CA2814049A1 - High-throughput single cell barcoding - Google Patents
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Abstract
Methods and compositions for high-throughput, single cell analyses are provided. The methods and compositions can be used for analysis of genomes and transcriptomes, as well as antibody discovery, HLA typing, haplotyping and drug discovery.
Description
HIGH-THROUGHPUT SINGLE CELL BARCODING
RELATED APPLICATION DATA
[001] This application claims priority to U.S. Provisional Patent Application No. 61/391,364, filed on October 8, 2010 and is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENT INTERESTS
RELATED APPLICATION DATA
[001] This application claims priority to U.S. Provisional Patent Application No. 61/391,364, filed on October 8, 2010 and is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENT INTERESTS
[002] This invention was made with government support under HG003170 awarded by the National Institutes of Health. The Government has certain rights in the invention.
FIELD
FIELD
[003] The present invention relates to methods and compositions for obtaining and analyzing nucleic acid sequences derived from many single cells at once.
BACKGROUND
BACKGROUND
[004] Classical single cell analysis is performed by isolating a single cell into a single well of a processing plate from which DNA and/or RNA can be amplified or where the cell can be subculture into a larger population, with both approaches performed until enough genomic material is achieved for subsequent downstream processing. A
limitation of such approaches is that it is not always possible to isolate single cells from a tissue section or a complex cellular mixture or population. Furthermore, in a clonally amplified cell population in culture, even if the cells should present the exact same genome, which they should in theory, the transcriptomic information is variable from one cell to another.
Also, culturing cells modifies their expression patterns, so it is often preferable to capture the transcriptomic information when the cells are in their original environment. In addition, the extreme low amounts of DNA and/or RNA obtained when isolating a single cell makes downstream processing steps quite challenging. Moreover, the processes by which DNA and/or RNA are amplified to large enough amounts to allow such analysis causes significant bias in the resulting material and, therefore, is not representative of the nucleic acids in the cell. Finally, classical approaches are limited in the amount of single cells that can be assayed in one analysis. For example, a complex population of 10,000 cells is to be studied, 10,000 cells would need to be sorted and separated (using, e.g., approximately 100 x 96 well plates), which requires substantial investment in costly automation equipment as well as significant processing time and additional costs.
limitation of such approaches is that it is not always possible to isolate single cells from a tissue section or a complex cellular mixture or population. Furthermore, in a clonally amplified cell population in culture, even if the cells should present the exact same genome, which they should in theory, the transcriptomic information is variable from one cell to another.
Also, culturing cells modifies their expression patterns, so it is often preferable to capture the transcriptomic information when the cells are in their original environment. In addition, the extreme low amounts of DNA and/or RNA obtained when isolating a single cell makes downstream processing steps quite challenging. Moreover, the processes by which DNA and/or RNA are amplified to large enough amounts to allow such analysis causes significant bias in the resulting material and, therefore, is not representative of the nucleic acids in the cell. Finally, classical approaches are limited in the amount of single cells that can be assayed in one analysis. For example, a complex population of 10,000 cells is to be studied, 10,000 cells would need to be sorted and separated (using, e.g., approximately 100 x 96 well plates), which requires substantial investment in costly automation equipment as well as significant processing time and additional costs.
[005] Early approaches included split pooled DNA synthesis. While split pooled DNA synthesis on beads can potentially be used to achieve uniquely bar-coded beads (Brenner et al.
(2000) Proc. Natl. Acad. Sci. USA 97:1665), the technical difficulties associated with such an approach and the incorporation inefficiency of nucleotide during chemical synthesis of the sequence, results in beads having very few oligonucleotide sequences with correct sequences and/or length. Even when nucleotide synthesis chemistry is quite efficient, there is, on average, 1% non-incorporation at each nucleotide cycle.
Consequently, attempts to synthesize a clonal bar-code on beads of proper length split pooled DNA synthesis were unsuccessful. For example, for a typical oligonucleotide of 50-60 nucleotides this error rate would result in less than 40% of the oligos on the beads having the correct sequence. Moreover, because the oligonucleotides are synthesized on a solid support it is impossible to identify the correct one, using purification approaches such as with HPLC purification or PAGE. Split pool synthesis was originally developed by Linx Therapeutics, who was acquired by Solexa who was acquired by Illumina based on the early work on split pool synthesis, but the technology was abandoned because of these issues. Thus, the efficient use of bar-coded beads has not been achieved. Beads with an internal dye gradient core (such as the one used by Luminex Corporation) can be used in application where the overall bead bar-code signal is used. While that approach is acceptable when an average signal intensity is desired, it is inadequate where the downstream use of these molecules requires unique identification of the cell.
Also "luminex beads" can only be generated in a limited amount which result in limited capability for probing more then a few hundreds of cells.
(2000) Proc. Natl. Acad. Sci. USA 97:1665), the technical difficulties associated with such an approach and the incorporation inefficiency of nucleotide during chemical synthesis of the sequence, results in beads having very few oligonucleotide sequences with correct sequences and/or length. Even when nucleotide synthesis chemistry is quite efficient, there is, on average, 1% non-incorporation at each nucleotide cycle.
Consequently, attempts to synthesize a clonal bar-code on beads of proper length split pooled DNA synthesis were unsuccessful. For example, for a typical oligonucleotide of 50-60 nucleotides this error rate would result in less than 40% of the oligos on the beads having the correct sequence. Moreover, because the oligonucleotides are synthesized on a solid support it is impossible to identify the correct one, using purification approaches such as with HPLC purification or PAGE. Split pool synthesis was originally developed by Linx Therapeutics, who was acquired by Solexa who was acquired by Illumina based on the early work on split pool synthesis, but the technology was abandoned because of these issues. Thus, the efficient use of bar-coded beads has not been achieved. Beads with an internal dye gradient core (such as the one used by Luminex Corporation) can be used in application where the overall bead bar-code signal is used. While that approach is acceptable when an average signal intensity is desired, it is inadequate where the downstream use of these molecules requires unique identification of the cell.
Also "luminex beads" can only be generated in a limited amount which result in limited capability for probing more then a few hundreds of cells.
[006] The present approach offers particular advantages over earlier approaches such as split pooled DNA synthesis on bead.
SUMMARY
SUMMARY
[007] The present approach efficiently produces bar-coded beads coated with clonal copies of the bar-coded oligonucleotides having the correct sequence. Moreover, the speed, ease and cost of production is also advantageous. And, unlike split pooled DNA
synthesis on beads, millions of uniquely bar-coded beads can be generated for single cell analysis.
synthesis on beads, millions of uniquely bar-coded beads can be generated for single cell analysis.
[008] In one aspect, the invention consists of an approach for bar-coding many single cells in a complex mixtures of cells. Each cell is provided with a unique individual bar-code for each cell. The unique bar-code allows each cell's nucleic acids (genome or transcriptome) to be associated with the original cell. Thus, for any given individual cell multiple different genes and transcripts can be identified and correlated to the same cell because the sequences share the same unique bar code.
[009] The unique bar-code is inserted into each individual cell in a way that each cell receives one unique bar-code and is present in a large enough amount to allow subsequent genomic or transcriptomic targeting. Once the bar-code is inserted, downstream manipulations are conducted to capture and then sequence all these unique bar-codes and the genome or transcriptome sequences of interest in one simultaneous reaction. The present approach, when coupled with high-throughput sequencing technology allows analyzing a large number of single cells and achieving the analysis in one single reaction assay. In principle, one can sequence any number of cells and any number of targeted regions per cell. The number of single cells that can be processed is limited only by practical constraints, such as the speed of high throughput sequencing; for example. In some embodiments, high-throughput sequencing technologies are used, such as the ones conducted of sequencing platform such as Illumina HiSeq or genome analyzer, Roche 454, Pacific Bioscience, Ion Torrents, Harvard Polonator, ABI Solid or other similar instruments in the field. Classic sequencing approaches, such as Sanger sequencing can be used; however, the true power in the technology is to be able to sequence a larger number of sequences from single cells simultaneously. High-throughput sequencing platforms are thus better-suited for most embodiments. If a sequencing platform generates 10 million reads per run, then one can sequence one unique transcript across 1 million cells to achieved a 10x coverage. In other embodiments, a partial transcriptome, for example targeting 10,000 unique transcripts, requires only 100 cells to be targeted for capture and sequencing.
[010] In some embodiments, full or targeted transcriptome RNA analysis is performed. Thus, in a single cell, only selected transcripts may be sequenced. In other applications, all or substantially all transcripts may be captured and sequenced. In yet other embodiments, full or partial genomic DNA analysis is performed.
[011] Analyses of multiple cells in heterogeneous cell populations is particularly useful when studying complex samples or mixtures. Complex samples or cell mixtures include, for example, metagenomic samples, normal and cancerous tissue sections, embryonic and stem cell colonies. Genome and transcriptome sequencing is desirable where sequences are highly divergent; for example, in certain cell types or in cells at certain stages.
Particularly suitable applications include molecular haplotyping, HLA typing, and T- and B- cell receptor profiling. Metagenomic samples refers to samples containing genomes from multiple origins, such as species. For example, the present approach may be applied to mixtures of bacterial species to allow sequencing of nucleic acids from multiple bacteria in one assay followed by correlating the sequences to the same bacterial cell.
Similarly, nucleic acid sequences of foreign cells living in the mouth can be determined and correlated to the same cell.
Particularly suitable applications include molecular haplotyping, HLA typing, and T- and B- cell receptor profiling. Metagenomic samples refers to samples containing genomes from multiple origins, such as species. For example, the present approach may be applied to mixtures of bacterial species to allow sequencing of nucleic acids from multiple bacteria in one assay followed by correlating the sequences to the same bacterial cell.
Similarly, nucleic acid sequences of foreign cells living in the mouth can be determined and correlated to the same cell.
[012] Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
1013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
[014] Figures 1A-1E schematically depict a method of amplifying each unique molecule composed of a degenerate barcode on a bead according to certain aspects of the invention.
(A) Attach barcoded template oligonucleotide. (B) saturate solid support with anchor primer. (C) Perform emulsion PCR. (D) Emulsion PCR completed. (E) Barcoded beads are recovered.
[015] Figures 2A-2D schematically depict emulsion PCR of a single cell. (A) Capture of cell and barcoded bead in an emulsion. (B) Lyse cell. (C) Anneal DNA and/or RNA to barcoded bead followed by primer extension and/or reverse transcription.
[016] Figures 3A-3D schematically depict downstream processing of recovered beads bound to barcoded fragments. (A) Example of one RNA template. (B) Second strand synthesis.
(C) Gene specific primer, restriction digest or universal adapter ligation.
(D) Recovered barcoded DNA, ready for high-throughput sequencing.
[017] Figure 4 depicts beads according to certain aspects of the invention. Cy 5 shows presence of an adenine nucleotide at position one of the bar-code. Cy3 the shows presence of a thymine nucleotide at position one of the bar-code Texas Red (TxRed) shows the presence of a cytosine nucleotide at position one of the bar-code.
Fluorescein isothiocyanate (FITC) shows the presence of a guanine at position one of the bar-code.
Sequentially sequencing each position of the bar-code provides the unique bar-code identifier. Each transcript captured by the beads can be correlated to a unique starting cell because each cell is represented by a unique bar-code.
[018] Figures 5A-5G depicts a method to generate multiple copies of a uniquely degenerate barcode for single cell analysis according to certain aspects of the invention. (A) Reverse DNA (i.e., starting) template. (B) Circularizing by ligation. (C) Rolling circle amplification using strand displacing polymerase and complementary primer. (D) Inserting into liposome or emulsion with restriction complementary sequence and restriction enzyme. (E) Resulting barcoded oligonucleotides. Each liposome or emulsion contains a unique, degenerate barcode. (F) Each liposome can be fused directly with a single cell (or each barcoded emulsion can be fused with one cell in emulsion). (G) Sequencing query of the barcode region of rolling circle amplification (Rolony) demonstrated clonality. Rolonies were ordered on a grid of 250 nanometer size features.
[019] Figures 6A-6B depicts a method to generate multiple copies of a uniquely degenerate barcode for single cell analysis according to certain aspects of the invention relating to targeting more than one nucleic acid sequence of interest. The left panel in Figures 6A
and 6B shows an oligo-dT sequence annealing primer, which can target polyA
tails of mRNAs found in a cell. The right panel in Figures 6A and 6B demonstrates using a "universal sequence" primer, which has a sequence complementary to an overhang common to several annealing primer sequences, to generate a bead having oligonucleotides that anneal to multiple different nucleic acid targets of interest (shown in red and blue at the 3' end of the oligonucleotide.) [020] Figures 7A-7E show bead clonality using different concentrations of primer. (Fig. 7A:
0.1 pM; Fig. 7B: 1pM, Fig. 7C: 10 pM, and Fig. 7D: 100 pM). Figure 7E shows the 100 pM sample overlaid with beads. See Example 1.
