, comprises: (a) providing a plurality of samples wherein each of the plurality of samples comprises a plurality of cells; (b) for each of the plurality of samples, contacting a coupling agent with the sample, wherein the coupling agent comprises a coupling group and a first reactive group, thereby associating the coupling agent to the surface of the plurality of cells; and (c) for each of the plurality of samples, contacting a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent, thereby generating a plurality of cells each associated with the sample-specific-indexing label, wherein the sample-specific-indexing sequence for each sample is different from other samples.
Also enclosed herein includes a method of analyzing nucleic acids. The method, in some embodiments, comprises: (a) providing a plurality of samples wherein each of the plurality of samples comprises a plurality of cells; (b) for each of the plurality of samples, contacting a coupling agent with the sample, wherein the coupling agent comprises a coupling group and a first reactive group, thereby associating the coupling agent to the surface of the plurality of cells; (c) for each of the plurality of samples, contacting a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent, thereby generating a plurality of cells each associated with the sample-specific-indexing label, wherein the sample-specific-indexing sequence for each sample is different from other samples; (d) pooling the plurality of cells associated with the sample-specific-indexing labels from the plurality of samples to form a pooled sample with a plurality of pooled cells; (e) partitioning the plurality of pooled cells into a plurality of partitions, thereby at least 25% of the plurality of partitions each comprises a single cell of the plurality of pooled cells; (f) analyzing target nucleic acids associated with the single cell, wherein the target nucleic acids comprise the indexing labels associated with the single cell, and (g) determining the sample origin of single cell based on the sample-specific-indexing sequence associated with the single cell.
In some embodiments of the method disclosed herein, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the plurality of partitions each comprises a single cell of the plurality of pooled cells.
In some embodiments, analyzing target nucleic acids comprises sequencing the target nucleic acids, products thereof (e.g., amplification products thereof), or a portion of the target nucleic acids or products thereof. In some embodiments, determining the sample origin of single cell comprises high temperature denaturation, fragment sorting, or a combination thereof.
In some embodiments, the target nucleic acids comprise cellular nucleic acids, viral nucleic acids, bacterial nucleic acids, mitochondrial nucleic acids, synthetic nucleic acids, or amplification product thereof, or a combination thereof. In some embodiments, the target nucleic acids comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. In some embodiments, the target nucleic acids comprise poly-adenylated messenger ribonucleic acid (mRNAs) of the single cell.
In some embodiments, analyzing target nucleic acids comprises barcoding in the plurality of partitions comprising a single cell, using a plurality of barcode molecules in a single partition, to generate barcoded target nucleic acids. In some embodiments, barcoding the target nucleic acids comprises barcoding (i) the indexing labels associated with the single cell and (ii) mRNAs of the single cells to generate (i-a) a barcoded indexing label and (ii-a) barcoded cDNAs. In some embodiments, barcoding target nucleic acids comprise a reverse transcription reaction, and the barcoded targeted nucleic acids comprises complementary deoxyribonucleic acid (cDNA). In some embodiments, a barcode molecule of the plurality of barcode molecules comprises a cell barcode sequence, a molecular label sequence, a primer sequence (e.g., a sequencing primer sequence), a primer binding site, a template switching oligonucleotide, or a combination thereof. In some embodiments, the barcode molecules of the plurality of barcode molecules in a single partition comprise an identical cell barde sequence and different molecular label sequence. In some embodiments, the molecular label sequences comprise unique molecule identifiers (UMIs).
The length of the molecular label sequence can vary, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more, or any number or any range between two of these values. In some embodiments, the molecular label sequence is or is about 2-40 nucleotides in length. The sequencing primer sequence can be, for example, a Read 1 sequence, a Read 2 sequence, or a portion thereof.
In some embodiments, in each of the plurality comprising a single cell, the plurality of barcode molecules are attached to, reversibly attached to, covalently attached to, or irreversibly attached to a bead. In some embodiments, barcoding in a single partition comprises partitioning the bead into the single partition. In some embodiments, partitioning the plurality of pooled cells into a plurality of partitions comprises co-partitioning the pooled cells and the bead into the single partitions. The bead can be, for example, a solid bead. In some embodiments, the bead is a magnetic bead or a polymer bead.
In some embodiments, the plurality of partitions compris droplets or microwells. In some embodiments, analyzing target nucleic acids comprises introducing a plurality of template switching oligonucleotides into the partition and barcoding the plurality of target nucleic acids by extending the plurality of barcode molecules using the target nucleic acids and the plurality of template switching oligonucleotides as templates to generate barcoded nucleic acids.
In some embodiments, analyzing target nucleic acids comprises introducing a plurality of extension primers to the partition and barcoding the target nucleic acids by extending the plurality of extension primers using the target nucleic acids as templates and the plurality of barcode molecules as template switching oligonucleotides to generate barcoded nucleic acids.
In some embodiments, the coupling group of the coupling agent is capable of forming a covalent bond with the surface of the plurality of cells. In some embodiments, the coupling agent and/or the indexing label further comprise a hydrophilic group, for example a hydrophilic group comprises PEG. In some embodiments, the coupling agent is NHS-PEG4-TCO. In some embodiments, coupling group of the coupling agent is capable of forming a covalent bond with —NH2 on the surface of the plurality of cells. The plurality of cells can comprise fixed cells, living cells, or any combination thereof.
In some embodiments, each of the indexing labels is a 5′ NHS-PEG5-Tz modified oligonucleotide. In some embodiments, each of the indexing labels further comprises a PCR handle sequence, a UMI, a capture sequence, or a combination thereof. In some embodiments, the capture sequence comprises a poly(dA) sequence. In some embodiments, the indexing labels are single-stranded DNA. In some embodiments, first reactive group and the second reactive group form the second covalent bond in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, one of the first reactive group and the second reactive group comprises a tetrazine (Tz) group and the other comprises a trans-cyclooctene (TCO) group.
The number of the samples in the plurality of samples can vary. For example, the plurality of samples can comprise, comprise at least, or comprise at most, 3, 6, 12, 15, 18, 21, 24, 27, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, or a number or a range between any two of these values, samples. In some embodiments, the plurality of samples comprise at least 12 samples. The samples can be, for example clinical samples, environmental samples, biological samples, or a combination thereof. In some embodiments, the plurality of cells comprises prokaryotic cells, eukaryotic cells, or a combination thereof.
The method can, for example, comprise washing the plurality of cells before step (a) and/or resuspending the cells in aqueous solution. In some embodiments, the method comprises washing the plurality of cells to remove unbound coupling agent after step (b) and before step (c) and/or resuspending the cells in aqueous solution. In some embodiments, the method comprises washing the cells associated with the sample-specific-indexing labels to remove unbound sample-specific-indexing labels after step (c) and/or resuspending the cells in aqueous solution.
Also disclosed herein include a kit for indexing a plurality of samples. The kit can, in some embodiments, comprise a coupling agent comprising a coupling group and a first reactive group; for each of the plurality of samples, a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent; and instructions to use the kit for indexing multiple samples according to any one of the methods disclosed herein for indexing multiple samples.
Also disclosed herein include a kit for analyzing nucleic acids in a plurality of samples. The kit can, in some embodiments, comprises a coupling agent each comprising a coupling group and a first reactive group; for each of the plurality of samples, a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent; a plurality of beads, wherein each bead is attached to, reversibly attached to, covalently attached to, or irreversibly attached to a plurality of barcode molecules, and wherein each barcode molecule of the plurality of barcode molecules comprises a cell barcode sequence, a molecular label sequence, a primer sequence, a primer binding site, a template switching oligonucleotide, or a combination thereof; and instructions to use the kit for indexing multiple samples according to any one of the methods disclosed herein for indexing multiple samples.
In some embodiments, the present disclosure provides a method for cell labeling for high-throughput single-cell RNA sequencing. The method can comprise:
In some embodiments, the cell labeling comprises reaction of the cell with a chemical reagent.
In some embodiments, the index sequence can additionally comprise a poly(A) sequence that can be capture with oligo-dT.
In some embodiments, the sample index can comprise a unique molecular index (UMI) sequence that can be used to quantification and data split.
In some embodiments, the sample index can be separated by high temperature denaturation.
In some embodiments, the sample index can be separated by fragment sorting.
In some embodiments, the sample index library is analyzed by sequencing.
The present disclosure also provides the chemical reagents used in the present method. The present disclosure also provides a product that includes reagents needed to carry out the present method of cell labeling.
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.
All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.
Provided herein include methods, reagents, compositions, systems and kits for cell labeling and high-throughput single-cell nucleic acid analysis. The methods, reagents, compositions, systems, and kits can be used for sample indexing to allow high-throughput single-cell analysis (e.g., single-cell RNA sequencing), and can also be used for fast and convenient cell labeling and sample pooling. The present methods provide an efficient way to label cells and index samples, and can have broad applicability in cell labeling without being limited by cell types. Combined with single-cell sequencing, the methods disclosed herein can greatly reduce the cost of single cell analysis, and simultaneously improve the throughput of sample processing. In addition, the present sample indexing method can be used to facilitate the wide application of high-throughput single cell transcriptome sequencing.
Disclosed herein include methods of indexing multiple samples comprising cells. The method, in some embodiments, comprises: (a) providing a plurality of samples wherein each of the plurality of samples comprises a plurality of cells; (b) for each of the plurality of samples, contacting a coupling agent with the sample, wherein the coupling agent comprises a coupling group and a first reactive group, thereby associating the coupling agent to the surface of the plurality of cells; and (c) for each of the plurality of samples, contacting a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent, thereby generating a plurality of cells each associated with the sample-specific-indexing label, wherein the sample-specific-indexing sequence for each sample is different from other samples. The characteristics of the cells to which the method can be applied to are not particularly limited. The state, type, or morphology of the cells can vary. For example, the cells can be living cells, fixed cells, healthy cells, cells in disease state, or any combination thereof.
The association of the coupling agent to the cell surface can be based on, or a result of, the covalent bond. The association of the coupling agent and/or the label to the cell surface can be independent of, or unaffected by, the cell type or any specific cell surface proteins (such as antibodies, antigens, structural proteins, or receptors). Accordingly, a wide variety of cells can be labeled using the present methods.
The surface of the cell can comprise a surface reactive group that reacts with the coupling group of the coupling agent to form a covalent bond. The surface reactive group and the coupling group can include, but are not limited to, known crosslinking groups and their counterparts. For example, the surface reactive group and the coupling group can be selected from nucleophilic groups, such as an amino group (—NH2), a hydroxy group (—OH), a sulfhydryl group (—SH), or a carboxylate group (—COOH), and corresponding electrophilic groups, such as activated carboxylate groups, including but not limited to acid chlorides, anhydrides, carbodiimide derivatives, and N-hydroxysuccinimide (NHS) esters. In some embodiments, the surface reactive group is an amino group (e.g., —NH2 on a side chain of an amino acid of a surface protein) and the coupling group is an NHS group. Other suitable coupling pairs for the surface reactive group and the coupling group can be selected according to known technologies. In some embodiments, the cell surface can be modified to introduce the surface reactive group prior to the attachment of the coupling agent.