[021] Figures 8A-8H shows bead clonality in emulsions. Fig. 8A shows overlay of uniquely bar-coded beads over white light, showing clonality of beads with an optimal amount of starting template. Fig. 8B shows one cycle sequencing of the bar-code on the fluorescence channels only. Fig. 8C shows white light only. Fig. 8D shows single bead capture in emulsion. Fig 8E shows bar-coded beads in presence of lysed cells in emulsion post-amplification. Fig. 8F and G are magnifications of Fig, 8E. Fig 8H shows introduction of fluorescent bar-codes in single cells [022] Figures 9A-9C shows sequences used in aspects of the invention. Fig. 9A
shows 5' and 3' sequences of a primer (SEQ ID NO:1) used in the Illumina system. Figure 9B
shows 5' and 3' sequence of a primer (SEQ ID NO:2) used in aspects of the invention, including anchor sequence primer, 20-nucleotide bar-code position (--BC(N20)--), and oligo dT
sequence. The cluster sequences facilitate sequencing in the Illumina system.
Figure 9C
shows a sample oligonucleotide attached to a bead having an anchor sequence primer, 20-nucleotide bar-code (--BC(N20)--), and oligo dT sequence (SEQ ID NO:3).
DETAILED DESCRIPTION
[023] In certain aspects, the methods and compositions described herein are useful for single cells analysis, such as, e.g., for the study of genomes, transcriptomes, proteomes, metabolic pathways and the like of complex cell samples. In other aspects, the methods and compositions described herein can be used for antibody discovery by pairing heavy and light chain in single B and T cells, as well as for HLA typing, and long range haplotyping. In still other aspects, the methods and compositions described herein can be used to monitor the impact of small molecule and drugs and their effect in complex normal or cancerous samples for the discovery of new drugs. In yet other aspect, the methods and composition can be used to detect and analyze pathogens such as bacteria or viruses in biological samples.
[024] In certain exemplary embodiments, methods are provided for creating clonal copies of barcode sequences (e.g., degenerate barcodes) and delivering the barcode sequences into a plurality of single cells. According to one aspect of the invention, a plurality of unique nucleic acid sequences comprising a degenerate barcode are amplified on a support (e.g., a bead) such that each discrete area of the support (e.g., each bead) will be coated with clonal copy of a starting nucleic acid sequence (Figure 1). Accordingly, each discrete area of a support; bead, for example, will be uniquely barcoded with a plurality of targeting barcode oligonucleotides. In certain exemplary embodiments, emulsion PCR is performed, wherein degenerate oligonucleotide sequences are attached to a bead using a dilution equivalent maximum of one molecule per bead. The bar-code oligonucleotide length is related to cell sample size of interest. Generally, bar-codes are at least 3 nucleotides long. Often, they are about 20 nucleotides. Thus, for example, a support-attached oligonucleotide having a total length of about 50-60 nucleotides, includes nucleotides encoding a sequencing primer, 20 nucleotides for the bar-code, and an annealing primer.
[025] In some embodiments, the support is a bead. The initial template oligonucleotide loaded on the beads has a sequencing primer region (which will be used to facilitate sequencing of the bar-code), a degenerate region (the actual bar-code) and an annealing primer region, which has a sequence complementary to the target nucleic acid sequence or sequences of interest. The annealing primer can be DNA or RNA (Figure 1A).
Some beads may contain oligonucleotides that bind to more than one target nucleic acid of interest.
[026] The beads are then saturated with an anchor primer. (Figure 1B). The anchor primer has the same sequence as the sequencing primer region of the template oligonucleotide. The anchor primer serves as the second PCR priming end, which allows attachment of the product generated during emulsion PCR to the beads. The beads are then amplified in emulsion PCR (Figure 1C) using a primer complementary to the annealing section of the starting molecule. When emulsion PCR is complete, the anchor primer is extended and contains a copy of the bar-code and the annealing primer. The bead can subsequently be purified from the emulsions and used in downstream applications.
[027] Once the bar-coded beads are prepared, they are used in a second emulsion PCR in the presence of a single cell. The cell is contained within its own unique emulsion, allowing simultaneous PCR in a single assay that contains many cells. (Figure 2). The beads and cells may be introduced to each other in any suitable way. For example, by transfection using liposomes, or by emulsification. Samples containing multiple beads and multiple cells are diluted to achieve a maximum of one bead and one cell per emulsion PCR
reaction. In Figure 2, an example of one bead-cell event is shown.
[028] In some embodiments, thousands to millions of the events shown in Figure 2 may be performed in a single assay, such as one assay performed in a single well.
Each single cell is sequestered into its own unique emulsion in the presence of one bar-coded beads.
The multiple reaction are in the same reaction volume for all the cells.
Because so many cells are analyzed in a single assay, the approach is equivalent to mixing millions of wells of PCR plates. Therefore a single assay is not limited in the amount of single cells to target, or the amount of transcript to target per single cells, provided each cell is uniquely bar-coded in either single emulsion per cell or through liposome transfection of a single bead or bar-code system. See Figure 2A. Millions of emulsions can be present in a single assay; i.e in a single well.
[029] Upon cell lysis, the nucleic acid target of interest is annealed to the complementary sequences on the bar-coded bead template. Figure 2B. Reverse transcription, for a RNA
target, or primer extension, for a DNA target, is performed, and appends a bar-code to the cell RNA or DNA target. Figure 2D. Within one cell, the same bar-code is added to all the target sequences. Thus, as shown in Figure 2D, Cell number 1, bead bar code number 1 has captured four examples of the target sequence (green, yellow, purple, and red).
Each independent cell in the reaction has a different bar code. Figure 2D.
[030] DNA from Beads with bar-coded fragments of interest are recovered and processed in downstream assays. When the bead has RNA attached, cDNA synthesis is performed, followed by PCR amplification using gene specific primer (or restriction cleavage, and/or adapter ligation, follow by PCR) similarly to what has been described previously (Kim et al. (2007) Science 316:1481). See Figure 3. Sequencing of DNA using high-throughput technology is then performed. The sequencing primer is used to sequence the bar-code, through the annealing primer into the target sequence. The target sequencing conveys transcript identity and expression levels, or other genomic or transcriptomic sequence of interest. The bar-code sequence allows each target sequence to be correlated to the single cell from which the sequences originated. While each transcript originating from one cell will have the same bar-code sequence, variation in genomic or transcriptomic information across the cell population is determined by assaying many single cells at the same time.
Because each single cell contains a unique bar-code different from the other single cells, the identified sequences having the same bar-code can be correlated to the same originating cell.
[031] In certain embodiments, multiple mRNAs from each single cell can be obtained and analyzed. For example, oligo-dT (or similar primers) may be used as the annealing primer. See Figures 6A and 6B. The oligo-dT sequence anneals to mRNA polyA
tails and thus capture simultaneously multiple messenger RNAs from a single cell.
This allows for complete or substantially complete transcriptome analysis of multiple single cells in a complex mixture. Characterizing the transcriptomes of multiple cells on a per-cell basis has particular application in studies investigating which cells are malignant than others cancer samples. Moreover, in patients undergoing cancer therapy, the present approach provides for monitoring mutation of each cell's genome and transcriptome before and after treatment; for example with a drug, or following surgery.
This information is particularly useful when coupled with medicines known to be affected by the sequence of a protein. For example, the EGFR inhibitor Erbitux (cetuximab) is ineffective when used with certain mutations of K-ras. The present approach can be used diagnostically to determine, down to the single cell level, how many cells in a tumor sample carry the mutations that make the cells Erbitux resistant. Information regarding the nucleic acid sequences of multiple proteins in each tumor cell is valuable in determining whether to continue or stop treatment with a given drug or switch to an alternative drug.
[032] In another embodiment, at least two oligonucleotides having different annealing primers are attached to the same bead, which allows several target nucleic acids in the same cell to be captured and sequenced. To produce beads containing different annealing primers a universal sequence is attached downstream of the bar-code primer. See Figures 6A and 6B. The universal sequence is complementary to an overhang region on a second primer that contains the annealing primer, which targets the gene of interest.
Multiple annealing primers, each targeting a different gene of interest, may be used. The universal sequence, common to the overhangs of all the annealing primers allows incorporation of the multiple annealing primer sequences onto the beads by PCR. See Figures 6A and 6B.
[033] Beads with multiple annealing primers targeting different nucleic acids of interest have particular use in immune cell applications. In one embodiment, specific sets of targeting oligonucleotides complementary to the heavy and light chains of the B cell antibody coding gene or its RNA are used to capture the pairing of each unique single cell's heavy and light chains that define each specific antibody. In another embodiment, sequences encoding T cell receptor components may be targeted and sequenced. See (Embleton et al. (1992) Nucleic Acids Res. 20(15):3831; Chapal et al. (1997) Biotechniques 23(3):518).
10341 In yet other embodiments, annealing primers are selected for analyzing small nucleotide polymorphisms (SNPs), and for long range haplotyping (Zhang et al., "Long-range polony haplotyping of individual human chromosome molecules," Nat Genet. 2006 Mar;38(3):382-7). These approaches provide specific information for each cell in multiple-cell biological samples.
[035] In certain immune related examples, bar-coding is not necessary if one uses strategies to attach the heavy and light chain prior to PCR or cleavage of the molecules from the beads, such as ligation, of CRE-LOX coupling or fragments of each unique bead, as described by Embleton etal. (1992) Nucleic Acids Res. 20(15):3831; Chapal etal. (1997) Biotechniques 23(3):518, but in such way that many single cell at once can be treated as described in the current invention.
[036] As used herein, the term "barcode" refers to a unique oligonucleotide sequence that allows a corresponding nucleic acid base and/or nucleic acid sequence to be identified. In certain aspects, the nucleic acid base and/or nucleic acid sequence is located at a specific position on a larger polynucleotide sequence (e.g., a polynucleotide covalently attached to a bead). In certain embodiments, barcodes can each have a length within a range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.
In certain aspects, the melting temperatures of barcodes within a set are within 10 C of one another, within 5 C of one another, or within 2 C of one another. In other aspects, barcodes are members of a minimally cross-hybridizing set. That is, the nucleotide sequence of each member of such a set is sufficiently different from that of every other member of the set that no member can form a stable duplex with the complement of any other member under stringent hybridization conditions. In one aspect, the nucleotide sequence of each member of a minimally cross-hybridizing set differs from those of every other member by at least two nucleotides. Barcode technologies are known in the art and are described in Winzeler et al. (1999) Science 285:901; Brenner (2000) Genome Biol.
1:1 Kumar et al. (2001) Nature Rev. 2:302; Giaever et al. (2004) Proc. Natl.
Acad. Sci.
USA 101:793; Eason et al. (2004) Proc. Natl. Acad. Sci. USA 101:11046; and Brenner (2004) Genome Biol. 5:240.
[037] "Complementary" or "substantially complementary" refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
Complementary nucleotides are, generally, A and T/U, or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res. 12:203.
10381 As used herein, the term "hybridization" refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term "hybridization" may also refer to triple-stranded hybridization.
The resulting (usually) double-stranded polynucleotide is a "hybrid" or "duplex."
"Hybridization conditions" will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM.
Hybridization temperatures can be as low as 5 C, but are typically greater than 22 C, more typically greater than about 30 C, and often in excess of about 37 C.
Hybridizations are usually performed under stringent conditions, i.e., conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
Generally, stringent conditions are selected to be about 5 C lower than the I'm for the specific sequence at s defined ionic strength and pH. Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25 C. For example, conditions of 5XSSPE (750 mM NaC1, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30 C are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999).
"Hybridizing specifically to" or "specifically hybridizing to" or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
[039] "Nucleoside" as used herein includes the natural nucleosides, including T-deoxy and 2'-hydroxyl forms, e.g. as described in Komberg and Baker, DNA Replication, 2nd Ed.
(Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990), or the like, with the proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.
Polynucleotides comprising analogs with enhanced hybridization or nuclease resistance properties are described in Uhlman and Peyman (cited above); Crooke et al., Exp. Opin.
Ther. Patents, 6: 855-870 (1996); Mesmaeker et al., Current Opinion in Structural Biology, 5:343-355 (1995); and the like. Exemplary types of polynucleotides that are capable of enhancing duplex stability include oligonucleotide phosphoramidates (referred to herein as "amidates"), peptide nucleic acids (referred to herein as "PNAs"), oligo-2'-0-alkylribonucleotides, polynucleotides containing C-5 propynylpyrimidines, locked nucleic acids (LNAs), and like compounds. Such oligonucleotides are either available commercially or may be synthesized using methods described in the literature.
[040] As used herein, the terms "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," "oligonucleotide fragment" and "polynucleotide"
are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid molecules include single stranded DNA
BRIEF DESCRIPTION OF THE DRAWINGS
1013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
[014] Figures 1A-1E schematically depict a method of amplifying each unique molecule composed of a degenerate barcode on a bead according to certain aspects of the invention.
(A) Attach barcoded template oligonucleotide. (B) saturate solid support with anchor primer. (C) Perform emulsion PCR. (D) Emulsion PCR completed. (E) Barcoded beads are recovered.
[015] Figures 2A-2D schematically depict emulsion PCR of a single cell. (A) Capture of cell and barcoded bead in an emulsion. (B) Lyse cell. (C) Anneal DNA and/or RNA to barcoded bead followed by primer extension and/or reverse transcription.
[016] Figures 3A-3D schematically depict downstream processing of recovered beads bound to barcoded fragments. (A) Example of one RNA template. (B) Second strand synthesis.
(C) Gene specific primer, restriction digest or universal adapter ligation.
(D) Recovered barcoded DNA, ready for high-throughput sequencing.
[017] Figure 4 depicts beads according to certain aspects of the invention. Cy 5 shows presence of an adenine nucleotide at position one of the bar-code. Cy3 the shows presence of a thymine nucleotide at position one of the bar-code Texas Red (TxRed) shows the presence of a cytosine nucleotide at position one of the bar-code.
Fluorescein isothiocyanate (FITC) shows the presence of a guanine at position one of the bar-code.
Sequentially sequencing each position of the bar-code provides the unique bar-code identifier. Each transcript captured by the beads can be correlated to a unique starting cell because each cell is represented by a unique bar-code.
[018] Figures 5A-5G depicts a method to generate multiple copies of a uniquely degenerate barcode for single cell analysis according to certain aspects of the invention. (A) Reverse DNA (i.e., starting) template. (B) Circularizing by ligation. (C) Rolling circle amplification using strand displacing polymerase and complementary primer. (D) Inserting into liposome or emulsion with restriction complementary sequence and restriction enzyme. (E) Resulting barcoded oligonucleotides. Each liposome or emulsion contains a unique, degenerate barcode. (F) Each liposome can be fused directly with a single cell (or each barcoded emulsion can be fused with one cell in emulsion). (G) Sequencing query of the barcode region of rolling circle amplification (Rolony) demonstrated clonality. Rolonies were ordered on a grid of 250 nanometer size features.
[019] Figures 6A-6B depicts a method to generate multiple copies of a uniquely degenerate barcode for single cell analysis according to certain aspects of the invention relating to targeting more than one nucleic acid sequence of interest. The left panel in Figures 6A
and 6B shows an oligo-dT sequence annealing primer, which can target polyA
tails of mRNAs found in a cell. The right panel in Figures 6A and 6B demonstrates using a "universal sequence" primer, which has a sequence complementary to an overhang common to several annealing primer sequences, to generate a bead having oligonucleotides that anneal to multiple different nucleic acid targets of interest (shown in red and blue at the 3' end of the oligonucleotide.) [020] Figures 7A-7E show bead clonality using different concentrations of primer. (Fig. 7A:
0.1 pM; Fig. 7B: 1pM, Fig. 7C: 10 pM, and Fig. 7D: 100 pM). Figure 7E shows the 100 pM sample overlaid with beads. See Example 1.
[021] Figures 8A-8H shows bead clonality in emulsions. Fig. 8A shows overlay of uniquely bar-coded beads over white light, showing clonality of beads with an optimal amount of starting template. Fig. 8B shows one cycle sequencing of the bar-code on the fluorescence channels only. Fig. 8C shows white light only. Fig. 8D shows single bead capture in emulsion. Fig 8E shows bar-coded beads in presence of lysed cells in emulsion post-amplification. Fig. 8F and G are magnifications of Fig, 8E. Fig 8H shows introduction of fluorescent bar-codes in single cells [022] Figures 9A-9C shows sequences used in aspects of the invention. Fig. 9A
shows 5' and 3' sequences of a primer (SEQ ID NO:1) used in the Illumina system. Figure 9B
shows 5' and 3' sequence of a primer (SEQ ID NO:2) used in aspects of the invention, including anchor sequence primer, 20-nucleotide bar-code position (--BC(N20)--), and oligo dT
sequence. The cluster sequences facilitate sequencing in the Illumina system.
Figure 9C
shows a sample oligonucleotide attached to a bead having an anchor sequence primer, 20-nucleotide bar-code (--BC(N20)--), and oligo dT sequence (SEQ ID NO:3).
DETAILED DESCRIPTION
[023] In certain aspects, the methods and compositions described herein are useful for single cells analysis, such as, e.g., for the study of genomes, transcriptomes, proteomes, metabolic pathways and the like of complex cell samples. In other aspects, the methods and compositions described herein can be used for antibody discovery by pairing heavy and light chain in single B and T cells, as well as for HLA typing, and long range haplotyping. In still other aspects, the methods and compositions described herein can be used to monitor the impact of small molecule and drugs and their effect in complex normal or cancerous samples for the discovery of new drugs. In yet other aspect, the methods and composition can be used to detect and analyze pathogens such as bacteria or viruses in biological samples.
[024] In certain exemplary embodiments, methods are provided for creating clonal copies of barcode sequences (e.g., degenerate barcodes) and delivering the barcode sequences into a plurality of single cells. According to one aspect of the invention, a plurality of unique nucleic acid sequences comprising a degenerate barcode are amplified on a support (e.g., a bead) such that each discrete area of the support (e.g., each bead) will be coated with clonal copy of a starting nucleic acid sequence (Figure 1). Accordingly, each discrete area of a support; bead, for example, will be uniquely barcoded with a plurality of targeting barcode oligonucleotides. In certain exemplary embodiments, emulsion PCR is performed, wherein degenerate oligonucleotide sequences are attached to a bead using a dilution equivalent maximum of one molecule per bead. The bar-code oligonucleotide length is related to cell sample size of interest. Generally, bar-codes are at least 3 nucleotides long. Often, they are about 20 nucleotides. Thus, for example, a support-attached oligonucleotide having a total length of about 50-60 nucleotides, includes nucleotides encoding a sequencing primer, 20 nucleotides for the bar-code, and an annealing primer.
[025] In some embodiments, the support is a bead. The initial template oligonucleotide loaded on the beads has a sequencing primer region (which will be used to facilitate sequencing of the bar-code), a degenerate region (the actual bar-code) and an annealing primer region, which has a sequence complementary to the target nucleic acid sequence or sequences of interest. The annealing primer can be DNA or RNA (Figure 1A).
Some beads may contain oligonucleotides that bind to more than one target nucleic acid of interest.
[026] The beads are then saturated with an anchor primer. (Figure 1B). The anchor primer has the same sequence as the sequencing primer region of the template oligonucleotide. The anchor primer serves as the second PCR priming end, which allows attachment of the product generated during emulsion PCR to the beads. The beads are then amplified in emulsion PCR (Figure 1C) using a primer complementary to the annealing section of the starting molecule. When emulsion PCR is complete, the anchor primer is extended and contains a copy of the bar-code and the annealing primer. The bead can subsequently be purified from the emulsions and used in downstream applications.
[027] Once the bar-coded beads are prepared, they are used in a second emulsion PCR in the presence of a single cell. The cell is contained within its own unique emulsion, allowing simultaneous PCR in a single assay that contains many cells. (Figure 2). The beads and cells may be introduced to each other in any suitable way. For example, by transfection using liposomes, or by emulsification. Samples containing multiple beads and multiple cells are diluted to achieve a maximum of one bead and one cell per emulsion PCR
reaction. In Figure 2, an example of one bead-cell event is shown.
[028] In some embodiments, thousands to millions of the events shown in Figure 2 may be performed in a single assay, such as one assay performed in a single well.
Each single cell is sequestered into its own unique emulsion in the presence of one bar-coded beads.
The multiple reaction are in the same reaction volume for all the cells.
Because so many cells are analyzed in a single assay, the approach is equivalent to mixing millions of wells of PCR plates. Therefore a single assay is not limited in the amount of single cells to target, or the amount of transcript to target per single cells, provided each cell is uniquely bar-coded in either single emulsion per cell or through liposome transfection of a single bead or bar-code system. See Figure 2A. Millions of emulsions can be present in a single assay; i.e in a single well.
[029] Upon cell lysis, the nucleic acid target of interest is annealed to the complementary sequences on the bar-coded bead template. Figure 2B. Reverse transcription, for a RNA
target, or primer extension, for a DNA target, is performed, and appends a bar-code to the cell RNA or DNA target. Figure 2D. Within one cell, the same bar-code is added to all the target sequences. Thus, as shown in Figure 2D, Cell number 1, bead bar code number 1 has captured four examples of the target sequence (green, yellow, purple, and red).
Each independent cell in the reaction has a different bar code. Figure 2D.
[030] DNA from Beads with bar-coded fragments of interest are recovered and processed in downstream assays. When the bead has RNA attached, cDNA synthesis is performed, followed by PCR amplification using gene specific primer (or restriction cleavage, and/or adapter ligation, follow by PCR) similarly to what has been described previously (Kim et al. (2007) Science 316:1481). See Figure 3. Sequencing of DNA using high-throughput technology is then performed. The sequencing primer is used to sequence the bar-code, through the annealing primer into the target sequence. The target sequencing conveys transcript identity and expression levels, or other genomic or transcriptomic sequence of interest. The bar-code sequence allows each target sequence to be correlated to the single cell from which the sequences originated. While each transcript originating from one cell will have the same bar-code sequence, variation in genomic or transcriptomic information across the cell population is determined by assaying many single cells at the same time.
Because each single cell contains a unique bar-code different from the other single cells, the identified sequences having the same bar-code can be correlated to the same originating cell.
[031] In certain embodiments, multiple mRNAs from each single cell can be obtained and analyzed. For example, oligo-dT (or similar primers) may be used as the annealing primer. See Figures 6A and 6B. The oligo-dT sequence anneals to mRNA polyA
tails and thus capture simultaneously multiple messenger RNAs from a single cell.
This allows for complete or substantially complete transcriptome analysis of multiple single cells in a complex mixture. Characterizing the transcriptomes of multiple cells on a per-cell basis has particular application in studies investigating which cells are malignant than others cancer samples. Moreover, in patients undergoing cancer therapy, the present approach provides for monitoring mutation of each cell's genome and transcriptome before and after treatment; for example with a drug, or following surgery.
This information is particularly useful when coupled with medicines known to be affected by the sequence of a protein. For example, the EGFR inhibitor Erbitux (cetuximab) is ineffective when used with certain mutations of K-ras. The present approach can be used diagnostically to determine, down to the single cell level, how many cells in a tumor sample carry the mutations that make the cells Erbitux resistant. Information regarding the nucleic acid sequences of multiple proteins in each tumor cell is valuable in determining whether to continue or stop treatment with a given drug or switch to an alternative drug.
[032] In another embodiment, at least two oligonucleotides having different annealing primers are attached to the same bead, which allows several target nucleic acids in the same cell to be captured and sequenced. To produce beads containing different annealing primers a universal sequence is attached downstream of the bar-code primer. See Figures 6A and 6B. The universal sequence is complementary to an overhang region on a second primer that contains the annealing primer, which targets the gene of interest.
Multiple annealing primers, each targeting a different gene of interest, may be used. The universal sequence, common to the overhangs of all the annealing primers allows incorporation of the multiple annealing primer sequences onto the beads by PCR. See Figures 6A and 6B.
[033] Beads with multiple annealing primers targeting different nucleic acids of interest have particular use in immune cell applications. In one embodiment, specific sets of targeting oligonucleotides complementary to the heavy and light chains of the B cell antibody coding gene or its RNA are used to capture the pairing of each unique single cell's heavy and light chains that define each specific antibody. In another embodiment, sequences encoding T cell receptor components may be targeted and sequenced. See (Embleton et al. (1992) Nucleic Acids Res. 20(15):3831; Chapal et al. (1997) Biotechniques 23(3):518).
10341 In yet other embodiments, annealing primers are selected for analyzing small nucleotide polymorphisms (SNPs), and for long range haplotyping (Zhang et al., "Long-range polony haplotyping of individual human chromosome molecules," Nat Genet. 2006 Mar;38(3):382-7). These approaches provide specific information for each cell in multiple-cell biological samples.
[035] In certain immune related examples, bar-coding is not necessary if one uses strategies to attach the heavy and light chain prior to PCR or cleavage of the molecules from the beads, such as ligation, of CRE-LOX coupling or fragments of each unique bead, as described by Embleton etal. (1992) Nucleic Acids Res. 20(15):3831; Chapal etal. (1997) Biotechniques 23(3):518, but in such way that many single cell at once can be treated as described in the current invention.
[036] As used herein, the term "barcode" refers to a unique oligonucleotide sequence that allows a corresponding nucleic acid base and/or nucleic acid sequence to be identified. In certain aspects, the nucleic acid base and/or nucleic acid sequence is located at a specific position on a larger polynucleotide sequence (e.g., a polynucleotide covalently attached to a bead). In certain embodiments, barcodes can each have a length within a range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.
In certain aspects, the melting temperatures of barcodes within a set are within 10 C of one another, within 5 C of one another, or within 2 C of one another. In other aspects, barcodes are members of a minimally cross-hybridizing set. That is, the nucleotide sequence of each member of such a set is sufficiently different from that of every other member of the set that no member can form a stable duplex with the complement of any other member under stringent hybridization conditions. In one aspect, the nucleotide sequence of each member of a minimally cross-hybridizing set differs from those of every other member by at least two nucleotides. Barcode technologies are known in the art and are described in Winzeler et al. (1999) Science 285:901; Brenner (2000) Genome Biol.
1:1 Kumar et al. (2001) Nature Rev. 2:302; Giaever et al. (2004) Proc. Natl.