After attachment of the coupling agent to the cell surface, the first reactive group of the coupling agent can be exposed (e.g., toward the outside of the cell) for reaction with the second reactive group of the label, thereby attaching the label to the cell surface through the coupling agent. In some embodiments, the first reactive group and the second reactive group form the second covalent bond in an inverse electron demand Diels-Alder (IEDDA) reaction. The IEDDA cycloaddition as a click chemistry conjugation reaction has been used for a variety of applications, including labeling of nanoparticles, antibodies, oligonucleotides, small molecules, and radiopharmaceuticals. For example, one of the first and the second reactive group can comprise a tetrazine (Tz) group, and the other can comprise a trans-cyclooctene (TCO) group. In some embodiments, the first reactive group comprises a TCO group and the second reactive group comprises a Tz group.
The coupling agent can comprise a hydrophilic group. The presence of the hydrophilic group can improve the aqueous solubility of the coupling agent to facilitate cell labeling. Suitable hydrophilic groups can include for example, hydrophilic polymers such as polyethylene glycol (PEG), poly(2-oxazoline), poly(vinyl alcohol), or polyacrylate. In some embodiments, the hydrophilic group comprises PEG. The PEG group can include one or more ethylene glycol (—CH2CH2O—) units. For example, the hydrophilic groups can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range between any two of these values, ethylene glycol units. In some embodiments, the hydrophilic group comprises 3, 4, 5, 6, 7, or 8 ethylene glycol units. In some embodiments, the hydrophilic group comprises 4 or 5 ethylene glycol units. In some embodiments, the coupling agent is NHS-PEG4-TCO.
The indexing label can comprise an identical sample-specific-indexing sequence and a reactive group capable of forming a covalent bond with the reactive group of the coupling agent. In some embodiments, the reactive group of the indexing label can be a function group (e.g., Tz or TCO) that forms a covalent bond with the reactive group of the coupling agent, for example in an inverse electron demand Diels-Alder (IEDDA) reaction as described herein. The indexing label can be referred to herein as indexing oligonucleotide, sample index oligo, sample-specific barcode, or sample tag.
The indexing label can comprise a sample-specific-indexing sequence (also referred to herein as sample barcode sequence). The sample-specific-indexing sequence can be used to identify a sample which the cell being labeled is from. The sample can be, for example, a cell line, a biological sample, an environmental sample, or a forensic sample.
A sample can comprise one or more types of cells. The number of types of cells in a sample can be different in different embodiments. The sample can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, or a number or a range between any two of these values, types of cells. In some embodiments, the sample comprises one type of cells. In some embodiments, the sample is a cell line.
The number of cells in a sample to be labeled can be different in different embodiments. The number of cells in different samples of the plurality of samples to be labeled can also be different in different embodiments. The number of cells in a sample can be, be about, be at least, be at least about, be at most, or be at most about, 100, 1000, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, or a number or a range between any two of these values. Cells of a sample can be identified by an identical sample-specific-indexing sequence. In some embodiments, at least two cells from a same sample are labeled with identical sample-specific-indexing sequences. Cells of different samples can be labeled with different sample-specific-indexing sequences. In some embodiments, at least two cells from different samples are labeled with different sample-specific-indexing sequences.
The sample-specific-indexing sequence can be, be about, be at least, be at least about, be at most, or be at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or a number or a range between any two of these values, nucleotides in length.
The indexing label can further comprise, for example, a PCR handle sequence, a random index sequence (e.g., an unique molecular index, or UMI), and/or a capture sequence (e.g., a poly(A) primer sequence). The random index sequence can be used to identify the molecular origin of the indexing labels and/or quantify the abundance of indexing labels on the labeled cell.
In some embodiments, the indexing label further comprises a poly(A) sequence, such as a poly(A) tail. An indexing label comprising a poly(A) sequence can be reverse transcribed, for example, by a reverse transcriptase.
The indexing label can be a single stranded DNA (ssDNA). In some embodiments, the indexing label is, is about, is at least, is at least about, is at most, or is at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length. In some embodiments, the indexing label comprises or consists of a sequence of SEQ ID NO:1.
The indexing label can further comprise a hydrophilic group. The presence of the hydrophilic group can improve the aqueous solubility of the label to facilitate cell labeling. Suitable hydrophilic groups can include for example, hydrophilic polymers such as polyethylene glycol (PEG), poly(2-oxazoline), poly(vinyl alcohol), or polyacrylate. In some embodiments, the hydrophilic groups comprise PEG. The PEG group can include one or more ethylene glycol (—CH2CH2O—) units. For example, the hydrophilic groups can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range between any two of these values, ethylene glycol units. In some embodiments, the hydrophilic group comprises 3, 4, 5, 6, 7, or 8 ethylene glycol units. In some embodiments, the hydrophilic group comprises 4 or 5 ethylene glycol units.
The indexing label can be prepared, for example, by attaching the indexing label to the reactive group through a coupling reaction. In some embodiments, the label is prepared by reacting an indexing label having a terminal amino group (e.g., 5′ modified NH2-C6-ssDNA) with an NHS activated molecule comprising the second reactive group (e.g., NHS-PEG5-Tz).
The cell (e.g., a living cell) can be treated with the coupling agent to generate the activated cell surface, and the label comprising the indexing label is subsequently attached to the activated cell surface (e.g., in an IEDDA reaction). In some embodiments, the cell having the activated cell surface is washed to remove the unbound coupling agent before attaching the label. In some embodiments, the cell is a living cell, and reacting the living cell with the coupling agent and the label can be carried out in a one-pot reaction. After labeling, the cells (e.g., living cells) can be washed and counted.
One or more types of cells, or cells from one or more samples, can be labeled using the present method. For example, different types of cells, or cells from different samples, can be labeled with labels having different sample-specific-indexing sequences according to the labeling methods disclosed herein.
A plurality of cells (e.g., living cells) labeled with labels having different sample-specific-indexing sequences can be pooled to generate pooled labeled cells for further analysis. The number of different sample-specific-indexing sequences in the pooled labeled cells can be, be about, be at least, be at least about, be at most, or be at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range between any two of these values.
Disclosed herein include methods of analyzing nucleic acids. In some embodiments, the method comprises a method of analyzing nucleic acids. The method, in some embodiments, comprises: (a) providing a plurality of samples wherein each of the plurality of samples comprises a plurality of cells; (b) for each of the plurality of samples, contacting a coupling agent with the sample, wherein the coupling agent comprises a coupling group and a first reactive group, thereby associating the coupling agent to the surface of the plurality of cells; (c) for each of the plurality of samples, contacting a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent, thereby generating a plurality of cells each associated with the sample-specific-indexing label, wherein the sample-specific-indexing sequence for each sample is different from other samples; (d) pooling the plurality of cells associated with the sample-specific-indexing labels from the plurality of samples to form a pooled sample with a plurality of pooled cells; (e) partitioning the plurality of pooled cells into a plurality of partitions, thereby at least 25% of the plurality of partitions each comprises a single cell of the plurality of pooled cells; (f) analyzing target nucleic acids associated with the single cell, wherein the target nucleic acids comprise the indexing labels associated with the single cell, and (g) determining the sample origin of single cell based on the sample-specific-indexing sequence associated with the single cell.
In some embodiments of the method, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the plurality of partitions each comprises a single cell of the plurality of pooled cells.
Analyzing target nucleic acids can, for example, comprise sequencing the target nucleic acids, products thereof (e.g., amplification products thereof), or a portion of the target nucleic acids or products thereof. In some embodiments, determining the sample origin of single cell comprises high temperature denaturation, fragment sorting, or a combination thereof. Target nucleic acids can, for example, comprise cellular nucleic acids, viral nucleic acids, bacterial nucleic acids, mitochondrial nucleic acids, synthetic nucleic acids, or amplification product thereof, or a combination thereof. In some embodiments, the target nucleic acids comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. In some embodiments, the target nucleic acids comprise poly-adenylated messenger ribonucleic acid (mRNAs) of the single cell.
The cells (e.g., living cells) can be labeled, for example, using the labeling method as described herein. In some embodiments, the indexing label comprises a sample-specific-indexing sequence and a random index sequence. In some embodiments, the indexing label further comprises a poly(A) sequence, such as a poly(A) tail. In some embodiments, the indexing label is a single-stranded DNA.
In some embodiments, the analyzing step of the present method comprises barcoding (i) the indexing label of the label of the plurality of labels covalently bonded to the single cell or single living cell and (ii) the plurality of target nucleic acids associated with the single cell or single live cell using a plurality of barcode molecules to generate (i-a) a barcoded indexing label and (ii-a) a plurality of barcoded nucleic acids. In some embodiments, the method further comprise analyzing (e.g., sequencing) the barcoded indexing label and the plurality of barcoded nucleic acids.
In some embodiments, the cells (e.g., living cells) are labeled with indexing labels each comprising a poly(A) sequence, such that target nucleic acids (e.g., RNAs) associated with the cells and the indexing label attached to the cells can be barcoded (e.g., by reverse transcription) and analyzed simultaneously.
The present disclosure provides an efficient and cost-effective cell labeling method, which can be used for multiplexed cellular analysis, including, for example, multiplexed single-cell RNA sequencing (scRNA-seq).
In some embodiments, the present methods includes labeling a cell surface by reacting NHS-reactive amines on the cell surface with NHS-PEG4-TCO, followed by an inverse electron-demand Diels-Alder (IEDDA) reaction to attach 5′NHS-PEG5-Tz modified sample-specific barcodes to the NHS-PEG4-TCO on the cell surface. The sample-specific oligo can include a PCR handle sequence, a sample barcode sequence, a UMI sequence (for molecular quantification), and/or a poly(A) tail. In some embodiments, the oligo label on the cell surface can be captured and reverse transcribed and amplified just like mRNA molecules. After sequencing, sample barcode mapping and quantification can be used to split the mixed sample sequencing data, thereby achieving scRNA-seq sample demultiplexing. The present disclosure can employ chemical modification methods to label living cells and can have a wide range of applicability.
A partition as used herein refers to a part, a portion, or a division sequestered from the rest of the parts, portions, or divisions. A partition can be formed through the use of wells, microwells, multi-well plates, microwell arrays, microfluidics, dilution, dispensing, droplets, or any other means of sequestering one fraction of a sample from another. In some embodiments, a partition is a well, a droplet or a microwell. In some embodiments, a partition is a microwell.
The plurality of partitions can comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, or 500000 partitions. In some embodiments, the plurality of partitions comprises at least 100 partitions.
The barcode molecules can be introduced to the partitions directly. The barcode molecules can be attached to a particle (e.g., a bead), and introducing the barcode molecules can comprise introducing the particle to a partition. In some embodiments, barcode molecules can be introduced into the partitions (e.g., microwells) by attaching or synthesizing the plurality of barcode molecules onto the surface of the partitions.
The plurality of partitions can comprise a plurality of microwells of a microwell array. The microwell array can comprise different numbers of microwells in different implementations. In some embodiments, the microwell array can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, or a number or a range between any two of these values, microwells. The microwells can be arranged into rows and columns. The number of microwells in a row (or a column) can be, be about, be at least, be at least about, be at most, or be at most about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, or a number or a range between any two of these values. Adjacent rows (or columns) of microwells can be aligned or staggered, for example.
The width, length, depth (or height), radius, or diameter of a microwell can vary. In some embodiments, the width, length, depth (or height), radius, or diameter of a microwell of the plurality of microwells can be, be about, be at least, be at least about, be at most, or be at most about, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, or a number or a range between any two of these values. For example, the width, the length, and/or depth of a microwell can be 10 μm to 200 μm, including 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μm, or a number or a range between any two of these values. The shape of a microwell can vary, for example circular, elliptical, square, rectangular, triangular, or hexagonal shape.
The volume of one, one or more, or each, of the plurality of microwells can be different in different embodiments. The volume of one, one or more, or each, of the plurality of microwells can be, be about, be at least, be at least about, be at most, or be at most about, 1 nm3, 2 nm3, 3 nm3, 4 nm3, 5 nm3, 6 nm3, 7 nm3, 8 nm3, 9 nm3, 10 nm3, 20 nm3, 30 nm3, 40 nm3, 50 nm3, 60 nm3, 70 nm3, 80 nm3, 90 nm3, 100 nm3, 200 nm3, 300 nm3, 400 nm3, 500 nm3, 600 nm3, 700 nm3, 800 nm3, 900 μm3, 1000 nm3, 10000 nm3, 100000 μm3, 1000000 nm3, 10000000 nm3, 100000000 μm3, 1000000000 nm3, 2 μm3, 3 μm3, 4 μm3, 5 μm3, 6 μm3, 7 μm3, 8 μm3, 9 μm3, 10 μm3, 20 μm3, 30 μm3, 40 μm3, 50 μm3, 60 μm3, 70 μm3, 80 μm3, 90 μm3, 100 μm3, 200 μm3, 300 μm3, 400 μm3, 500 μm3, 600 μm3, 700 μm3, 800 μm3, 900 μm3, 1000 μm3, 10000 μm3, or a number or a range between any two of these values. The volume of one, one or more, or each, of the plurality of microwells can be, be about, be at least, be at least about, be at most, or be at most about, 1 nanolieter (nl), 2 nl, 3 nl, 4 nl, 5 nl, 6 nl, 7 nl, 8 nl, 9 nl, 10 nl, 11 nl, 12 nl, 13 nl, 14 nl, 15 nl, 16 nl, 17 nl, 18 nl, 19 nl, 20 nl, 21 nl, 22 nl, 23 nl, 24 nl, 25 nl, 26 nl, 27 nl, 28 nl, 29 nl, 30 nl, 31 nl, 32 nl, 33 nl, 34 nl, 35 nl, 36 nl, 37 nl, 38 nl, 39 nl, 40 nl, 41 nl, 42 nl, 43 nl, 44 nl, 45 nl, 46 nl, 47 nl, 48 nl, 49 nl, 50 nl, 51 nl, 52 nl, 53 nl, 54 nl, 55 nl, 56 nl, 57 nl, 58 nl, 59 nl, 60 nl, 61 nl, 62 nl, 63 nl, 64 nl, 65 nl, 66 nl, 67 nl, 68 nl, 69 nl, 70 nl, 71 nl, 72 nl, 73 nl, 74 nl, 75 nl, 76 nl, 77 nl, 78 nl, 79 nl, 80 nl, 81 nl, 82 nl, 83 nl, 84 nl, 85 nl, 86 nl, 87 nl, 88 nl, 89 nl, 90 nl, 91 nl, 92 nl, 93 nl, 94 nl, 95 nl, 96 nl, 97 nl, 98 nl, 99 nl, 100 nl, or a number or a range between any two of these values. For example, the volume of one, one or more, or each, of the plurality of microwells is about 1 nm3 to about 10000 μm3.
The microwell array comprising a plurality of microwells can be formed from any suitable material. In some embodiments, a microwell array comprising a plurality of microwells can be formed from a material selected from silicon, glass, ceramic, elastomers such as polydimethylsiloxane (PDMS) and thermoset polyester, thermoplastic polymers such as polystyrene, polycarbonate, poly(methyl methacrylate) (PMMA), poly-ethylene glycol diacrylate (PEGDA), Teflon, polyurethane (PU), composite materials such as cyclic-olefin copolymer, and combinations thereof.
Partitions (e.g., microwells or droplets) can be introduced with samples, free reagents, and/or reagents encapsulated in microcapsules. The reagents can comprise restriction enzymes, ligase, polymerase, fluorophores, oligonucleotide barcodes, oligonucleotide probes, adapters, buffers, dNTPs, ddNTPs, and other reagents required for performing the methods described herein.
Cells (e.g., cells labeled using the labeling methods disclosed herein) and/or particles (e.g., beads, including barcoding beads disclosed herein) can be partitioned (separately partitioned or co-partitioned) into a plurality of partitions. As a result of partitioning, the percentage of the plurality of partitions comprising a desired number of cell(s) (e.g., a single cell), a single particle (e.g., a bead) or both of a desired number of cell(s) and a single particle can vary. For example, the percentage of the plurality of partitions comprising the desired number of cell(s) and/or a single particle can be, be about, be at least, be at least about, be at most, or be at most about, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values. In some embodiments, at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% of the plurality of partitions comprise a desired number of cell(s). In some embodiments, at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% of the plurality of partitions comprise a desired number of cell(s) and a single particle.
The percentage of the plurality of partitions comprising no cell can vary. For example, the percentage of the plurality of partitions comprising no cell can be, be about, be at least, be at least about, be at most, or be at most about, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a number or a range between any two of these values. In some embodiments, at most 50% of partitions of the plurality of partitions can comprise no cell of the plurality of cells.
The percentage of the plurality of partitions comprising more than the desired number of cell(s) can be different in different embodiments. For example, the percentage of the plurality of partitions comprising more than the desired number of cell(s) can be, be about, be at least, be at least about, be at most, or be at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a number or a range between any two of these values. In some embodiments, at most 10% of partitions of the plurality of partitions can comprise more than the desired number of cell(s).
As described herein, cells can be associated with target nucleic acids. For example, a cell can comprise one or more target nucleic acids (e.g., mRNA) or can be labeled with one or more target nucleic acids (e.g., directly, or indirectly through a binding moiety, such as an antibody conjugated with the nucleic acid). The target nucleic acids associated with the cell can be from, on the surface of, or binding to the surface of the cell. A target nucleic acid can have a sequence (e.g., an mRNA sequence, excluding the poly(A) tail).
The target nucleic acids associated with the cell can comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and/or any combination or hybrid thereof. The target nucleic acids can be single-stranded or double-stranded, or contain portions of both double-stranded or single-stranded sequences. The target nucleic acids can contain any combination of nucleotides, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine and any nucleotide derivative thereof. As used herein, the term “nucleotide” can include naturally occurring nucleotides and nucleotide analogs, including both synthetic and naturally occurring species. The target nucleic acids can be genomic DNA (gDNA), mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA), ribozyme, riboswitch or viral RNA, or any nucleic acids that may be obtained from a sample.
The plurality of target nucleic acids can, for example, comprise DNA, gDNA, RNA, and/or mRNA. In some embodiments, the plurality of target nucleic acids comprises mRNA, for example a poly-adenylated mRNA.
In some embodiments of the methods disclosed herein, analyzing target nucleic acids comprises barcoding in the plurality of partitions comprising a single cell, using a plurality of barcode molecules in a single partition, to generate barcoded target nucleic acids. In some embodiments, barcoding the target nucleic acids comprises barcoding (i) the indexing labels associated with the single cell and (ii) mRNAs of the single cells to generate (i-a) a barcoded indexing label and (ii-a) barcoded cDNAs. In some embodiments, barcoding target nucleic acids comprise a reverse transcription reaction, and the barcoded targeted nucleic acids comprises cDNA. In some embodiments, a barcode molecule of the plurality of barcode molecules comprises a cell barcode sequence, a molecular label sequence, a primer sequence (e.g., a sequencing primer sequence), a primer binding site, a template switching oligonucleotide, or a combination thereof. In some embodiments, the barcode molecules of the plurality of barcode molecules in a single partition comprise an identical cell barde sequence and different molecular label sequence. In some embodiments, the molecular label sequences comprise UMIs.
In the methods disclosed herein, barcode molecules can be introduced into the partitions for barcoding indexing labels and target nucleic acids. For example, the amount of the barcode molecules added directly to a partition can be, be about, be at least, be at least about, be at most, or be at most about, 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng, 0.9 ng, 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1000 ng, 2000 ng, 3000 ng, 4000 ng, 5000 ng, 6000 ng, 7000 ng, 8000 ng, 9000 ng, 10000 ng, 2000 ng, 3000 ng, 4000 ng, 5000 ng, 6000 ng, 7000 ng, 8000 ng, 90000 ng or a number or a range between any two of these values.
The barcode molecules introduced into the partitions (e.g., microwells or droplets) can be associated with particles (e.g., beads). In some embodiments, introducing the plurality of barcode molecules to the partition comprises introducing a particle comprising the plurality of barcode molecules to the partition. The particles can provide a surface upon which molecules, such as oligonucleotides, can be synthesized or attached. In some embodiments, the plurality of barcode molecules are attached to, reversibly attached to, covalently attached to, or irreversibly attached to the particle.
The particle can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or a number or a range between any two of these values, barcode molecules. The attachment of barcode molecules to the particle can be covalent or non-covalent via non-covalent bonds such as ionic bonds, hydrogen bonds, or van der Waals interactions. The attachment can be direct to the surface of a particle or indirect through other oligonucleotide sequences attached to the surface of a particle.
The particle (e.g., a bead) can be dissolvable, degradable, or disruptable. A particle can be a gel particle such as a hydrogel particle. In some embodiments, the gel particle is degradable upon application of a stimulus. The stimulus can comprise a thermal stimulus, a chemical stimulus, a biological stimulus, a photo-stimulus, or a combination thereof. The particle can be a solid particle and/or a magnetic particle. In some embodiments, the particle is a magnetic particle. The magnetic particle can comprise a paramagnetic material coated or embedded in the magnetic particle (e.g. on a surface, in an intermediate layer, and/or mixed with other materials of the magnetic particle). A paramagnetic material refers to a material having a magnetic susceptibility slightly greater than 1 (e.g. between about 1 and about 5). A magnetic susceptibility is a measure of how much a material can become magnetized in an applied magnetic field. Paramagnetic materials include, but not limited to, magnesium, molybdenum, lithium, aluminum, nickel, tantalum, titanium, iron oxide, gold, copper, or a combination thereof. In some embodiments, the magnetic particle comprising barcode molecules can be immobilized or retained in a partition (such as a microwell or a well) by an external magnetic field, thereby retaining the barcode molecules in a partition. The magnetic particle comprising barcode molecules can be mobilized or released when the external magnetic field is removed.