Acad. Sci.
USA 101:793; Eason et al. (2004) Proc. Natl. Acad. Sci. USA 101:11046; and Brenner (2004) Genome Biol. 5:240.
[037] "Complementary" or "substantially complementary" refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
Complementary nucleotides are, generally, A and T/U, or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res. 12:203.
10381 As used herein, the term "hybridization" refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term "hybridization" may also refer to triple-stranded hybridization.
The resulting (usually) double-stranded polynucleotide is a "hybrid" or "duplex."
"Hybridization conditions" will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM.
Hybridization temperatures can be as low as 5 C, but are typically greater than 22 C, more typically greater than about 30 C, and often in excess of about 37 C.
Hybridizations are usually performed under stringent conditions, i.e., conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
Generally, stringent conditions are selected to be about 5 C lower than the I'm for the specific sequence at s defined ionic strength and pH. Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25 C. For example, conditions of 5XSSPE (750 mM NaC1, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30 C are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999).
"Hybridizing specifically to" or "specifically hybridizing to" or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
[039] "Nucleoside" as used herein includes the natural nucleosides, including T-deoxy and 2'-hydroxyl forms, e.g. as described in Komberg and Baker, DNA Replication, 2nd Ed.
(Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990), or the like, with the proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.
Polynucleotides comprising analogs with enhanced hybridization or nuclease resistance properties are described in Uhlman and Peyman (cited above); Crooke et al., Exp. Opin.
Ther. Patents, 6: 855-870 (1996); Mesmaeker et al., Current Opinion in Structural Biology, 5:343-355 (1995); and the like. Exemplary types of polynucleotides that are capable of enhancing duplex stability include oligonucleotide phosphoramidates (referred to herein as "amidates"), peptide nucleic acids (referred to herein as "PNAs"), oligo-2'-0-alkylribonucleotides, polynucleotides containing C-5 propynylpyrimidines, locked nucleic acids (LNAs), and like compounds. Such oligonucleotides are either available commercially or may be synthesized using methods described in the literature.
[040] As used herein, the terms "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," "oligonucleotide fragment" and "polynucleotide"
are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid molecules include single stranded DNA
(ssDNA), double stranded DNA (dsDNA), single stranded RNA (ssRNA) and double stranded RNA (dsRNA). Different nucleic acid molecules may have different three-dimensional structures, and may perform various functions, known or unknown.
Non-limiting examples of nucleic acid molecules include a gene, a gene fragment, a genomic gap, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), miRNA, small nucleolar RNA (snoRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers.
Nucleic acid molecules useful in the methods described herein may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
[041] An oligonucleotide sequence refers to a linear polymer of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof.
The term "oligonucleotide" usually refers to a shorter polymer, e.g., comprising from about 3 to about 100 monomers, and the term "polynucleotide" usually refers to longer polymers, e.g., comprising from about 100 monomers to many thousands of monomers, e.g., 10,000 monomers, or more An "oligonucleotide fragment" refers to an oligonucleotide sequence that has been cleaved into two or more smaller oligonucleotide sequences.
Oligonucleotides comprising probes or primers usually have lengths in the range of from 12 to 60 nucleotides, and more usually, from 18 to 40 nucleotides.
Oligonucleotides and polynucleotides may be natural or synthetic. Oligonucleotides and polynucleotides include deoxyribonucleosides, ribonucleosides, and non-natural analogs thereof, such as anomeric forms thereof, peptide nucleic acids (PNAs), and the like, provided that they are capable of specifically binding to a target genome by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
[042] Usually nucleosidic monomers are linked by phosphodiester bonds.
Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T"
denotes deoxythymidine, and "U" denotes the ribonucleoside, uridine, unless otherwise noted.
Usually oligonucleotides comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed in methods and processes described herein. For example, where processing by an enzyme is called for, usually oligonucleotides consisting solely of natural nucleotides are required. Likewise, where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA/DNA
duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references. Oligonucleotides and polynucleotides may be single stranded or double stranded.
[043] Nucleic acid molecules may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcyto sine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethy1-2-thiouridine, carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like.
Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
[044] In certain exemplary embodiments, large polynucleotides are provided. In certain aspects, isolation techniques that maximize the lengths of polynucleotides (e.g., DNA
molecules) obtained are used. For example, in situ lysis or deproteinization (e.g., with EDTA, detergent, protease, any combinations thereof and the like) after agarose embedding (as routinely performed for pulsed field gel electrophoresis) can be used to obtain polynucleotides.
[045] Nucleic acid molecules may be isolated from natural sources or purchased from commercial sources. Oligonucleotide sequences may also be prepared by any suitable method, e.g., standard phosphoramidite methods such as those described by Beaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859) or the triester method according to Matteucci et al. (1981)1 Am. Chem. Soc. 103:3185), or by other chemical methods using either a commercial automated oligonucleotide synthesizer or high-throughput, high-density array methods known in the art (see U.S. Patent Nos. 5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference in its entirety for all purposes). Pre-synthesized oligonucleotides may also be obtained commercially from a variety of vendors.
[046] Nucleic acid molecules may be obtained from one or more biological samples. As used herein, a "biological sample" may be a single cell or many cells. A biological sample may comprise a single cell type or a combination of two or more cell types. A
biological sample further includes a collection of cells that perform a similar function such as those found, for example, in a tissue. Accordingly, certain aspects of the invention are directed to biological samples containing one or more tissues. As used herein, a tissue includes, but is not limited to, epithelial tissue (e.g., skin, the lining of glands, bowel, skin and organs such as the liver, lung, kidney), endothelium (e.g., the lining of blood and lymphatic vessels), mesothelium (e.g., the lining of pleural, peritoneal and pericardial spaces), mesenchyme (e.g., cells filling the spaces between the organs, including fat, muscle, bone, cartilage and tendon cells), blood cells (e.g. erythrocytes, granulocytes, neutrophils, eosinophils, basophils, monocytes, T-lymphocytes (also known as T-cells), B-lymphocytes (also known as B-cells), plasma cells, megakaryocytes and the like), neurons, germ cells (e.g., spermatozoa, oocytes), amniotic fluid cells, placenta, stem cells and the like. A tissue sample includes microscopic samples as well as macroscopic samples. In certain aspects, a sample can be obtained from one or more of single cells in culture, metagenomic samples, embryonic stem cells, induced pluripotent stem cells, cancer samples, tissue sections, biopsies and the like, and any combinations of these.
[047] In certain aspects, nucleic acid sequences derived or obtained from one or more organisms are provided. As used herein, the term "organism" includes, but is not limited to, a human, a non-human primate, a cow, a horse, a sheep, a goat, a pig, a dog, a cat, a rabbit, a mouse, a rat, a gerbil, a frog, a toad, a fish (e.g., Danio rerio) a roundworm (e.g., C. elegans) and any transgenic species thereof. The term "organism" further includes, but is not limited to, a yeast (e.g., S. cerevisiae) cell, a yeast tetrad, a yeast colony, a bacterium, a bacterial colony, a virion, virosome, virus-like particle and/or cultures thereof, and the like.
[048] Isolation, extraction or derivation of nucleic acid sequences may be carried out by any suitable method. Isolating nucleic acid sequences from a biological sample generally includes treating a biological sample in such a manner that nucleic acid sequences present in the sample are extracted and made available for analysis. Any isolation method that results in extracted nucleic acid sequences may be used in the practice of the present invention. It will be understood that the particular method used to extract nucleic acid sequences will depend on the nature of the source.
[049] Methods of DNA extraction are well-known in the art. A classical DNA
isolation protocol is based on extraction using organic solvents such as a mixture of phenol and chloroform, followed by precipitation with ethanol (J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2" Ed., Cold Spring Harbour Laboratory Press:
New York, N.Y.). Other methods include: salting out DNA extraction (P.
Sunnucks et al., Genetics, 1996, 144: 747-756; S. M. Aljanabi and I. Martinez, Nucl. Acids Res. 1997, 25: 4692-4693), trimethylammonium bromide salts DNA extraction (S. Gustincich et al., BioTechniques, 1991, 11: 298-302) and guanidinium thiocyanate DNA extraction (J. B.
W. Hammond et al., Biochemistry, 1996, 240: 298-300). A variety of kits are commercially available for extracting DNA from biological samples (e.g., BD
Biosciences Clontech (Palo Alto, CA): Epicentre Technologies (Madison, WI);
Gentra Systems, Inc. (Minneapolis, MN); MicroProbe Corp. (Bothell, WA); Organon Teknika (Durham, NC); and Qiagen Inc. (Valencia, CA)).
[050] Methods of RNA extraction are also well known in the art (see, for example, J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" 1989, 2" Ed., Cold Spring Harbour Laboratory Press: New York) and several kits for RNA extraction from bodily fluids are commercially available (e.g., Ambion, Inc. (Austin, TX); Amersham Biosciences (Piscataway, NJ); BD Biosciences Clontech (Palo Alto, CA); BioRad Laboratories (Hercules, CA); Dynal Biotech Inc. (Lake Success, NY); Epicentre Technologies (Madison, WI); Gentra Systems, Inc. (Minneapolis, MN); GIBCO BRL
(Gaithersburg, MD); Invitrogen Life Technologies (Carlsbad, CA); MicroProbe Corp. (Bothell, WA);
Organon Teknika (Durham, NC); Promega, Inc. (Madison, WI); and Qiagen Inc.
(Valencia, CA)).
[051] In certain exemplary embodiments, oligonucleotide sequences are immobilized on a solid support. The support can be simple square grids, checkerboard (e.g., offset) grids, hexagonal arrays and the like. Suitable supports include, but are not limited to, slides, beads, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, plates (e.g., 96-well, 48-well, 24-well, 12-well, eight-well, six-well, four-well, single-well and the like), cell surfaces (e.g., S. aureus cells) and the like. In various embodiments, a solid support may be biological, non-biological, organic, inorganic, or any combination thereof.
[052] In certain exemplary embodiments, beads and bead-based arrays are provided. As used herein, the term "bead" refers to a discrete particle that may be spherical (e.g., microspheres) or have an irregular shape. Beads may be as small as approximately 0.1 i.trn in diameter or as large approximately several millimeters in diameter.
Beads may comprise a variety of materials including, but not limited to, paramagnetic materials, ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon and the like.
[053] In accordance with certain examples, a support (e.g., a bead) may have functional groups attached to its surface which can be used to bind one or more reagents described herein to the bead. One or more reagents can be attached to a support (e.g., a bead) by hybridization, covalent attachment, magnetic attachment, affinity attachment and the like.
Beads coated with a variety of attaachments are commercially available (Dynabeads, Invitrogen). Supports (e.g., beads) may also be functionalized using, for example, solid-phase chemistries known in the art (see, e.g., U.S. Pat. No. 5,919,523).
[054] As used herein, the term "attach" refers to both covalent interactions and noncovalent interactions. A covalent interaction is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (i.e., a single bond), two pairs of electrons (i.e., a double bond) or three pairs of electrons (i.e., a triple bond).
Covalent interactions are also known in the art as electron pair interactions or electron pair bonds. Noncovalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (i.e., via short-range noncovalent forces), hydrophobic interactions, ionic bonds and the like. A review of noncovalent interactions can be found in Alberts et al., in Molecular Biology of the Cell, 3d edition, Garland Publishing, 1994.
[055] In certain exemplary embodiments, methods for amplifying nucleic acid sequences are provided. Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant.
Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods 1:241; and U.S. Patent Nos.
4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl.
Acad. Sci.
U.S.A. 91:360-364), self sustained sequence replication (Guatelli et al.
(1990) Proc. Natl.
Acad. Sci. US.A. 87:1874), transcriptional amplification system (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. US.A. 86:1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J Biol. Chem.
275:2619; and Williams et al. (2002) 1 Biol. Chem. 277:7790), the amplification methods described in U.S. Patent Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA) or any other nucleic acid amplification method using techniques well known to those of skill in the art.
[056] "Polymerase chain reaction," or "PCR," refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A
Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature greater than 90 C, primers annealed at a temperature in the range 50-75 C, and primers extended at a temperature in the range 72-78 C.
[057] The term "PCR" encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 microliters. "Reverse transcription PCR," or "RT-PCR," means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al., U.S. Patent No. 5,168,038. "Real-time PCR" means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Patent No.
5,210,015 ("Taqman"); Wittwer et al., U.S. Patent Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Patent No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). "Nested PCR" means a two-stage PCR wherein the amplicon of a first PCR
becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, "initial primers" in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and "secondary primers" mean the one or more primers used to generate a second, or nested, amplicon. "Multiplexed PCR" means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al.
(1999) Anal.
Biochem., 273:221-228 (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. "Quantitative PCR" means a PCR
designed to measure the abundance of one or more specific target sequences in a sample or specimen. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references: Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17:9437-9447 (1989);
Zimmerman et al., Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446 (1989);
and the like.
10581 In certain exemplary embodiments, methods of determining the sequence identities of nucleic acid sequences are provided. Determination of the sequence of a nucleic acid sequence of interest (e.g., immune cell nucleic acid sequences) can be performed using variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. Serial No. 12/027,039, filed February 6, 2008; Porreca et al (2007) Nat.
Methods 4:931), polymerized colony (POLONY) sequencing (U.S. Patent Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425); nanogrid rolling circle sequencing (ROLONY) (U.S. Serial No. 12/120,541, filed May 14, 2008), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout) and the like. High-throughput sequencing methods, e.g., on cyclic array sequencing using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrents, Complete Genomics, Pacific Bioscience, Helicos, Polonator platforms (Worldwide Web Site: Polonator.org), and the like, can also be utilized. High-throughput sequencing methods are described in U.S. Serial No.
61/162,913, filed March 24, 2009. A variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172).
[059] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more disorders or diseases associated with an infectious agent are provided.
Infectious agents include, but are not limited to, viruses, bacteria, fungi, parasites, infectious proteins and the like.
[060] Viruses include, but are not limited to, DNA or RNA animal viruses. As used herein, RNA viruses include, but are not limited to, virus families such as Picornaviridae (e.g., polioviruses), Reoviridae (e.g., rotaviruses), Togaviridae (e.g., encephalitis viruses, yellow fever virus, rubella virus), Orthomyxoviridae (e.g., influenza viruses), Paramyxoviridae (e.g., respiratory syncytial virus, measles virus, mumps virus, parainfluenza virus), Rhabdoviridae (e.g., rabies virus), Coronaviridae, Bunyaviridae, Flaviviridae, Filoviridae, Arenaviridae, Bunyaviridae and Retroviridae (e.g., human T
cell lymphotropic viruses (HTLV), human immunodeficiency viruses (HIV)). As used herein, DNA viruses include, but are not limited to, virus families such as Papovaviridae (e.g., papilloma viruses), Adenoviridae (e.g., adenovirus), Herpesviridae (e.g., herpes simplex viruses), and Poxviridae (e.g., variola viruses).
[061] Bacteria include, but are not limited to, gram positive bacteria, gram negative bacteria, acid-fast bacteria and the like.
[062] As used herein, gram positive bacteria include, but are not limited to, Actinomedurae, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium, Enterococcus faecalis, Listeria monocytogenes, Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epiderm, Streptococcus mutans, Streptococcus pneumoniae and the like.
[063] As used herein, gram negative bacteria include, but are not limited to, Afipia felis, Bacteriodes, Bartonella bacilliformis, Bortadella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella, Calymmatobacterium granulomatis, Campylobacter, Escherichia coli, Francisella tularensis, Gardnerella vaginalis, Haemophilius aegyptius, Haemophilius ducreyi, Haemophilius influenziae, Heliobacter pylori, Legionella pneumophila, Leptospira interrogans, Neisseria meningitidia, Porphyromonas gingivalis, Providencia sturti, Pseudomonas aeruginosa, Salmonella enteridis, Salmonella typhi, Serratia marcescens, Shigella boydii, Streptobacillus moniliformis, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis and the like.
[064] As used herein, acid-fast bacteria include, but are not limited to, Myobacterium avium, Myobacterium leprae, Myobacterium tuberculosis and the like.
[065] As used herein, other bacteria not falling into the other three categories include, but are not limited to, Bartonella henseiae, Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasma pneumoniae, Rickettsia akari, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia tsutsugamushi, Rickettsia typhi, Ureaplasma urealyticum, Diplococcus pneumoniae, Ehrlichia chafensis, Enterococcus faecium, Meningococci and the like.
[066] As used herein, fungi include, but are not limited to, Aspergilli, Candidae, Candida albicans, Coccidioides immitis, Cryptococci, and combinations thereof.
[067] As used herein, parasitic microbes include, but are not limited to, Balantidium coli, Cryptosporidium parvum, Cyclospora cayatanensis, Encephalitozoa, Entamoeba histolytica, Enterocytozoon bieneusi, Giardia lamblia, Leishmaniae, Plasmodii, Toxoplasma gondii, Trypanosomae, trapezoidal amoeba and the like.
[068] As used herein, parasites include worms (e.g., helminthes), particularly parasitic worms including, but not limited to, Nematoda (roundworms, e.g., whipworms, hookworms, pinworms, ascarids, filarids and the like), Cestoda (e.g., tapeworms) [069] As used herein, infectious proteins include prions. Disorders caused by prions include, but are not limited to, human disorders such as Creutzfeldt-Jakob disease (CJD) (including, e.g., iatrogenic Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease (vCJD), familial Creutzfeldt-Jakob disease (fCJD), and sporadic Creutzfeldt-Jakob disease (sCJD)), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (fFI), sporadic fatal insomnia (sFI), kuru, and the like, as well as disorders in animals such as scrapie (sheep and goats), bovine spongiform encephalopathy (BSE) (cattle), transmissible mink encephalopathy (TME) (mink), chronic wasting disease (CWD) (elk, mule deer), feline spongiform encephalopathy (cats), exotic ungulate encephalopathy (EUE) (nyala, oryx, greater kudu), spongiform encephalopathy of the ostrich and the like.
[070] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more cellular proliferative disorders are provided. Cellular proliferative disorders are intended to include disorders associated with rapid proliferation. As used herein, the term "cellular proliferative disorder" includes disorders characterized by undesirable or inappropriate proliferation of one or more subset(s) of cells in a multicellular organism.
The term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR
Medical Dictionary 1st edition (1995), incorporated herein by reference in its entirety for all purposes). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal. Id. Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor).
[071] The language "treatment of cellular proliferative disorders" is intended to include the prevention of the induction, onset, establishment or growth of neoplasms in a subject or a reduction in the growth of pre-existing neoplasms in a subject. The language also can describe inhibition of the invasion of neoplastic cells into neighboring tissues or the metastasis of a neoplasm from one site to another. Examples of the types of neoplasms intended to be encompassed by the present invention include but are not limited to those neoplasms associated with cancers of the breast, skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, neural tissue, head and neck, colon, stomach, bronchi, and/or kidneys.
[072] Cellular proliferative disorders can further include disorders associated with hyperproliferation of vascular smooth muscle cells such as proliferative cardiovascular disorders, e.g., atherosclerosis and restenosis. Cellular proliferation disorders can also include disorders such as proliferative skin disorders, e.g., X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic dermatitis. Cellular proliferative disorders can further include disorders such as autosomal dominant polycystic kidney disease (ADPKD), mastocystosis, and cellular proliferation disorders caused by infectious agents such as viruses.
[073] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more autoimmune disorders are provided. As used herein, the term "autoimmune disorder" is a disease or disorder caused by a subject producing an inappropriate immune response against its own tissues. As used herein, an autoimmune disorder includes, but is not limited to, disorders such as Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid sundrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Balo disease, Bechet disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis herpetiformis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST
syndrome, Crohn's disease, Degos disease, dermatomyositis, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves disease, Guillain-Barre, Hashimoto thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes, juvenile arthritis, lichen planus, lupus, Meniere disease, mixed connective tissue disease, multiple sclerosis, myasthemia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud phenomenon, Reiter syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren syndrome, stiff-person syndrome, Takayasu arthritis, temporal arteritis/giant cell arteritis, ulcerative colitis, vasculitis, vitiligo, Wegener granulomatosis and the like (See the American Autoimmune Related Diseases Association, Inc. website: aarda.org).
[074] It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.
[075] EXAMPLE 1 Preparing Bar-Coded Beads a. Loading the Template Bar-Code Oligonucleotide Onto Beads [076] Beads (1 M; Cl carboxylic 1 micron beads) were resuspended by vortexing and transferred in a volume of 80 IA to a 1.5 ml silicon tube (Ambion). The beads were washed twice with 2X (Bind and Wash Buffer contains 10 mM Tris-HC1 ph7.5, 1 mM
EDTA, 2M NaCI; "B&W"). Beads were isolated using magnets between washes.
Nucleotide sequences are listed in Table 4. The washed beads were resuspended in 100 11.1 B&W to which oligo dT bar-code template oligonucleotide (HSCT_BC_anchorl) were added at the concentrations as shown in Table 1.
Tube Primer Stock Primer Concentration volume (A) 1 100 pM 80 2 10 pM 80 3 1 pM 80 4 0.1 pN 80 Table 1.
[077] The template oligonucleotide and beads were incubated on a rotator for 20 minutes, then washed twice with 200 p1 of lx B&W then resuspended in 100 pi of 2x B&W.
b. Saturating the Beads with Anchor Primer [078] The beads, pre-loaded with the template oligonucleotide as in Example 1(a) above, were incubated on a rotator for 20 minutes with Anchor primer mix (1mM
HSCT_Bead_anchor 1 and lx B&W buffer) to coat the beads with the anchor primer then washed twice with 200 p,1 of lx B&W, once in 200 p,1 of TE, then resuspended in 100 1 of TE. The anchor primer has a 5' biotin which binds to the streptavidin coated beads.
Typically, 30% of the beads have an oligonucleotide. On those beads 100% or substantially all of the anchor primers are typically extended.
c. Emulsion PCR to Synthesis the Oligonucleotide From the Anchor Primer [079] Aqueous Mix and Oil Mix were prepared as described in Table 2.
AQUEOUS MIX
Component Volume for 1 Volume for tube (p,1) 4.5 tubes(p,1) x PCR 96 432 buffer (Enymatics) 50 mM 242 1089 MgC12 25 mM 135 607 dNTP mix 2 mM 6 27 HSCT dA-rev_emulsion primer OIL MIX
Component Volume for 1 Volume for tube ( 1) 4.5 tubes( 1) Tegosoft 4.4 19.8 DEC
Mineral oil 1.2 5.4 ABIL WE09 425 1.9 Table 2.
[080] Both solutions are mixed by vortexing. The oil mix is allowed to degass then 5.5 ml portions were placed in 50 ml Teflon-coated aluminum test tubes.
[081] The emulsions were made by adding 800 [L1 PCR mix, 100 p.1 Enzymatics Taq (5U/ 1), quickly vortexing and spinning, then immediately adding 60 ml of bar-code anchored beads, followed by vortex and spinning. The 960 ml mix was transferred to a tube of oil and vortexed for 2.25 min at 2200 rpm, which was followed by emulsion PCR
using the following PCR protocol steps:
a. 94 C for 5 min b. 94 C for 15 sec c. 58 C for 30 sec d. 70 C for 75 sec e. Cycle to step b 119 times f. 72 C for 2 min g. Incubate at ¨ 10 C until ready to use.
[082] Formation of an emulsion was confirmed by verifying under a microscope that a creamy white consistency was obtained when an emulsifier/oil mixture (240 p.1 emulsifier: 960 1 oil, or 480 1 emulsifier: 7200 oil) was added to an aqueous layer (384 p,1) and vortexed at 4 C for 5 minutes. Results are show in Figures 7A-7E.
[083] In a similar experiment, Dynal M270 3-micron beads were used under similar conditions and similar results were achieved.
[084] Bar-coding was also achieved as follows.
AQUEOUS MIX
Component Final Volume per concentration tube(i1) dH20 520.4 x PCR lx 80 buffer (Enymatics) 25 mM 2mM 64 dNTP mix 2 mM 10 M 4 HSCT dA-rev_emulsion primer 30% (w/v) 0.06 1.6 BSA (Sigma) OIL MIX
Component Volume for 1 Volume for tube ( 1) 4.5 tubes(1.11) Tegosoft 4.4 19.8 DEC
Mineral oil 1.2 5.4 ABIL WE09 425 1.9 Table 3.
[085] The aqueous mix was vortexed, then 0.6m! of mix was added per 1.5 ml tube (Ambion;
non-stick). 50 1 of M280 HSCT Anchor bead was added per tube, then the tubes were sonicated for 3 cycles of 10 seconds. After sonication, the tubes were placed on ice, and 80 vtl of Taq Polymerase (5U/ 1) was added per tube. The tubes were again vortexed and placed on ice. 800 pi of the mixture was added to the oil phase, the tubes were vortexed and PCR was performed as described in Example 1 part c. In similar experiments well/plates were used. Each well contained 55111/well of the mixture.
Sequence Name SEQ ID NO: Sequence HSCT BC anchor 1 4 /52-Bio/ACA CTC TTT CCC TAC ACG ACG CTC
TTC
CGA TCT NNN NNN NNN NNN NNN NNN NNC AGC
TTT TTT TTT TTT TTT TTT TTT TTT T
HSCT_Bead_anchor 5 /52-Bio/ACA CTC TTT CCC TAC ACG ACG CTC
TTC
HSCT clonaltest B 6 /5Phos/AGA TCG GAA GAG CGT CGT GTA
C seq H¨SCT_dA_rev_emul 7 AAA AAA AAA AAA AAA AAA AAA AAA ACG AC
sion primer HSCT BC_anchor_r 8 AAA AAA AAA AAA AAA AAA AAA AAA AGC TGN
ev(no-bio) NNN NNN NNN NNN NNN NNN NAG ATC GGA AGA
GCG TCG TGT AGG GAA AGA GTG T
Bead attached to 9 BEAD/52-Bar-coded Bio/ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Oligonucleotide NNN NNN NNN NNN NNN NNN NNC AGC TTT TTT
TTT TTT TTT TTT TTT TTT T
ATGTGCTGCGAGAAGGCTAGA/5Phos/
[086] Table 4 shows the sequences used in Example 1.