A particle can, for example, be immobilized or retained in a partition (e.g., a microwell) through an interaction between two members of a binding pair. For example, the partition (e.g., microwell) can be coated with a capture moiety (e.g., a member of a binding pair) capable of binding with a binding moiety (the other member of the binding pair) comprised in or conjugated to a particle, such that the binding of the two moieties results in the attachment of the particle to the partition, thereby immobilizing or retaining the particle in the partition. For example, the surface of a partition can be coated with streptavidin. The biotinylated particle can be attached to the surface of the partition via streptavidin-biotin interaction.
Particles can be of uniform size or heterogeneous size. For example, one or more of the particles can have a diameter of about, at least, at least about, at most, or at most about, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm.
In some embodiments, a particle can be sized such that at most one particle, not two particles, can fit one partition. A size or dimension (e.g., length, width, depth, radius, or diameter) of a particle can be different in different embodiments. For example, a size or dimension of one, or each, particle can be, be about, be at least, be at least about, be at most, or be at most about, 20 nanometer (nm), 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800 nm, 810 nm, 820 nm, 830 nm, 840 nm, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, 1000 nm, 2 micrometer (μm), 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, or a number or a range between any two of these values. In some embodiments, a size or dimension of one, or each, particle is about 1 nm to about 100 μm. In some embodiments, the particle can have a dimension about 10 μm to about 100 μm (e.g., 30 μm).
The volume of one, or each, particle can vary. The volume of one, or each, particle can be, be about, be at least, be at least about, be at most, or be at most about, 1 nm3, 2 nm3, 3 nm3, 4 nm3, 5 nm3, 6 nm3, 7 nm3, 8 nm3, 9 nm3, 10 nm3, 20 nm3, 30 nm3, 40 nm3, 50 nm3, 60 nm3, 70 nm3, 80 nm3, 90 nm3, 100 nm3, 200 nm3, 300 nm3, 400 nm3, 500 nm3, 600 nm3, 700 nm3, 800 nm3, 900 μm3, 1000 nm3, 10000 nm3, 100000 μm3, 1000000 nm3, 10000000 nm3, 100000000 μm3, 1000000000 nm3, 2 μm3, 3 μm3, 4 μm3, 5 μm3, 6 μm3, 7 μm3, 8 μm3, 9 μm3, 10 μm3, 20 μm3, 30 μm3, 40 μm3, 50 μm3, 60 μm3, 70 μm3, 80 μm3, 90 μm3, 100 μm3, 200 μm3, 300 μm3, 400 μm3, 500 μm3, 600 μm3, 700 μm3, 800 μm3, 900 μm3, 1000 μm3, 10000 μm3, 100000 μm3, 1000000 μm3, or a number or a range between any two of these values. The volume of one, or each, particle can be, be about, be at least, be at least about, be at most, or be at most about, 1 nanoliter (nL), 2 nL, 3 nL, 4 nL, 5 nL, 6 nL, 7 nL, 8 nL, 9 nL, 10 nL, 11 nL, 12 nL, 13 nL, 14 nL, 15 nL, 16 nL, 17 nL, 18 nL, 19 nL, 20 nL, 21 nL, 22 nL, 23 nL, 24 nL, 25 nL, 26 nL, 27 nL, 28 nL, 29 nL, 30 nL, 31 nL, 32 nL, 33 nL, 34 nL, 35 nL, 36 nL, 37 nL, 38 nL, 39 nL, 40 nL, 41 nL, 42 nL, 43 nL, 44 nL, 45 nL, 46 nL, 47 nL, 48 nL, 49 nL, 50 nL, 51 nL, 52 nL, 53 nL, 54 nL, 55 nL, 56 nL, 57 nL, 58 nL, 59 nL, 60 nL, 61 nL, 62 nL, 63 nL, 64 nL, 65 nL, 66 nL, 67 nL, 68 nL, 69 nL, 70 nL, 71 nL, 72 nL, 73 nL, 74 nL, 75 nL, 76 nL, 77 nL, 78 nL, 79 nL, 80 nL, 81 nL, 82 nL, 83 nL, 84 nL, 85 nL, 86 nL, 87 nL, 88 nL, 89 nL, 90 nL, 91 nL, 92 nL, 93 nL, 94 nL, 95 nL, 96 nL, 97 nL, 98 nL, 99 nL, 100 nL, or a number or a range between any two of these values. In some embodiments, the volume of one, or each, particle is about 1 nm3 to about 1000000 μm3.
The number of particles introduced into a plurality of partitions can be different in different embodiments. In some embodiments, the number of particles introduced into a plurality of partitions is, is about, is at least, is at least about, is at most, or is at most, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, or a number or a range between any two of these values.
In some embodiments, particles are introduced to the partitions such that the percentage of partitions each occupied with one particle is, is about, is at least, is at least about, is at most, or is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values. In some embodiments, at least 80% of the plurality of partitions is each occupied with one particle. In some embodiments, particles are introduced to the partitions such that the percentage of partitions with no particle is, is about, is at least, is at least about, is at most, or is at most about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a number or a range between any two of these values. In some embodiments, at most 20% of the plurality of partitions contain no particle.
Barcode molecules (e.g., barcode molecules attached to particles) can be partitioned, for example, in microwells or wells. The term “barcode” as used herein can be a verb or a noun. When used as a noun, the term “barcode” or “barcode molecule” refers to a label that can be attached to a polynucleotide, or any variant thereof, to convey information about the polynucleotide. For example, a barcode can be a polynucleotide sequence attached to fragments of the target nucleic acids associated with a cell in the partition. The barcode can then be sequenced alone or with the fragments of the target nucleic acids associated with the cell. The presence of the same barcode on multiple sequences or different barcodes on different sequences can provide information about the cell origin and/or the molecular origin of the sequences. When used as a verb, the term “barcode” refers to a process of attaching a barcode or a barcode molecule to a target nucleic acid associated with the cell.
Barcode molecules can be generated from a variety of different formats, including pre-designed polynucleotide barcodes, randomly synthesized barcode sequences, microarray-based barcode synthesis, random N-mers, or combinations thereof as will be understood by a person skilled in the art.
The plurality of barcode molecules can comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or a number or a range between any two of these values.
A barcode molecule (or a segment of a barcode molecule, such as a cell barcode sequence or a molecular barcode sequence) can be in any suitable length. For example, a barcode molecule (or a segment of a barcode molecule) can be about 2 to about 500 nucleotides in length, about 2 to about 100 nucleotides in length, about 2 to about 50 nucleotides in length, about 2 to about 40 nucleotides in length, about 4 to about 20 nucleotides in length, or about 6 to 16 nucleotides in length. In some embodiments, the barcode molecule (or a segment of a barcode molecule) can be, be about, be at least, be at least about, be at most, or be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100, 150, 200, 250, 300, 400, or 500 nucleotides in length, or a number or a range between any two of these values.
The barcode molecules used herein can comprise a cell barcode sequence and a molecular barcode sequence (e.g., a UMI). A barcode molecule can also comprise other sequences, such as a target binding sequence or region capable of hybridizing to target nucleic acids (e.g. poly(dT) sequence), other recognition or binding sequences, a template switching oligonucleotide (e.g., GGG, such as rGrGrG), and primer sequences (e.g. sequencing primer sequence, such as Read 1 or a PCR primer sequence) for subsequent processing (e.g. PCR amplification) and/or sequencing.
The configuration of the various sequences comprised in a barcode molecule (e.g. cell barcode sequence, UMI, primer sequence, target binding sequence or region, and/or any additional sequences) can vary depending on, for example, the particular configuration desired and/or the order in which the various components of the sequence are added as will be understood to a person skilled in the art. In some embodiments, a barcode molecule has a configuration of 5′-primer sequence-cell barcode sequence-UMI-target binding sequence-3′. In some embodiments, a barcode molecule has a configuration of 5′-primer sequence-cell barcode sequence-UMI-template switching oligonucleotide-3′.
In some embodiments, the barcode molecules can comprise a cell barcode sequence. Cell barcode sequences can be used to identify the barcoded indexing label and the barcoded nucleic acids originate from the cell. Barcoded nucleic acids that originate from the same cell (or the same partition) can have an identical cell barcode sequence. A cell barcode sequence can be referred to as a partition specific barcode, such as a microwell specific barcode. The cell barcode sequence of the barcode molecules in a partition can be identical or different.
In some embodiments, the cell barcode sequences can serve to track the target nucleic acids associated with the cell throughout the processing (e.g., location of the cells in a plurality of partitions, such as microwells) when the cell barcode sequence associated with the target nucleic acids is determined during sequencing.
The number (or percentage) of barcode molecules introduced in a partition with cell barcode sequences having an identical sequence can be different in different embodiments. In some embodiments, the number of barcode molecules introduced in a partition with cell barcode sequences having an identical sequence is, is about, is at least, is at least about, is at most, or is at most about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values. In some embodiments, the percentage of barcode molecules introduced in a partition with cell barcode sequences having an identical sequence is, is about, is at least, is at least about, is at most, or is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values. For example, the cell barcode sequences of at least two barcode molecules introduced in a partition comprise an identical sequence. In some embodiments, at least two of the cell barcode sequences of the plurality of barcode molecules in the same partition are identical.
A cell barcode sequence can be unique (or substantially unique) to a partition. The number of unique cell barcode sequences can be different in different embodiments. In some embodiments, the number of unique cell barcode sequences is, is about, is at least, is at least about, is at most, or is at most about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values. In some embodiments, the percentage of unique cell barcode sequences is, is about, is at least, is at least about, is at most, or is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values, of the cell barcode sequences of the barcode molecules introduced in a partition. For example, the cell barcode sequences of barcode molecules introduced in two partitions can comprise different sequences. In some embodiments, the cell barcode sequences of at least one barcode molecules in at least two different partitions are different.
In some embodiments, barcode molecules are introduced to the plurality of partitions such that different sets of a plurality of barcode molecules introduced in different partitions have different cell barcode sequences and a same set of plurality of barcode molecules introduced in a same partition have same cell barcode sequence. For example, target nucleic acids associated with a cell in a partition will be barcoded with the same cell barcode sequences.
The length of a cell barcode sequence of a barcode molecule (or a cell barcode sequence of each barcode molecule or all cell barcode sequences of the plurality of barcode molecules) can be different in different embodiments. In some embodiments, a cell barcode sequence of a barcode molecule (or each cell barcode sequence of each barcode molecule or all cell barcode sequences of the plurality of barcode molecules) is, is about, is at least, is at least about, is at most, or is at most about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length.
A barcode can, for example, comprise a molecular barcode sequence or molecular label. Molecular barcode sequences can be UMIs. Molecular barcode sequences can be used to identify molecular origins of the barcoded indexing label and the barcoded nucleic acids. Molecular barcode sequences (e.g., UMIs) are short sequences used to uniquely tag each molecule in a sample in some embodiments. The molecular barcode sequences of the barcode molecules partitioned into a partition can be identical or different.