[087] The final sequence attached to the bead in Example 1 is shown in SEQ ID
NO:9. The bead is connected 5' to 3' to the oligonucleotide which encodes the anchor primer sequence, the bar code (N20) and an oligo dT primer.
Introduction of One Unique Bar-Coded Bead per Cell [088] Figure 4 demonstrates introduction of beads carrying unique bar-coded oligonucleotides into individual cells. Here, beads post-emulsion PCR are sequenced for one base of their bar-code to show that each beads have a unique bar-code and demonstrate clonality. Each nucleotide is queried by a different fluorophores as describe previously (Porreca et al.
(2006) Curr. Protoc. MoL Biol. Chapter 7:Unit 78). Cy 5 shows presence of an adenine nucleotide at position one of the bar-code. Cy3 the shows presence of a thymine nucleotide at position one of the bar-code Texas Red (Txred) shows the presence of a cytosine nucleotide at position one of the bar-code. Fluorescein isothiocyanate (FITC) shows the presence of a guanine at position one of the bar-code. The image overlay of all four fluorophores for a single position on the bar-codes is shown and demonstrates clonality. Clonality refers to each single bead harboring one unique bar-code, which has been successfully amplified onto the bead. If the beads had contained multiple bar-codes;
that is, had been non-clonal (for example, having multiple bar-code templates loaded on the bead by accident), the overlay would have demonstrated more than one fluorophore color per bead when querying a single position on the bar-code during sequencing.
Complete sequencing of the bar-code, which allows correlation to the cell, is by multiple successive round of sequencing for each nucleotide position.
[089] White light microscopy analysis of the beads and emulsion reaction shows that the starting template and the bead in emulsion were correctly diluted to achieve a maximum of one bead or less per emulsion and one template or less per bead.
Introduction of Unique Bar-Coded Oligonucleotides on a Grid Support [090] Multiple copies of the same unique bar-code for single cell analysis were made by rolling circle amplification (RCA) product (Rolony) from a circularized starting bar-code unique oligonucleotide (Figure 5). See U.S. Published Application No. 20090018024.
The uniquely bar-coded Rolony is cleaved into targeting bar-coded oligonucleotides when incubated in presence of a complementary restriction compatible DNA fragment and restriction enzyme. Cleavage may also be performed for example, in liposomes or inside emulsions. Liposomes containing bar-coded oligonucleotides were then fused to cells, allowing the annealing primer to anneal to the target nucleic acid of interest in each cell, as described in the bead-based approach. Figure 5 shows the query of the Rolony (similar to the query of the bar-coded beads, but ordered on a grid) to demonstrate efficiency at generating uniquely bar-coded clonal Rolony. Figure 5 demonstrates the rolony are clonally amplified, because for each query of a single position only one fluorophore overlays for that position. Subsequent sequencing of the other nucleotide positions can be performed to identify the complete bar-codes (used to correlated to the single originating cell) and to identify the captured transcripts.
Non-limiting examples of nucleic acid molecules include a gene, a gene fragment, a genomic gap, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), miRNA, small nucleolar RNA (snoRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers.
Nucleic acid molecules useful in the methods described herein may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
[041] An oligonucleotide sequence refers to a linear polymer of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof.
The term "oligonucleotide" usually refers to a shorter polymer, e.g., comprising from about 3 to about 100 monomers, and the term "polynucleotide" usually refers to longer polymers, e.g., comprising from about 100 monomers to many thousands of monomers, e.g., 10,000 monomers, or more An "oligonucleotide fragment" refers to an oligonucleotide sequence that has been cleaved into two or more smaller oligonucleotide sequences.
Oligonucleotides comprising probes or primers usually have lengths in the range of from 12 to 60 nucleotides, and more usually, from 18 to 40 nucleotides.
Oligonucleotides and polynucleotides may be natural or synthetic. Oligonucleotides and polynucleotides include deoxyribonucleosides, ribonucleosides, and non-natural analogs thereof, such as anomeric forms thereof, peptide nucleic acids (PNAs), and the like, provided that they are capable of specifically binding to a target genome by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
[042] Usually nucleosidic monomers are linked by phosphodiester bonds.
Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T"
denotes deoxythymidine, and "U" denotes the ribonucleoside, uridine, unless otherwise noted.
Usually oligonucleotides comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed in methods and processes described herein. For example, where processing by an enzyme is called for, usually oligonucleotides consisting solely of natural nucleotides are required. Likewise, where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA/DNA
duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references. Oligonucleotides and polynucleotides may be single stranded or double stranded.
[043] Nucleic acid molecules may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcyto sine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethy1-2-thiouridine, carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like.
Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
[044] In certain exemplary embodiments, large polynucleotides are provided. In certain aspects, isolation techniques that maximize the lengths of polynucleotides (e.g., DNA
molecules) obtained are used. For example, in situ lysis or deproteinization (e.g., with EDTA, detergent, protease, any combinations thereof and the like) after agarose embedding (as routinely performed for pulsed field gel electrophoresis) can be used to obtain polynucleotides.
[045] Nucleic acid molecules may be isolated from natural sources or purchased from commercial sources. Oligonucleotide sequences may also be prepared by any suitable method, e.g., standard phosphoramidite methods such as those described by Beaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859) or the triester method according to Matteucci et al. (1981)1 Am. Chem. Soc. 103:3185), or by other chemical methods using either a commercial automated oligonucleotide synthesizer or high-throughput, high-density array methods known in the art (see U.S. Patent Nos. 5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference in its entirety for all purposes). Pre-synthesized oligonucleotides may also be obtained commercially from a variety of vendors.
[046] Nucleic acid molecules may be obtained from one or more biological samples. As used herein, a "biological sample" may be a single cell or many cells. A biological sample may comprise a single cell type or a combination of two or more cell types. A
biological sample further includes a collection of cells that perform a similar function such as those found, for example, in a tissue. Accordingly, certain aspects of the invention are directed to biological samples containing one or more tissues. As used herein, a tissue includes, but is not limited to, epithelial tissue (e.g., skin, the lining of glands, bowel, skin and organs such as the liver, lung, kidney), endothelium (e.g., the lining of blood and lymphatic vessels), mesothelium (e.g., the lining of pleural, peritoneal and pericardial spaces), mesenchyme (e.g., cells filling the spaces between the organs, including fat, muscle, bone, cartilage and tendon cells), blood cells (e.g. erythrocytes, granulocytes, neutrophils, eosinophils, basophils, monocytes, T-lymphocytes (also known as T-cells), B-lymphocytes (also known as B-cells), plasma cells, megakaryocytes and the like), neurons, germ cells (e.g., spermatozoa, oocytes), amniotic fluid cells, placenta, stem cells and the like. A tissue sample includes microscopic samples as well as macroscopic samples. In certain aspects, a sample can be obtained from one or more of single cells in culture, metagenomic samples, embryonic stem cells, induced pluripotent stem cells, cancer samples, tissue sections, biopsies and the like, and any combinations of these.
[047] In certain aspects, nucleic acid sequences derived or obtained from one or more organisms are provided. As used herein, the term "organism" includes, but is not limited to, a human, a non-human primate, a cow, a horse, a sheep, a goat, a pig, a dog, a cat, a rabbit, a mouse, a rat, a gerbil, a frog, a toad, a fish (e.g., Danio rerio) a roundworm (e.g., C. elegans) and any transgenic species thereof. The term "organism" further includes, but is not limited to, a yeast (e.g., S. cerevisiae) cell, a yeast tetrad, a yeast colony, a bacterium, a bacterial colony, a virion, virosome, virus-like particle and/or cultures thereof, and the like.
[048] Isolation, extraction or derivation of nucleic acid sequences may be carried out by any suitable method. Isolating nucleic acid sequences from a biological sample generally includes treating a biological sample in such a manner that nucleic acid sequences present in the sample are extracted and made available for analysis. Any isolation method that results in extracted nucleic acid sequences may be used in the practice of the present invention. It will be understood that the particular method used to extract nucleic acid sequences will depend on the nature of the source.
[049] Methods of DNA extraction are well-known in the art. A classical DNA
isolation protocol is based on extraction using organic solvents such as a mixture of phenol and chloroform, followed by precipitation with ethanol (J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2" Ed., Cold Spring Harbour Laboratory Press:
New York, N.Y.). Other methods include: salting out DNA extraction (P.
Sunnucks et al., Genetics, 1996, 144: 747-756; S. M. Aljanabi and I. Martinez, Nucl. Acids Res. 1997, 25: 4692-4693), trimethylammonium bromide salts DNA extraction (S. Gustincich et al., BioTechniques, 1991, 11: 298-302) and guanidinium thiocyanate DNA extraction (J. B.
W. Hammond et al., Biochemistry, 1996, 240: 298-300). A variety of kits are commercially available for extracting DNA from biological samples (e.g., BD
Biosciences Clontech (Palo Alto, CA): Epicentre Technologies (Madison, WI);
Gentra Systems, Inc. (Minneapolis, MN); MicroProbe Corp. (Bothell, WA); Organon Teknika (Durham, NC); and Qiagen Inc. (Valencia, CA)).
[050] Methods of RNA extraction are also well known in the art (see, for example, J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" 1989, 2" Ed., Cold Spring Harbour Laboratory Press: New York) and several kits for RNA extraction from bodily fluids are commercially available (e.g., Ambion, Inc. (Austin, TX); Amersham Biosciences (Piscataway, NJ); BD Biosciences Clontech (Palo Alto, CA); BioRad Laboratories (Hercules, CA); Dynal Biotech Inc. (Lake Success, NY); Epicentre Technologies (Madison, WI); Gentra Systems, Inc. (Minneapolis, MN); GIBCO BRL
(Gaithersburg, MD); Invitrogen Life Technologies (Carlsbad, CA); MicroProbe Corp. (Bothell, WA);
Organon Teknika (Durham, NC); Promega, Inc. (Madison, WI); and Qiagen Inc.
(Valencia, CA)).
[051] In certain exemplary embodiments, oligonucleotide sequences are immobilized on a solid support. The support can be simple square grids, checkerboard (e.g., offset) grids, hexagonal arrays and the like. Suitable supports include, but are not limited to, slides, beads, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, plates (e.g., 96-well, 48-well, 24-well, 12-well, eight-well, six-well, four-well, single-well and the like), cell surfaces (e.g., S. aureus cells) and the like. In various embodiments, a solid support may be biological, non-biological, organic, inorganic, or any combination thereof.
[052] In certain exemplary embodiments, beads and bead-based arrays are provided. As used herein, the term "bead" refers to a discrete particle that may be spherical (e.g., microspheres) or have an irregular shape. Beads may be as small as approximately 0.1 i.trn in diameter or as large approximately several millimeters in diameter.
Beads may comprise a variety of materials including, but not limited to, paramagnetic materials, ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon and the like.
[053] In accordance with certain examples, a support (e.g., a bead) may have functional groups attached to its surface which can be used to bind one or more reagents described herein to the bead. One or more reagents can be attached to a support (e.g., a bead) by hybridization, covalent attachment, magnetic attachment, affinity attachment and the like.
Beads coated with a variety of attaachments are commercially available (Dynabeads, Invitrogen). Supports (e.g., beads) may also be functionalized using, for example, solid-phase chemistries known in the art (see, e.g., U.S. Pat. No. 5,919,523).
[054] As used herein, the term "attach" refers to both covalent interactions and noncovalent interactions. A covalent interaction is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (i.e., a single bond), two pairs of electrons (i.e., a double bond) or three pairs of electrons (i.e., a triple bond).
Covalent interactions are also known in the art as electron pair interactions or electron pair bonds. Noncovalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (i.e., via short-range noncovalent forces), hydrophobic interactions, ionic bonds and the like. A review of noncovalent interactions can be found in Alberts et al., in Molecular Biology of the Cell, 3d edition, Garland Publishing, 1994.
[055] In certain exemplary embodiments, methods for amplifying nucleic acid sequences are provided. Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant.
Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods 1:241; and U.S. Patent Nos.
4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl.
Acad. Sci.
U.S.A. 91:360-364), self sustained sequence replication (Guatelli et al.
(1990) Proc. Natl.
Acad. Sci. US.A. 87:1874), transcriptional amplification system (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. US.A. 86:1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J Biol. Chem.
275:2619; and Williams et al. (2002) 1 Biol. Chem. 277:7790), the amplification methods described in U.S. Patent Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA) or any other nucleic acid amplification method using techniques well known to those of skill in the art.
[056] "Polymerase chain reaction," or "PCR," refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A
Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature greater than 90 C, primers annealed at a temperature in the range 50-75 C, and primers extended at a temperature in the range 72-78 C.
[057] The term "PCR" encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 microliters. "Reverse transcription PCR," or "RT-PCR," means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al., U.S. Patent No. 5,168,038. "Real-time PCR" means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Patent No.