In some embodiments, the molecular barcode sequences of the plurality of barcode molecules are different. The number (or percentage) of molecular barcode sequences of barcode molecules introduced in a partition (e.g., a microwell or a well) with different sequences can be different in different embodiments. In some embodiments, the number of molecular barcode sequences of barcode molecules introduced in a partition with different sequences is, is about, is at least, is at least about, is at most, or is at most about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values. In some embodiments, the percentage of molecular barcode sequences of barcode molecules introduced in a partition with different sequences is, is about, is at least, is at least about, is at most, or is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values. For example, the molecular barcode sequences of two barcode molecules of the plurality of barcode molecules introduced in a partition can comprise different sequences.
The number of barcode molecules introduced in a partition with molecular barcode sequences having an identical sequence can be different in different embodiments. In some embodiments, the number of barcode molecules introduced in a partition with molecular barcode sequences having an identical sequence is, is about, is at least, is at least about, is at most, or is at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any two of these values. For example, the molecular barcode sequences of two barcode molecules introduced in a partition can comprise an identical sequence.
The number of unique molecular barcode sequences can vary. For example, the number of unique molecular barcode sequences can be, be about, be at least, be at least about, be at most, or be at most about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values.
In some embodiments, at least two of the molecular barcode sequences of the plurality of barcode molecules in a partition comprise different molecular barcode sequences (e.g., unique molecular identifiers).
The length of a molecular barcode sequence of a barcode molecule (or a molecular barcode sequence of each barcode molecule) can be different in different embodiments. In some embodiments, a molecular barcode sequence of a barcode molecule (or a molecular barcode sequence of each barcode molecule) is, is about, is at least, is at least about, is at most, or is at most about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length.
In some embodiments, a barcode molecule comprises a primer sequence. The primer sequence can be a sequencing primer sequence (or a sequencing primer binding sequence) or a PCR primer sequence (or PCR primer binding sequence). For example, the sequencing primer can be a Read 1 sequence. In some embodiments, the barcode molecule comprises a PCR primer binding sequence, which allows for PCR amplification of a barcoded nucleic acid.
The length of the primer sequence can vary, for example the primer sequence can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length. The number (or percentage) of barcode molecules in a partition (e.g., a microwell) each comprising a primer sequence (or each comprising an identical primer sequence) can be different in different embodiments. In some embodiments, the number of barcode molecules in a partition (e.g., a microwell) each comprising a primer sequence (such as a PCR primer binding sequence) is, is about, is at least, is at least about, is at most, or is at most about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values. In some embodiments, the percentage of barcode molecules in a partition (e.g., a microwell) each comprising a primer sequence (or each comprising an identical primer sequence) is, is about, is at least, is at least about, is at most, or is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values.
In some embodiments, each of the plurality of barcode molecules comprises a primer sequence (e.g., a sequencing primer sequence, including but not limited to, a Read 1 sequence, a Read 2 sequence, or a portion thereof).
In some embodiments, a barcode molecule comprises a target binding sequence or region capable of hybridizing to the target nucleic acids, a particular type of target nucleic acids (e.g. mRNA), and/or specific target nucleic acids (e.g. specific gene of interest). In some embodiments, the target binding sequence comprises a poly(dT) sequence and/or a sequence capable of hybridizing to the plurality of target nucleic acids.
The length of a target binding sequence can vary. For example, the target binding sequence can be, be about, be at least, be at least about, be at most, or be at most about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length. The target binding sequence can be 12-18 deoxythymidines in length. In some embodiments, the target binding sequence can be 20 nucleotides or longer to enable their annealing in reverse transcription reactions at higher temperatures as will be understood by a person of skill in the art.
In some embodiments, barcode molecules comprising target binding sequences are introduced into the partitions together with other reagents such as the reverse transcription reagents. The number of the barcode molecules introduced into a partition comprising a target binding sequence can vary. For example, the number of barcode molecules introduced into a partition comprising a target binding sequence (e.g., poly(dT) sequence) can be, be about, be at least, be at least about, be at most, or be at most about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values.
In some embodiments, the target binding sequence can be on a 3′ end of a barcode molecule of the plurality of barcode molecules introduced in a partition. Barcode molecules each comprising a poly(dT) target binding sequence can be used to capture (e.g., hybridize to) a poly(A) sequence in an indexing label and/or 3′ end of polyadenylated mRNA transcripts in a target nucleic acid for a downstream 3′ gene expression library construction.
In some embodiments, the target binding sequence comprises a poly(dT) sequence which is a single-stranded sequence of deoxythymidine (dT) used for first-strand cDNA synthesis catalyzed by reverse transcriptase. In some embodiments, the target binding sequence comprises a poly(dT) sequence is introduced into the partitions as extension primers to synthesize the first-strand cDNA using the target nucleic acid (e.g. RNA) as a template.
In some embodiments, the poly(dT) of the barcode molecules introduced into a partition are identical (e.g., same number of dTs). In some embodiments, the poly(dT) of the barcode molecules introduced into a partition are different (e.g. different numbers of dTs). The percentage of the barcode molecules of the plurality of barcode molecules introduced into a partition with an identical poly(dT) sequence can vary. In some embodiments, the percentage of the barcode molecules of the plurality of barcode molecules introduced into a partition with an identical poly(dT) sequence is, is about, is at least, is at least about, is at most, is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values.
In some embodiments, the target binding regions of all barcode molecules of the plurality of barcode molecules comprise poly(dT) capable of hybridizing to a poly(A) sequence in an indexing label and/or poly(A) tails of mRNA molecules (or poly(dA) regions or tails of DNA). In some embodiments, the target binding regions of some barcode molecules of the plurality of barcode molecules comprise gene-specific or target-specific primer sequences. For example, a barcode molecule of the plurality of barcode molecules can also comprise a target binding region capable of hybridizing to a specific target nucleic acid associated with the cell, thereby capturing specific targets or analytes of interest. For example, the target binding region capable of hybridizing to a specific target nucleic acid can be a gene-specific primer sequence. The gene-specific primer sequences can be designed based on known sequences of a target nucleic acid of interest. The gene-specific primer sequences can span a nucleic acid region of interest, or adjacent (upstream or downstream) of a nucleic acid region of interest.
The length of the gene-specific primer sequence can vary. For example, a gene-specific primer sequence can be, be about, be at least, be at least about, be at most, or be at most about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length. In some embodiments, the gene-specific primer sequence is at least 10 nucleotides in length.
The number of the barcode molecules introduced into a partition comprising a gene-specific primer sequence can vary. For example, the number of barcode molecules introduced into a partition comprising a gene-specific primer sequence can be, be about, be at least, be at least about, be at most, or be at most about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, or a number or a range between any two of these values. In some embodiments, the barcode molecules introduced into a partition comprises a set of different gene-specific primer sequences each capable of binding to a specific target nucleic acid sequence.
The number of different gene-specific primer sequences of the barcode molecules introduced into a partition can vary. For example, the number of different gene-specific primer sequences of the barcode molecules introduced into a partition can be, be about, be at least, be at least about, be at most, or be at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 50000, 1000000, or a number or a range between any two of these values.
The number of target nucleic acids of interest (e.g. genes of interest) that the barcode molecules introduced into a partition are capable of binding can vary. For example, the number of target nucleic acids of interest (e.g. genes of interest) the barcode molecules introduced into a partition are capable of binding can be, be about, be at least, be at least about, be at most, or be at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 50000, 1000000, or a number or a range between any two of these values. In some embodiments, one barcode molecule introduced into a partition can bind to a molecule (or a copy) of a target nucleic acid. Barcode molecules introduced into a partition can bind to molecules (or copies) of a target nucleic acid or a plurality of target nucleic acids.
In some embodiments, the barcode molecules of the plurality of barcode molecules each comprise a poly(dT) sequence, a gene-specific primer sequence, and/or both. The poly(dT) sequence and the gene-specific primer sequence can be on a same barcode molecule or different barcode molecules of the plurality of barcode molecules introduced into a partition.
The ratio of the number of barcode molecules introduced into a partition comprising a poly(dT) sequence and the number of barcode molecules introduced into a partition comprising a gene-specific primer sequence can vary. For example, the ratio can be, be about, be at least, be at least about, be at most, be at most about, 1:100, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83, 1:82, 1:81, 1:80, 1:79, 1:78, 1:77, 1:76, 1:75, 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, or a number or a range between any two of these values.
In some embodiments, a barcode molecule (or each barcode molecule of the plurality of barcode molecules) comprises a template switching oligonucleotide (TSO). A primer comprising a target binding region, such as a poly(dT) sequence, can hybridize to an indexing label and/or a target nucleic acid (e.g., an mRNA) and be extended by, for example, reverse transcription to generate an extended primer comprising a reverse complement of the indexing label and/or the target nucleic acid, or a portion thereof (e.g., cDNA). The extended primer or cDNA can be further extended to include the reverse complement of a TSO oligonucleotide or barcode molecule. The resulting barcoded indexing label or barcoded nucleic acid includes the barcodes of the barcode molecule on the 3′-end.
In some embodiments, a barcode molecule does not comprise a TSO. A barcode molecule comprising a target binding region, such as a poly(dT) sequence, can hybridize to an indexing label and/or a target nucleic acid (e.g., an mRNA) and be extended by, for example, reverse transcription to generate an extended primer comprising a reverse complement of the target nucleic acid, or a portion thereof (e.g., cDNA). The extended primer or cDNA can be further extended to include the reverse complement of a template switching oligonucleotide. The resulting barcoded indexing label or barcoded nucleic acid includes the barcodes of the barcode molecule on the 5′-end. The resulting barcoded indexing label or barcoded nucleic acid (e.g., extended cDNA) can comprise a PCR primer binding sequence introduced in the reverse complement of the template switching oligonucleotide.
A TSO is an oligonucleotide that hybridizes to untemplated C nucleotides added by a reverse transcriptase during reverse transcription. The TSO can hybridize to the 3′ end of a cDNA molecule. The TSO can include one or more nucleotides with guanine (G) bases on the 3′-end of the TSO, with which the one or more cytosine (C) bases added by a reverse transcriptase to the 3′-end of a cDNA can hybridize. The series of G bases can comprise 1G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The series of G bases can be ribonucleotides. The reverse transcriptase can further extend the cDNA using the TSO as the template to generate a barcoded cDNA comprising the TSO. The length of a TSO can vary. For example, a TSO can be, be about, be at least, be at least about, be at most, or be at most about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length.
The number of the barcode molecules introduced into a partition comprising a TSO can vary. In some embodiments, the number of barcode molecules introduced into a partition comprising a TSO is, is about, is at least, is at least about, is at most, or is at most about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000, 100000000, 200000000, 300000000, 400000000, 500000000, 600000000, 700000000, 800000000, 900000000, 1000000000, or a number or a range between any two of these values.
The TSO of the barcode molecules introduced into a partition can be identical. In some embodiments, the TSO of the barcode molecules introduced into a partition is different. The percentage of the barcode molecules of the plurality of barcode molecules introduced into a partition with an identical TSO sequence can be different in different embodiments. In some embodiments, the percentage of the barcode molecules of the plurality of barcode molecules introduced into a partition with an identical TSO sequence is, is about, is at least, is at least about, is at most, is at most about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100%, or a number or a range between any two of these values.