5,210,015 ("Taqman"); Wittwer et al., U.S. Patent Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Patent No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). "Nested PCR" means a two-stage PCR wherein the amplicon of a first PCR
becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, "initial primers" in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and "secondary primers" mean the one or more primers used to generate a second, or nested, amplicon. "Multiplexed PCR" means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al.
(1999) Anal.
Biochem., 273:221-228 (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. "Quantitative PCR" means a PCR
designed to measure the abundance of one or more specific target sequences in a sample or specimen. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references: Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17:9437-9447 (1989);
Zimmerman et al., Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446 (1989);
and the like.
10581 In certain exemplary embodiments, methods of determining the sequence identities of nucleic acid sequences are provided. Determination of the sequence of a nucleic acid sequence of interest (e.g., immune cell nucleic acid sequences) can be performed using variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. Serial No. 12/027,039, filed February 6, 2008; Porreca et al (2007) Nat.
Methods 4:931), polymerized colony (POLONY) sequencing (U.S. Patent Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425); nanogrid rolling circle sequencing (ROLONY) (U.S. Serial No. 12/120,541, filed May 14, 2008), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout) and the like. High-throughput sequencing methods, e.g., on cyclic array sequencing using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrents, Complete Genomics, Pacific Bioscience, Helicos, Polonator platforms (Worldwide Web Site: Polonator.org), and the like, can also be utilized. High-throughput sequencing methods are described in U.S. Serial No.
61/162,913, filed March 24, 2009. A variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172).
[059] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more disorders or diseases associated with an infectious agent are provided.
Infectious agents include, but are not limited to, viruses, bacteria, fungi, parasites, infectious proteins and the like.
[060] Viruses include, but are not limited to, DNA or RNA animal viruses. As used herein, RNA viruses include, but are not limited to, virus families such as Picornaviridae (e.g., polioviruses), Reoviridae (e.g., rotaviruses), Togaviridae (e.g., encephalitis viruses, yellow fever virus, rubella virus), Orthomyxoviridae (e.g., influenza viruses), Paramyxoviridae (e.g., respiratory syncytial virus, measles virus, mumps virus, parainfluenza virus), Rhabdoviridae (e.g., rabies virus), Coronaviridae, Bunyaviridae, Flaviviridae, Filoviridae, Arenaviridae, Bunyaviridae and Retroviridae (e.g., human T
cell lymphotropic viruses (HTLV), human immunodeficiency viruses (HIV)). As used herein, DNA viruses include, but are not limited to, virus families such as Papovaviridae (e.g., papilloma viruses), Adenoviridae (e.g., adenovirus), Herpesviridae (e.g., herpes simplex viruses), and Poxviridae (e.g., variola viruses).
[061] Bacteria include, but are not limited to, gram positive bacteria, gram negative bacteria, acid-fast bacteria and the like.
[062] As used herein, gram positive bacteria include, but are not limited to, Actinomedurae, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium, Enterococcus faecalis, Listeria monocytogenes, Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epiderm, Streptococcus mutans, Streptococcus pneumoniae and the like.
[063] As used herein, gram negative bacteria include, but are not limited to, Afipia felis, Bacteriodes, Bartonella bacilliformis, Bortadella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella, Calymmatobacterium granulomatis, Campylobacter, Escherichia coli, Francisella tularensis, Gardnerella vaginalis, Haemophilius aegyptius, Haemophilius ducreyi, Haemophilius influenziae, Heliobacter pylori, Legionella pneumophila, Leptospira interrogans, Neisseria meningitidia, Porphyromonas gingivalis, Providencia sturti, Pseudomonas aeruginosa, Salmonella enteridis, Salmonella typhi, Serratia marcescens, Shigella boydii, Streptobacillus moniliformis, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis and the like.
[064] As used herein, acid-fast bacteria include, but are not limited to, Myobacterium avium, Myobacterium leprae, Myobacterium tuberculosis and the like.
[065] As used herein, other bacteria not falling into the other three categories include, but are not limited to, Bartonella henseiae, Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasma pneumoniae, Rickettsia akari, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia tsutsugamushi, Rickettsia typhi, Ureaplasma urealyticum, Diplococcus pneumoniae, Ehrlichia chafensis, Enterococcus faecium, Meningococci and the like.
[066] As used herein, fungi include, but are not limited to, Aspergilli, Candidae, Candida albicans, Coccidioides immitis, Cryptococci, and combinations thereof.
[067] As used herein, parasitic microbes include, but are not limited to, Balantidium coli, Cryptosporidium parvum, Cyclospora cayatanensis, Encephalitozoa, Entamoeba histolytica, Enterocytozoon bieneusi, Giardia lamblia, Leishmaniae, Plasmodii, Toxoplasma gondii, Trypanosomae, trapezoidal amoeba and the like.
[068] As used herein, parasites include worms (e.g., helminthes), particularly parasitic worms including, but not limited to, Nematoda (roundworms, e.g., whipworms, hookworms, pinworms, ascarids, filarids and the like), Cestoda (e.g., tapeworms) [069] As used herein, infectious proteins include prions. Disorders caused by prions include, but are not limited to, human disorders such as Creutzfeldt-Jakob disease (CJD) (including, e.g., iatrogenic Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease (vCJD), familial Creutzfeldt-Jakob disease (fCJD), and sporadic Creutzfeldt-Jakob disease (sCJD)), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (fFI), sporadic fatal insomnia (sFI), kuru, and the like, as well as disorders in animals such as scrapie (sheep and goats), bovine spongiform encephalopathy (BSE) (cattle), transmissible mink encephalopathy (TME) (mink), chronic wasting disease (CWD) (elk, mule deer), feline spongiform encephalopathy (cats), exotic ungulate encephalopathy (EUE) (nyala, oryx, greater kudu), spongiform encephalopathy of the ostrich and the like.
[070] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more cellular proliferative disorders are provided. Cellular proliferative disorders are intended to include disorders associated with rapid proliferation. As used herein, the term "cellular proliferative disorder" includes disorders characterized by undesirable or inappropriate proliferation of one or more subset(s) of cells in a multicellular organism.
The term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR
Medical Dictionary 1st edition (1995), incorporated herein by reference in its entirety for all purposes). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal. Id. Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor).
[071] The language "treatment of cellular proliferative disorders" is intended to include the prevention of the induction, onset, establishment or growth of neoplasms in a subject or a reduction in the growth of pre-existing neoplasms in a subject. The language also can describe inhibition of the invasion of neoplastic cells into neighboring tissues or the metastasis of a neoplasm from one site to another. Examples of the types of neoplasms intended to be encompassed by the present invention include but are not limited to those neoplasms associated with cancers of the breast, skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, neural tissue, head and neck, colon, stomach, bronchi, and/or kidneys.
[072] Cellular proliferative disorders can further include disorders associated with hyperproliferation of vascular smooth muscle cells such as proliferative cardiovascular disorders, e.g., atherosclerosis and restenosis. Cellular proliferation disorders can also include disorders such as proliferative skin disorders, e.g., X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic dermatitis. Cellular proliferative disorders can further include disorders such as autosomal dominant polycystic kidney disease (ADPKD), mastocystosis, and cellular proliferation disorders caused by infectious agents such as viruses.
[073] In certain exemplary embodiments, methods of prognosing, diagnosing and/or monitoring one or more autoimmune disorders are provided. As used herein, the term "autoimmune disorder" is a disease or disorder caused by a subject producing an inappropriate immune response against its own tissues. As used herein, an autoimmune disorder includes, but is not limited to, disorders such as Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid sundrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Balo disease, Bechet disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis herpetiformis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST
syndrome, Crohn's disease, Degos disease, dermatomyositis, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves disease, Guillain-Barre, Hashimoto thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes, juvenile arthritis, lichen planus, lupus, Meniere disease, mixed connective tissue disease, multiple sclerosis, myasthemia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud phenomenon, Reiter syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren syndrome, stiff-person syndrome, Takayasu arthritis, temporal arteritis/giant cell arteritis, ulcerative colitis, vasculitis, vitiligo, Wegener granulomatosis and the like (See the American Autoimmune Related Diseases Association, Inc. website: aarda.org).
[074] It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.
[075] EXAMPLE 1 Preparing Bar-Coded Beads a. Loading the Template Bar-Code Oligonucleotide Onto Beads [076] Beads (1 M; Cl carboxylic 1 micron beads) were resuspended by vortexing and transferred in a volume of 80 IA to a 1.5 ml silicon tube (Ambion). The beads were washed twice with 2X (Bind and Wash Buffer contains 10 mM Tris-HC1 ph7.5, 1 mM
EDTA, 2M NaCI; "B&W"). Beads were isolated using magnets between washes.
Nucleotide sequences are listed in Table 4. The washed beads were resuspended in 100 11.1 B&W to which oligo dT bar-code template oligonucleotide (HSCT_BC_anchorl) were added at the concentrations as shown in Table 1.
Tube Primer Stock Primer Concentration volume (A) 1 100 pM 80 2 10 pM 80 3 1 pM 80 4 0.1 pN 80 Table 1.
[077] The template oligonucleotide and beads were incubated on a rotator for 20 minutes, then washed twice with 200 p1 of lx B&W then resuspended in 100 pi of 2x B&W.
b. Saturating the Beads with Anchor Primer [078] The beads, pre-loaded with the template oligonucleotide as in Example 1(a) above, were incubated on a rotator for 20 minutes with Anchor primer mix (1mM
HSCT_Bead_anchor 1 and lx B&W buffer) to coat the beads with the anchor primer then washed twice with 200 p,1 of lx B&W, once in 200 p,1 of TE, then resuspended in 100 1 of TE. The anchor primer has a 5' biotin which binds to the streptavidin coated beads.
Typically, 30% of the beads have an oligonucleotide. On those beads 100% or substantially all of the anchor primers are typically extended.
c. Emulsion PCR to Synthesis the Oligonucleotide From the Anchor Primer [079] Aqueous Mix and Oil Mix were prepared as described in Table 2.
AQUEOUS MIX
Component Volume for 1 Volume for tube (p,1) 4.5 tubes(p,1) x PCR 96 432 buffer (Enymatics) 50 mM 242 1089 MgC12 25 mM 135 607 dNTP mix 2 mM 6 27 HSCT dA-rev_emulsion primer OIL MIX
Component Volume for 1 Volume for tube ( 1) 4.5 tubes( 1) Tegosoft 4.4 19.8 DEC
Mineral oil 1.2 5.4 ABIL WE09 425 1.9 Table 2.
[080] Both solutions are mixed by vortexing. The oil mix is allowed to degass then 5.5 ml portions were placed in 50 ml Teflon-coated aluminum test tubes.
[081] The emulsions were made by adding 800 [L1 PCR mix, 100 p.1 Enzymatics Taq (5U/ 1), quickly vortexing and spinning, then immediately adding 60 ml of bar-code anchored beads, followed by vortex and spinning. The 960 ml mix was transferred to a tube of oil and vortexed for 2.25 min at 2200 rpm, which was followed by emulsion PCR
using the following PCR protocol steps:
a. 94 C for 5 min b. 94 C for 15 sec c. 58 C for 30 sec d. 70 C for 75 sec e. Cycle to step b 119 times f. 72 C for 2 min g. Incubate at ¨ 10 C until ready to use.
[082] Formation of an emulsion was confirmed by verifying under a microscope that a creamy white consistency was obtained when an emulsifier/oil mixture (240 p.1 emulsifier: 960 1 oil, or 480 1 emulsifier: 7200 oil) was added to an aqueous layer (384 p,1) and vortexed at 4 C for 5 minutes. Results are show in Figures 7A-7E.
[083] In a similar experiment, Dynal M270 3-micron beads were used under similar conditions and similar results were achieved.
[084] Bar-coding was also achieved as follows.
AQUEOUS MIX
Component Final Volume per concentration tube(i1) dH20 520.4 x PCR lx 80 buffer (Enymatics) 25 mM 2mM 64 dNTP mix 2 mM 10 M 4 HSCT dA-rev_emulsion primer 30% (w/v) 0.06 1.6 BSA (Sigma) OIL MIX
Component Volume for 1 Volume for tube ( 1) 4.5 tubes(1.11) Tegosoft 4.4 19.8 DEC
Mineral oil 1.2 5.4 ABIL WE09 425 1.9 Table 3.
[085] The aqueous mix was vortexed, then 0.6m! of mix was added per 1.5 ml tube (Ambion;
non-stick). 50 1 of M280 HSCT Anchor bead was added per tube, then the tubes were sonicated for 3 cycles of 10 seconds. After sonication, the tubes were placed on ice, and 80 vtl of Taq Polymerase (5U/ 1) was added per tube. The tubes were again vortexed and placed on ice. 800 pi of the mixture was added to the oil phase, the tubes were vortexed and PCR was performed as described in Example 1 part c. In similar experiments well/plates were used. Each well contained 55111/well of the mixture.