The method described herein can comprise barcoding indexing labels and target nucleic acids associated with a cell in the partition (e.g., microwell) using the barcode molecules to generate a barcoded indexing label and a barcoded nucleic acids (e.g., target nucleic acids each hybridized with a barcode molecule, single-stranded barcoded nucleic acids, or double-stranded barcoded nucleic acids).
The method can, in some embodiments, further comprises releasing the indexing label and the plurality of target nucleic acids associated with the one or more cells in the partition prior to barcoding the indexing label and the plurality of target nucleic acids. In some embodiments, releasing the indexing label and the plurality of target nucleic acids associated with the one or more cells comprises lysing the plurality of cells. For example, prior to barcoding the indexing label and the target nucleic acids, the method can comprise lysing the cells to release the content of the cells within the partition. Lysis agents can be contacted with the cells or cell suspension concurrently. Non-limiting examples of lysis agents include bioactive reagents, such as lysis enzymes, or surfactant based lysis solutions including non-ionic surfactants (e.g., Triton X-100 and Tween 20) and ionic surfactants (e.g., sodium dodecyl sulfate (SDS)). Lysis methods including, but not limited to, thermal, acoustic, electrical, or mechanical cellular disruption can also be used.
Barcoding the indexing label and the plurality of target nucleic acids can comprise a reverse transcription reaction, for example, to generate a barcoded indexing label and a plurality of barcoded nucleic acids comprising cDNAs. In some embodiments, barcoding the indexing label and the plurality of target nucleic acids comprises extending the plurality of barcode molecules using the indexing label and the plurality of target nucleic acids as templates to generate the barcoded indexing label and the plurality of barcoded nucleic acids comprising a plurality of single-stranded barcoded nucleic acids. In some embodiments, the plurality of single-stranded barcoded nucleic acids can be hybridized to the plurality of target nucleic acids in the partition.
In some embodiments, barcoding target nucleic acids associated with a cell in the partition can comprise extending the barcode molecules using the target nucleic acids as templates to generate partially single-stranded/partially double-stranded barcoded nucleic acids hybridized to the target nucleic acids in the partition (or after target nucleic acids hybridized with barcode molecules are pooled). The partially single-stranded/partially double-stranded barcoded nucleic acids hybridized to target nucleic acids can be separated by denaturation (e.g., heat denaturation or chemical denaturation using for example, sodium hydroxide) to generate single-stranded barcoded nucleic acids of the plurality of barcoded nucleic acids. The single-stranded barcoded nucleic acids can comprise a barcode molecule and an oligonucleotide complementary to the target nucleic acids. In some embodiments, the single-stranded barcoded nucleic acids are generated by reverse transcription using a reverse transcriptase. In some embodiments, the single-stranded barcoded nucleic acids is generated by using a DNA polymerase.
In some embodiments, the method further comprises introducing a plurality of TSO into the partition. Barcoding the plurality of target nucleic acids can comprise extending the plurality of barcode molecules using the plurality of target nucleic acids and the plurality of template switching oligonucleotides as templates to generate the plurality of barcoded nucleic acids comprising a plurality of single-stranded barcoded nucleic acids.
For example, the single-stranded barcoded nucleic acids can be cDNA produced by extending a barcode molecule using a target RNA associated with the cell as a template. The single-stranded barcoded nucleic acids can be further extended using a TSO. The TSO can be introduced into the partitions together with the reverse transcription reagents. For example, a reverse transcriptase can be used to generate a cDNA by extending a barcode molecule hybridized to an RNA. After extending the barcode molecule to the 5′-end of the RNA, the reverse transcriptase can add one or more nucleotides with cytosine (C) bases (e.g. two or three) to the 3′-end of the cDNA. The TSO can include one or more nucleotides with guanine (G) bases (e.g. two or more) on the 3′-end of the TSO. The nucleotides with G bases can be ribonucleotides. The G bases at the 3′-end of the TSO can hybridize to the cytosine bases at the 3′-end of the cDNA. The reverse transcriptase can further extend the cDNA using the TSO as the template to generate a cDNA with the reverse complement of the TSO sequence on its 3′-end. The barcoded nucleic acid can include the barcode sequences (e.g., cell barcode sequence and molecular barcode sequence (e.g., UMI)) on the 5′-end and a TSO sequence at its 3′-end.
In some embodiments, barcoding the target nucleic acids comprises extending the barcode molecules using the target nucleic acids as templates and the barcode molecules as TSO to generate single-stranded barcoded nucleic acids that are hybridized to the target nucleic acids. In some embodiments, the present method further comprises introducing a plurality of extension primers to the partition. Barcoding the plurality of target nucleic acids can comprise extending the plurality of extension primers using the plurality of target nucleic acids as templates and the plurality of barcode molecules as template switching oligonucleotides to generate the plurality of barcoded nucleic acids comprising a plurality of single-stranded barcoded nucleic acids.
In some embodiments, the barcode molecules are not attached to a particle and the barcode molecules can comprise TSO. Extension primers (e.g. oligonucleotides comprising a poly(dT) sequence) can be introduced into the partitions which hybridize to a target nucleic acid (e.g. the poly-adenylated mRNA). The extension primers can be extended using the target nucleic acids as a template. For example, a reverse transcriptase can be used to generate a cDNA by extending an extension primer hybridized to an RNA. After extending the extension primers to the 5′-end of the RNA, the reverse transcriptase can add one or more C bases (e.g. two or three) to the 3′-end of the cDNA. The TSO or barcode molecule can include one or more G bases (e.g. two or more) on the 3′-end of the TSO. The nucleotides with guanine bases can be ribonucleotides. The G bases at the 3′-end of the TSO or barcode molecule can hybridize to the cytosine bases at the 3′-end of the cDNA. The reverse transcriptase can switch template from the mRNA to the TSO or barcode molecule. The reverse transcriptase can further extend the cDNA using the TSO or barcode molecule as the template to generate a cDNA further comprising the reverse complement of the TSO or barcode molecule. In this case, the barcode sequences (e.g., cell barcode sequence and molecular barcode sequence (e.g., UMI)) are on the 3′-end of the generated cDNA.
In some embodiments, each of the plurality of single-stranded barcoded nucleic acids is hybridized to one of the plurality of target nucleic acids and one of the plurality of template switching oligonucleotides in the partition.
The single-stranded barcoded nucleic acids can be separated from the template target nucleic acids by digesting the template target nucleic acids (e.g., using RNase), by chemical treatment (e.g., using sodium hydroxide), by hydrolyzing the template target nucleic acids, or via a denaturation or melting process by increasing the temperature, adding organic solvents, or increasing pH. Following the melting process, the target nucleic acids can be removed (e.g. washed away) and the single-stranded barcoded nucleic acids can be retained in the partition (e.g. through attachment to the partitions or through attachments to particles which can be retained in the partitions). In some embodiments, the method further comprises removing the plurality of target nucleic acids and the plurality of template switching oligonucleotides hybridized to the single-stranded barcoded nucleic acids. In some embodiments, removing the plurality of target nucleic acids comprises denaturation, thermal denaturation, digesting, or hydrolyzing the plurality of target nucleic acids.
In some embodiments, each of the plurality of single-stranded barcoded nucleic acid comprises a sequence of a barcode molecule of the plurality of barcode molecules (e.g., an actual sequence of the barcode molecule), a sequence of a target nucleic acid of the plurality of target nucleic acids (e.g. a reverse complement of the target nucleic acid), and/or a sequence of an extension primer of the plurality of extension primers (e.g., an actual sequence of the extension primer).
The method can further comprise amplifying the barcoded indexing labels and the plurality of barcoded nucleic acids to generate a double-stranded barcoded indexing labels and a plurality of double-stranded barcoded nucleic acids in the partition using the single-stranded barcoded indexing labels and the single-stranded barcoded nucleic acids as templates. The amplifying step can be used to amplify the product of first strand synthesis and/or RT reaction as described here.
For example, barcoding target nucleic acids associated with the cell in the partition (e.g., microwell) can comprise amplifying the barcoded nucleic acids (such as a single-stranded barcoded nucleic acid, or a cDNA generated by using a barcode molecule as disclosed herein). The amplification can comprise generating barcoded nucleic acids comprising double-stranded barcoded nucleic acids in the partition using the single-stranded barcoded nucleic acids as templates. The double-stranded barcoded nucleic acids can be generated from the single-stranded barcoded nucleic acids retained in the partition using, for example, second-strand synthesis or one-cycle PCR. Amplification of the barcoded nucleic acids can include additional cycles of PCR reactions.
The generated double-stranded barcoded nucleic acid can be denaturized or melted to generate two single-stranded barcoded nucleic acids: one single-stranded barcoded nucleic acid retained in the partition (e.g., attached to the particle) and the other single-stranded barcoded nucleic acid released into the solution from the retained single-stranded barcoded nucleic acid that can then be pooled to provide a pooled mixture outside the partitions. Both single-stranded barcoded nucleic acids (e.g. retained in the partitions or pooled outside the partitions) have a sequence comprising a sequence of a barcode molecule (e.g. cell barcode sequence and molecular barcode sequence (e.g., UMI)) and a sequence of a target nucleic acid or a reverse complement thereof.
In some embodiments, amplifying the plurality of barcoded nucleic acids comprises amplifying the plurality of barcoded nucleic acids in the partition to generate the plurality of double-stranded barcoded nucleic acids. The plurality of target nucleic acids in a partition can be barcoded and the plurality of barcoded nucleic acids generated are then amplified in the same partition. Further, the plurality of target nucleic acids in a partition can be barcoded and the plurality of barcoded nucleic acids generated are then amplified in the same reaction. For example, the reaction can be a one-step RT-PCR reaction.
Each of the plurality of barcode molecules can comprise a primer sequence. In some embodiments, the primer sequence can comprise a PCR primer sequence. Amplifying the plurality of barcoded nucleic acids can comprise amplifying the plurality of barcoded nucleic acids using the primer sequences in single-stranded barcoded nucleic acids of the plurality of single-stranded barcoded nucleic acids, or products thereof.
In some embodiments, the barcoding process comprises reverse transcription using an mRNA associated with the cell (and optionally a TSO) as template to generate a barcoded cDNA molecule (optionally with a reverse complement of a TSO) and amplification of the barcoded cDNA by PCR.
The methods disclosed herein can comprise pooling the barcoded indexing labels and the plurality of barcoded nucleic acids, or products thereof, in each of the plurality of partitions to generate pooled barcoded indexing labels and pooled barcoded nucleic acids. Subjecting the plurality of barcoded nucleic acids, or products thereof, to sequencing can comprise subjecting the pooled barcoded nucleic acids, or products thereof, to sequencing. In some embodiments, pooling the plurality of barcoded nucleic acids, or products thereof, comprises pooling the plurality of double-stranded barcoded nucleic acids in each of the plurality of partitions to generate the pooled barcoded nucleic acids. For example, the method can comprise pooling the barcoded nucleic acids after barcoding the target nucleic acids and before sequencing the barcoded nucleic acids to obtain pooled barcoded nucleic acids.
In some embodiments, pooling barcoded nucleic acids occurs after generating double-stranded barcoded nucleic acids (e.g., after second strand synthesis) or after generating amplified barcoded nucleic acids. The amplified barcoded nucleic acids can be subject to sequencing library construction prior to sequencing. In some embodiments, synthesis of single-stranded barcoded nucleic acids and double-stranded barcoded nucleic acids occur after the pooling of target nucleic acids hybridized with barcode molecules.
In some embodiments the barcode molecules are attached to particles, only single-stranded barcoded nucleic acids released into bulk (e.g., after amplification of the barcoded nucleic acids) are collected by pooling, and the particles are not pooled (e.g. not removed from the partitions) but retained in the partitions (e.g. by an external magnetic field applied on magnetic beads), thereby allowing one to trace the origin of the pooled barcoded nucleic acids, for example, to its original location in the partitions.
The pooled barcoded nucleic acids can be single-stranded or double-stranded (e.g. generated from the single-stranded pooled barcoded nucleic acids by PCR amplification). The pooled barcoded nucleic acids (e.g. amplified barcoded cDNA) can be purified, and optionally further amplified, prior to sequencing library construction. The pooled barcoded nucleic acids with desired length can be selected.
The barcoded indexing label and the barcoded nucleic acids (e.g. pooled barcoded nucleic acids) can be further processed prior to sequencing to generate processed barcoded indexing label and processed barcoded nucleic acids. For example, the method herein can include amplification of barcoded nucleic acids, fragmentation of amplified barcoded nucleic acids, end repair of fragmented barcoded nucleic acids, A-tailing of fragmented barcoded nucleic acids that have been end-repaired (e.g., to facilitate ligation to adapters), and attaching (e.g., by ligation and/or PCR) with a second sequencing primer sequence (e.g., a Read 2 sequence), sample indexes (e.g. short sequences specific to a given sample library), and/or flow cell binding sequences (e.g., P5 and/or P7). Additional PCR amplification can also be performed. This process can also be referred to as sequencing library construction. In some embodiments, separate sequencing libraries are constructed for the barcoded indexing labels and the barcoded nucleic acids.
PCR amplification can be carried out to generate sufficient mass for the subsequent library construction processes. In some embodiments, the present method comprises performing a polymerase chain reaction in bulk on the pooled barcoded nucleic acids, or the fragmented barcoded nucleic acids, to generate amplified barcoded nucleic acids. For example, the method can comprise performing a polymerase chain reaction in bulk, subsequent to the pooling, on the pooled barcoded nucleic acids, thereby generating amplified barcoded nucleic acids. In some embodiments, performing the polymerase chain reaction in bulk is subsequent to fragmenting the pooled barcoded nucleic acids. The amplification for library preparation can be a separate process from the amplification of the first strand barcoded nucleic acid generated by, for example, the RT reaction as described herein (such as a one-step RT-PCR reaction).
In some embodiments, the method comprises fragmenting the pooled barcoded nucleic acids to generate fragmented barcoded nucleic acids to generate fragmented barcoded nucleic acids prior to subjecting the plurality of barcoded nucleic acids, or products thereof, to sequencing. For example, the method can comprise fragmenting (e.g., via enzymatic fragmentation, mechanical force, chemical treatment, etc.) the pooled barcoded nucleic acids to generate fragmented barcoded nucleic acids. Fragmentation can be carried out by any suitable process such as physical fragmentation, enzymatic fragmentation, or a combination of both. For example, the barcoded nucleic acids can be sheared physically using acoustics, nebulization, centrifugal force, needles, or hydrodynamics. The barcoded nucleic acids can also be fragmented using enzymes, such as restriction enzymes and endonucleases.
Fragmentation yields fragments of a desired size for subsequent sequencing. The desired sizes of the fragmented nucleic acids are determined by the limitations of the next generation sequencing instrumentation and by the specific sequencing application as will be understood by a person skilled in the art. For example, when using Illumina technology, the fragmented nucleic acids can have a length of between about 50 bases to about 1,500 bases. In some embodiments, the fragmented barcoded nucleic acids have about 100 bp to 700 bp in length.
Fragmented barcoded nucleic acids can undergo end-repair and A-tailing (to add one or more adenine bases) to form an A overhang. This A overhang allows adapter containing one or more thymine overhanging bases to base pair with the fragmented barcoded nucleic acids.
Fragmented barcoded nucleic acids can be further processed by adding additional sequences (e.g. adapters) for use in sequencing based on specific sequencing platforms. Adapters can be attached to the fragmented barcoded nucleic acids by ligation using a ligase and/or PCR. For example, fragmented barcoded nucleic acids can be processed by adding a second sequencing primer sequence. The second sequencing primer sequence can comprise a Read 2 sequence. An adapter comprising the second primer sequence can be ligated to the fragmented barcoded nucleic acids after, for example, end-repair and A tailing, using a ligase. The adaptor can include one or more thymine (T) bases that can hybridize to the one or more A bases added by A tailing. An adaptor can be, for example, partially double-stranded or double stranded. In some embodiments, the amplified barcoded nucleic acids comprise a sequencing primer sequence.
The adapter can also include platform-specific sequences for fragment recognition by specific sequencing instrument. In some embodiments, the amplified barcoded nucleic acids comprise a sequence for attaching the amplified barcoded nucleic acids to a flow well. For example, the amplified barcoded nucleic acids can comprise an adapter that comprises a sequence for attaching the fragmented barcoded nucleic acids to a flow well of Illumina platforms, such as a P5 sequence, a P7 sequence, or a portion thereof. Different adapter sequences can be used for different next generation sequencing instrument as will be understood by a person skilled in the art.
The adapter can also contain sample indexes to identify samples and to permit multiplexing. Sample indexes enable multiple samples to be sequenced together (i.e. multiplexed) on the same instrument flow cell as will be understood by a person skilled in the art. Adapters can comprise a single sample index or a dual sample indexes depending on the implementations such as the number of libraries combined and the level of accuracy desired.
In some embodiments, the amplified barcoded nucleic acids generated from sequencing library construction can include a P5 sequence, a sample index, a Read 1 sequence, a cell barcode sequence, a molecular barcode sequence (e.g., UMI), a poly(dT) sequence, a target biding region, a sequence of a target nucleic acid or a portion thereof, a Read 2 sequence, a sample index, and/or a P7 sequence (e.g., from 5′-end to 3′-end). In some embodiments, the amplified barcoded nucleic acids can include a P5 sequence, a sample index, a Read 1 sequence, a cell barcode sequence, a molecular barcode sequence (e.g., UMI), a sequence of a template switching oligonucleotide, a sequence of a target nucleic acid or a portion thereof, a Read 2 sequence, a sample index, and/or a P7 sequence (e.g., from 5′-end to 3′-end).
Sequencing the barcoded nucleic acids, or products thereof, can comprise sequencing products of the barcoded nucleic acids. Products of the barcoded nucleic acids can include the processed nucleic acids generated by any step of the sequencing library construction process, such as amplified barcoded nucleic acids, fragmented barcoded nucleic acids, fragmented barcoded nucleic acids comprising additional sequences such as the second sequencing primer sequence and/or adapter sequences described herein.
The method disclosed herein can comprise sequencing the barcoded indexing labels and the barcoded nucleic acids or products thereof to obtain nucleic acid sequences of the barcoded indexing label and the barcoded nucleic acids. The barcoded nucleic acids generated by the method disclosed herein can comprise barcoded nucleic acids pooled, from each partition, into a pooled mixture outside the partitions. The barcoded nucleic acids retained in a partition and the pooled barcoded nucleic acids in a pooled mixture outside the partitions can be sequenced using a same or different sequencing techniques.
In some embodiments, sequencing the plurality of barcoded nucleic acids (or the barcoded indexing labels) or products thereof comprises sequencing the pooled barcoded nucleic acids (or the pooled barcoded indexing labels) to obtain nucleic acid sequences of the pooled barcoded nucleic acids (or the pooled barcoded indexing labels). As used herein, a “sequence” can refer to the sequence, a complementary sequence thereof (e.g., a reverse, a compliment, or a reverse complement), the full-length sequence, a subsequence, or a combination thereof. The nucleic acids sequences of the pooled barcoded nucleic acids (or the pooled barcoded indexing labels) can each comprise a sequence of a barcode molecule (e.g., the cell barcode sequence and the molecular barcode sequence (e.g., UMI)) and a sequence of a target nucleic acid (or an indexing label) associated with the cell or a reverse complement thereof.
Pooled barcoded nucleic acids (or pooled barcoded indexing labels) can be sequenced using any suitable sequencing method identifiable. For example, sequencing the pooled barcoded nucleic acids can be performed using high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, sequencing-by-ligation, sequencing-by-hybridization, next generation sequencing, massively-parallel sequencing, primer walking, and any other sequencing methods known in the art and suitable for sequencing the barcoded nucleic acids generated using the methods herein described.
Method disclosed herein can comprise determining a profile of the cells simultaneously from multiple samples, for example from the sequence of the barcode nucleic acids. The obtained nucleic acid sequences of the plurality of barcoded nucleic acids (e.g. nucleic acid sequences of pooled barcoded nucleic acids) can be subjected to any downstream post-sequencing data analysis as will be understood by a person of skill in the art. The sequence data can undergo a quality control process to remove adapter sequences, low-quality reads, uncalled bases, and/or to filter out contaminants. The high-quality data obtained from the quality control can be mapped or aligned to a reference genome or assembled de novo.
Profile analysis, for example gene expression quantification and differential expression analysis, can be carried out to identify genes whose expression differs in different cells. Barcoded nucleic acids from a cell can have an identical cell barcode sequence in the sequencing data and can be identified. Barcoded nucleic acids from different cells can have different cell barcode sequences in the sequencing data and can be identified. Barcoded nucleic acids with an identical cell barcode sequence, an identical target sequence, and different molecular barcode sequences in the sequencing data can be quantified and used to determine the expression of the target.
The method can, for example, comprise determining a profile (e.g. an expression profile, a transcription profile, an omics profile, or a multi-omics profile) of the one or more cells from the sequences of the barcoded nucleic acids. In some embodiments, the profile comprises a single omics profile, such as a transcriptome profile. In some embodiments, the profile comprises a multi-omics profile, which can include profiles of genome (e.g. a genomics profile), proteome (e.g. a proteomics profile), transcriptome (e.g. a transcriptomics profile), epigenome (e.g. an epigenomics profile), metabolome (e.g. a metabolomics profile), and/or microbiome (e.g. microbiome profile). In some embodiments, the multi-omics profile comprises a genomics profile, a proteomics profile, a transcriptomics profile, an epigenomics profile, a metabolomics profile, a chromatics profile, a protein expression profile, a cytokine secretion profile, or a combination thereof.
The profile can comprise an expression of a target nucleic acid of the plurality of target nucleic acids. For example, the expression of the target nucleic acid can comprise an abundance of the target nucleic acid. The abundance of the target nucleic acid can comprise an abundance of molecules of the target nucleic acid barcoded using the barcode molecules. The abundance of the molecules of the target nucleic acid can comprise a number of occurrences of the molecules of the target nucleic acid. In some embodiments, the number of occurrences of the molecules of the target nucleic acid is, is indicated by, or is determined using, a number of the barcoded nucleic acids comprising a sequence of the target nucleic acid and different molecular barcode sequences in the sequences of the barcoded nucleic acids. In some embodiments, the profile includes an RNA expression profile and/or a protein expression profile. The expression profile can comprise an RNA expression profile, an mRNA expression profile, and/or a protein expression profile. A profile can also be a profile of one or more target nucleic acids (e.g. gene markers) or a selection of genes associated with the cell. In some embodiments, target nucleic acids associate with a cell (e.g., a living cell) can be analyzed in a high-throughput manner by the present method.
The cells can be obtained from any organism of interest. A cell can be, for example, a mammalian cell, and particularly a human cell such as T cells, B cells, natural killer cells, stem cells, or cancer cells.
Cells described herein can be obtained from, derived from, cultured from, or progenies of cells cultured from a cell sample. A cell sample comprising cells can be obtained from any source including a clinical sample and a derivative thereof, a biological sample and a derivative thereof, a forensic sample and a derivative thereof, and a combination thereof. A cell sample can be collected from any bodily fluids including, but not limited to, blood, urine, serum, lymph, saliva, anal, and vaginal secretions, perspiration and semen of any organism. A cell sample can be products of experimental manipulation including purification, cell culturation, cell isolation, cell separation, cell quantification, sample dilution, or any other cell sample processing approaches. A cell sample can be obtained by dissociation of any biopsy tissues of any organism including, but not limited to, skin, bone, hair, brain, liver, heart, kidney, spleen, pancreas, stomach, intestine, bladder, lung, esophagus.
In some embodiments, the cell sample is a clinical sample or a derivative thereof, a biological sample or a derivative thereof, an environmental sample or a derivative thereof, a forensic sample or a derivative thereof, or a combination thereof. In some embodiments, the cell sample is collected from blood, urine, serum, lymph, saliva, anal, and vaginal secretions, perspiration, and/or semen of any organism. In some embodiments, the cell sample is obtained from skin, bone, hair, brain, liver, heart, kidney, spleen, pancreas, stomach, intestine, bladder, lung, and/or esophagus of any organism.
In some embodiments, the cells are cultured cells, such as cells from a cultured cell line. In some embodiments, the cells comprise immune cells, fibroblast cells, stem cells, or cancer cells. In some embodiments, the cells are obtained from, cultured from, or progenies of cells cultured from a cell sample of a disease or disorder disclosed herein.
The cells can be cancer cells. Examples of cancer cells include, but are not limited to, bladder cancer cells (e.g., CRL-1472, CRL-1473, CRL-1749, CRL-2169, HTB-2, HTB-4, HTB-5, HTB-9), breast cancer cells (e.g., MCF-7, CRL-1897, CRL-1902, CRL-2983, CRL-2988, CRL-3127, CRL-3166, CRL-1897, CRL-3180), colon cancer cells (e.g., CCL-229, CCL-233, CCL-235, CCL-237, CCL-248, CCL-255, CRL-5792, HTB-37, HTB-39), endometrial cancer cells (e.g., CRL-1671), gastric cancer cells (e.g., CRL-1739, CRL-5822, CRL-5971, CRL-5973, CRL-5974, HTB-103, MKN-28, SNU638), leukemia cells (e.g., NB4, CCL-119, CCL-240, CCL-243, CRL-1582, CRL-1873, CRL-2724, TIB-202), liver cancer cells (e.g., CRL-2234, CRL-2236, CRL-2237, CRL-2238, CRL-8024, CRL-10741, HTB-52, HB-8065), lung cancer cells (e.g., CCL-256, CCL-257, CRL-5803, CRL-5872, CRL-5875, CRL-5877, CRL-5908, HTB-183), lymphoma cells (e.g., U937), small cell lung cancer cells (CRL-11350), non-small cell lung cancer cells (e.g., A549, CRL-5803, CRL-5893, CRL-5908, CRL-9609, HTB-178), kidney cancer cells (e.g., CRL-7569, CRL-7629, HTB-46, HTB-47) ovarian (e.g., SKOV3, CRL-1572, HTB-75, HTB-78), pancreatic cancer cells (e.g., CRL-1682, CRL-1687, CRL-1918, CRL-1997, CRL-2172, CRL-2547, HTB-79, HTB-80), prostate cancer cells (e.g., CRL-1740, CRL-3031, CRL-3033, CRL-3314, CRL-3315, CRL-3470, HTB-81), and skin cancer cells (e.g., A-375, HTB-66, HTB-69, HTB-71, CRL-7724).
The cells can be, for example, cells suitable for studying a cardiovascular disease (e.g., CRL-1395, CRL-1444, CRL-1476, CRL-1730, CRL-1999, CRL-2018, and CRL-2581), diabetes (e.g., CRL-3237, CRL-3242, CRL-11506, PCS-210-010), an infectious disease (e.g. CCL-86, CCL-156, CCL-214), a neurodegenerative disease (e.g., ACS-5001, ACS-1013, CRL-2541, HTB-11), or a respiratory disease (e.g., PCS-301-011, PCS-301-013, CRL-1848, CRL-4051, CRL-9609). In some embodiments, the cells comprise A549 cells, NB4 cells, U937 cells, or a combination thereof. In some embodiments, the cells comprise living cells.
Disclosed herein include a kit for indexing a plurality of samples. The kit can, in some embodiments, comprise a coupling agent comprising a coupling group and a first reactive group; for each of the plurality of samples, a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent; and instructions to use the kit for indexing multiple samples according to any one of the methods disclosed herein for indexing multiple samples. Also disclosed herein include a kit for analyzing nucleic acids in a plurality of samples. The kit can, in some embodiments, comprises a coupling agent each comprising a coupling group and a first reactive group; for each of the plurality of samples, a plurality of indexing labels each comprising an identical sample-specific-indexing sequence and a second reactive group capable of forming a covalent bond with the first reactive group of the coupling agent; a plurality of beads, wherein each bead is attached to, reversibly attached to, covalently attached to, or irreversibly attached to a plurality of barcode molecules, and wherein each barcode molecule of the plurality of barcode molecules comprises a cell barcode sequence, a molecular label sequence, a primer sequence, a primer binding site, a template switching oligonucleotide, or a combination thereof; and instructions to use the kit for indexing multiple samples according to any one of the methods disclosed herein for indexing multiple samples.
The kit can, in some embodiments, further comprise a plurality of partitions comprising, for example, at least 100 partitions (e.g., droplets or wells). The kit can, in some embodiments, comprise one or more reagents for use in the method disclosed herein. For example, the kit can further comprise one or more of cell lysis agents, enzymes (such as reverse transcriptase, polymerase), and chemical reagents.
Some aspects of the embodiments discussed above are disclosed in further detail in the following example, which are not in any way intended to limit the scope of the present disclosure.
For multiplexed single cell RNA-seq, modified sample-specific oligos were used as sample barcodes to label cell from different samples.
5′NHS-PEG5-Tz modified sample-specific oligos were used to tag multiple samples before sample pooling for multiplexed scRNA-seq. The sample-specific oligo sequence consists of a PCR handle sequence, a well position specific barcode, a random DNA sequence as unique molecular index (UMI) and an poly(A) primer sequence.
A sample index oligonucleotide is a single-stranded DNA (ssDNA) having a sequence as follows:
NH2-C6-ssDNA was incubated with NHS-PEG5-Tz to prepare ssDNA-Tz. The efficiency of small molecule modification during label preparation was evaluated using mass spectrometry (
Cell labeling was carried out as follows:
1. AG49, NB4, U937 cell lines were washed with PBS, resuspended in PBS, and centrifuged at 400×g. The supernatant was removed.
2. Pre-labeling of cells. NHS-PEG4-TCO was used as a pre-labeling reagent to label and resuspend the washed cells. The pre-labeling reaction was carried out for 10 minutes in the dark at room temperature. An equal volume of FBS was then added to quench the reaction for 10 minutes.
3. Washing with PBS. After quenching, the cells were centrifuged for 2 min at 400×g to discard the supernatant, and then washed twice with PBS, the same as step 2.
4. Sample specific oligonucleotide tag. 20 μL of the specific ssDNA-Tz reagent was added to the cell pellet and resuspend. The labeling reaction was carried out for 30 minutes in the dark at room temperature, and the cells were resuspended every 15 minutes. During this period, quenching reagent was prepared with PBS 5 μL of quenching reagent was added to the cells after labeling, and reacted for 10 minutes at room temperature in the dark.
6. Washing the cells. After quenching, the cells were centrifuged for 2 minutes at 400×g to discard the supernatant, and were then washed twice with PBS, the same as 2.
7. Counting and pooling the cells from three cell line. A cell counter was used to detect the concentration and viability of the cells. The three cell lines were pooled in a 1:1:1 ratio, and were then analyzed with single-cell sequencing process.
GEXSCOPE® Single Cell RNAseq Library Construction kit (Singleron Biotechnologies) was used to demonstrate the technical feasibility and the utility of the present disclosure in high-throughput multiplexed single-cell RNA sequencing. The experiment was conducted according to manufacturer's instructions with modifications described below.
Briefly, cells were labeled as described in the above steps. After sample pooling, cell suspensions were loaded onto the microchip to partition single cells into individual wells on the chip. Cell barcoding magnetic beads were then loaded to the microchip and washed. Each cell-barcoding magnetic bead contains oligos with a unique cell barcode sequence combined with oligo-dT on the surface. Each oligo on the bead also has a unique molecule index sequence (UMI); the number of UMIs detected in the sequence can be used to accurately quantify different RNA molecules. Only one bead can fall into each well on the microchip based on the diameters of the beads and well (about 30 μm and 40 μm, respectively). Then 100 μL reaction mixture was loaded into the chip, and the chip was incubated on ice for 10 minutes to lyse the cells. After the cells are lysed, the magnetic beads, together with captured RNAs, were taken out of the microchip and subject to reverse transcription.
After the reverse transcription is completed, sample labels were recovered through heat shock reaction. The magnetic beads were washed with wash buffer, and were then resuspend in TE buffer. The beads were placed at 95ºC for 5 minutes, and were then quickly placed on the magnetic stand to recover the supernatant. The supernatant was used as a template for PCR amplification to construct tag library. The remaining magnetic beads were also used as a template for PCR amplification. The cDNA was then used to construct a transcriptome sequencing library. The resulting RNA-seq library was sequenced on an Illumina Nova-Seq with PE150 mode and analyzed with CeleScope bioinformatics workflow (Singleron Biotechnologies), as shown in
In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/079903 | Mar 2021 | WO | international |
This application is a U.S. national phase application under 35 U.S.C. § 371 of International Application No. PCT/CN2022/080093, filed on Mar. 10, 2022, which claims the benefit of priority to PCT Application No. PCT/CN2021/079903, filed on Mar. 10, 2021, the content of each which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/080093 | 3/10/2022 | WO |