Sequence Name SEQ ID NO: Sequence HSCT BC anchor 1 4 /52-Bio/ACA CTC TTT CCC TAC ACG ACG CTC
TTC
CGA TCT NNN NNN NNN NNN NNN NNN NNC AGC
TTT TTT TTT TTT TTT TTT TTT TTT T
HSCT_Bead_anchor 5 /52-Bio/ACA CTC TTT CCC TAC ACG ACG CTC
TTC
HSCT clonaltest B 6 /5Phos/AGA TCG GAA GAG CGT CGT GTA
C seq H¨SCT_dA_rev_emul 7 AAA AAA AAA AAA AAA AAA AAA AAA ACG AC
sion primer HSCT BC_anchor_r 8 AAA AAA AAA AAA AAA AAA AAA AAA AGC TGN
ev(no-bio) NNN NNN NNN NNN NNN NNN NAG ATC GGA AGA
GCG TCG TGT AGG GAA AGA GTG T
Bead attached to 9 BEAD/52-Bar-coded Bio/ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Oligonucleotide NNN NNN NNN NNN NNN NNN NNC AGC TTT TTT
TTT TTT TTT TTT TTT TTT T
ATGTGCTGCGAGAAGGCTAGA/5Phos/
[086] Table 4 shows the sequences used in Example 1.
[087] The final sequence attached to the bead in Example 1 is shown in SEQ ID
NO:9. The bead is connected 5' to 3' to the oligonucleotide which encodes the anchor primer sequence, the bar code (N20) and an oligo dT primer.
Introduction of One Unique Bar-Coded Bead per Cell [088] Figure 4 demonstrates introduction of beads carrying unique bar-coded oligonucleotides into individual cells. Here, beads post-emulsion PCR are sequenced for one base of their bar-code to show that each beads have a unique bar-code and demonstrate clonality. Each nucleotide is queried by a different fluorophores as describe previously (Porreca et al.
(2006) Curr. Protoc. MoL Biol. Chapter 7:Unit 78). Cy 5 shows presence of an adenine nucleotide at position one of the bar-code. Cy3 the shows presence of a thymine nucleotide at position one of the bar-code Texas Red (Txred) shows the presence of a cytosine nucleotide at position one of the bar-code. Fluorescein isothiocyanate (FITC) shows the presence of a guanine at position one of the bar-code. The image overlay of all four fluorophores for a single position on the bar-codes is shown and demonstrates clonality. Clonality refers to each single bead harboring one unique bar-code, which has been successfully amplified onto the bead. If the beads had contained multiple bar-codes;
that is, had been non-clonal (for example, having multiple bar-code templates loaded on the bead by accident), the overlay would have demonstrated more than one fluorophore color per bead when querying a single position on the bar-code during sequencing.
Complete sequencing of the bar-code, which allows correlation to the cell, is by multiple successive round of sequencing for each nucleotide position.
[089] White light microscopy analysis of the beads and emulsion reaction shows that the starting template and the bead in emulsion were correctly diluted to achieve a maximum of one bead or less per emulsion and one template or less per bead.
Introduction of Unique Bar-Coded Oligonucleotides on a Grid Support [090] Multiple copies of the same unique bar-code for single cell analysis were made by rolling circle amplification (RCA) product (Rolony) from a circularized starting bar-code unique oligonucleotide (Figure 5). See U.S. Published Application No. 20090018024.
The uniquely bar-coded Rolony is cleaved into targeting bar-coded oligonucleotides when incubated in presence of a complementary restriction compatible DNA fragment and restriction enzyme. Cleavage may also be performed for example, in liposomes or inside emulsions. Liposomes containing bar-coded oligonucleotides were then fused to cells, allowing the annealing primer to anneal to the target nucleic acid of interest in each cell, as described in the bead-based approach. Figure 5 shows the query of the Rolony (similar to the query of the bar-coded beads, but ordered on a grid) to demonstrate efficiency at generating uniquely bar-coded clonal Rolony. Figure 5 demonstrates the rolony are clonally amplified, because for each query of a single position only one fluorophore overlays for that position. Subsequent sequencing of the other nucleotide positions can be performed to identify the complete bar-codes (used to correlated to the single originating cell) and to identify the captured transcripts.
Claims (20)
1. A method for high-throughput sequencing of nucleic acids from a plurality of biological samples comprising (a) generating at least two bar-codes attached to a solid support, (b) delivering each of the at least two bar-codes to individual samples of the plurality of biological samples, (c) sequencing a nucleic acid from at least two of the plurality of biological samples; and (d) correlating the nucleic acid sequence to a single sample of the plurality of biological samples through bar-code sequencing identification.
2. The method of claim 1 wherein the analysis is of a genome.
3. The method of claim 1 wherein the analysis is of a transcriptome.
4. The method of claim 1 wherein the solid support is a bead.
5. The method of claim 1 wherein the bar-codes are made by Rolony generation.
6. The method of claim 1 wherein delivery is by transfection, by emulsification, or using liposomes.
7. The method of claim 1 wherein the delivery of each of the at least two bar-codes to individual samples of the plurality of biological samples is in a single assay.
8. The method of claim 1 wherein the biological samples are cells.
9. The method of claim 8 wherein the cells are selected from the group consisting of cells in in vitro culture, stem cells, tumor cells, tissue biopsy cells, blood cells, and tissue section cells.
10. The method of claim 1 wherein the samples are metagenomic samples.
11. The method of claim 9 wherein the blood cells are T-lymphocytes or B-lymphocytes.
12. The method of claim 9 wherein the stem cells are embryonic stem cells or induced pluripotent stem cells.
13. The method of claim 1 wherein the samples are selected from the group consisting of bacteria, viruses, fungi, and hybdridomas.
14. The method of claim 1 wherein the nucleic acid is a DNA or an RNA.
15. A method of preparing bar-code oligonucleotides on a solid support comprising steps of (i) attaching an initial template oligonucleotide to a solid support, wherein the initial template oligonucleotide comprises a nucleotide sequence encoding (a) a sequencing primer, (b) a bar-code, and (c) an annealing primer, (ii) saturating the solid support with a plurality of anchor primers, and (iii) performing emulsion PCR.
16. The method of claim 15 wherein the solid support is a bead.
17. A solid support comprising a plurality of oligonucleotides wherein each oligonucleotide comprises a nucleotide sequence encoding:
(i) a sequencing primer, (ii) a bar-code, and (iii) an annealing primer which targets a nucleic acid of interest.
(i) a sequencing primer, (ii) a bar-code, and (iii) an annealing primer which targets a nucleic acid of interest.
18. The solid support of claim 17 wherein the solid support is a bead.
19. The solid support of claim 17 wherein the annealing primer comprises an oligo (dT) primer.
20. The solid support of claim 17 wherein at least two of the plurality of oligonucleotides encode annealing primers which target different nucleic acids of interest.
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Families Citing this family (286)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006507921A (en) | 2002-06-28 | 2006-03-09 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Method and apparatus for fluid dispersion |
EP2266687A3 (en) | 2003-04-10 | 2011-06-29 | The President and Fellows of Harvard College | Formation and control of fluidic species |
BRPI0414004A (en) | 2003-08-27 | 2006-10-24 | Harvard College | electronic control of fluidic species |
US7968287B2 (en) | 2004-10-08 | 2011-06-28 | Medical Research Council Harvard University | In vitro evolution in microfluidic systems |
JP2008535644A (en) | 2005-03-04 | 2008-09-04 | プレジデント・アンド・フエローズ・オブ・ハーバード・カレツジ | Method and apparatus for the formation of multiple emulsions |
WO2007081386A2 (en) | 2006-01-11 | 2007-07-19 | Raindance Technologies, Inc. | Microfluidic devices and methods of use |
US9562837B2 (en) | 2006-05-11 | 2017-02-07 | Raindance Technologies, Inc. | Systems for handling microfludic droplets |
EP2481815B1 (en) | 2006-05-11 | 2016-01-27 | Raindance Technologies, Inc. | Microfluidic devices |
US8772046B2 (en) | 2007-02-06 | 2014-07-08 | Brandeis University | Manipulation of fluids and reactions in microfluidic systems |
US9029085B2 (en) * | 2007-03-07 | 2015-05-12 | President And Fellows Of Harvard College | Assays and other reactions involving droplets |
US8592221B2 (en) | 2007-04-19 | 2013-11-26 | Brandeis University | Manipulation of fluids, fluid components and reactions in microfluidic systems |
WO2009085215A1 (en) | 2007-12-21 | 2009-07-09 | President And Fellows Of Harvard College | Systems and methods for nucleic acid sequencing |
EP4047367A1 (en) | 2008-07-18 | 2022-08-24 | Bio-Rad Laboratories, Inc. | Method for detecting target analytes with droplet libraries |
US20110218123A1 (en) * | 2008-09-19 | 2011-09-08 | President And Fellows Of Harvard College | Creation of libraries of droplets and related species |
EP4335932A2 (en) | 2008-11-07 | 2024-03-13 | Adaptive Biotechnologies Corporation | Methods of monitoring conditions by sequence analysis |
US8748103B2 (en) | 2008-11-07 | 2014-06-10 | Sequenta, Inc. | Monitoring health and disease status using clonotype profiles |
US9506119B2 (en) | 2008-11-07 | 2016-11-29 | Adaptive Biotechnologies Corp. | Method of sequence determination using sequence tags |
US8628927B2 (en) | 2008-11-07 | 2014-01-14 | Sequenta, Inc. | Monitoring health and disease status using clonotype profiles |
US9528160B2 (en) | 2008-11-07 | 2016-12-27 | Adaptive Biotechnolgies Corp. | Rare clonotypes and uses thereof |
US9365901B2 (en) | 2008-11-07 | 2016-06-14 | Adaptive Biotechnologies Corp. | Monitoring immunoglobulin heavy chain evolution in B-cell acute lymphoblastic leukemia |
EP2373812B1 (en) | 2008-12-19 | 2016-11-09 | President and Fellows of Harvard College | Particle-assisted nucleic acid sequencing |
PT2387627E (en) | 2009-01-15 | 2016-06-03 | Adaptive Biotechnologies Corp | Adaptive immunity profiling and methods for generation of monoclonal antibodies |
US9056299B2 (en) | 2009-03-13 | 2015-06-16 | President And Fellows Of Harvard College | Scale-up of flow-focusing microfluidic devices |
EA023190B9 (en) | 2009-04-02 | 2018-03-30 | Флуидигм Корпорейшн | Multi-primer amplification method for barcoding of target nucleic acids |
EP3409792B1 (en) | 2009-06-25 | 2023-09-20 | Fred Hutchinson Cancer Center | Method of measuring adaptive immunity |
US20120211084A1 (en) | 2009-09-02 | 2012-08-23 | President And Fellows Of Harvard College | Multiple emulsions created using jetting and other techniques |
AU2010315580B2 (en) | 2009-10-27 | 2014-11-06 | President And Fellows Of Harvard College | Droplet creation techniques |
US8835358B2 (en) | 2009-12-15 | 2014-09-16 | Cellular Research, Inc. | Digital counting of individual molecules by stochastic attachment of diverse labels |
US9315857B2 (en) | 2009-12-15 | 2016-04-19 | Cellular Research, Inc. | Digital counting of individual molecules by stochastic attachment of diverse label-tags |
JP5934657B2 (en) | 2010-02-12 | 2016-06-15 | レインダンス テクノロジーズ, インコーポレイテッド | Digital specimen analysis |
US9366632B2 (en) | 2010-02-12 | 2016-06-14 | Raindance Technologies, Inc. | Digital analyte analysis |
US10351905B2 (en) | 2010-02-12 | 2019-07-16 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
US9399797B2 (en) | 2010-02-12 | 2016-07-26 | Raindance Technologies, Inc. | Digital analyte analysis |
US10787701B2 (en) | 2010-04-05 | 2020-09-29 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US20190300945A1 (en) | 2010-04-05 | 2019-10-03 | Prognosys Biosciences, Inc. | Spatially Encoded Biological Assays |
KR101866401B1 (en) | 2010-04-05 | 2018-06-11 | 프로그노시스 바이오사이언스, 인코포레이티드 | Spatially encoded biological assays |
ES2523140T3 (en) | 2010-09-21 | 2014-11-21 | Population Genetics Technologies Ltd. | Increased confidence in allele identifications with molecular count |
EP2622103B2 (en) | 2010-09-30 | 2022-11-16 | Bio-Rad Laboratories, Inc. | Sandwich assays in droplets |
EP2652155B1 (en) * | 2010-12-16 | 2016-11-16 | Gigagen, Inc. | Methods for massively parallel analysis of nucleic acids in single cells |
JP6069224B2 (en) | 2011-01-31 | 2017-02-01 | アプライズ バイオ, インコーポレイテッド | Methods for identifying multiple epitopes in a cell |
EP3412778A1 (en) | 2011-02-11 | 2018-12-12 | Raindance Technologies, Inc. | Methods for forming mixed droplets |
EP3736281A1 (en) * | 2011-02-18 | 2020-11-11 | Bio-Rad Laboratories, Inc. | Compositions and methods for molecular labeling |
US9260753B2 (en) | 2011-03-24 | 2016-02-16 | President And Fellows Of Harvard College | Single cell nucleic acid detection and analysis |
GB201106254D0 (en) | 2011-04-13 | 2011-05-25 | Frisen Jonas | Method and product |
US9074204B2 (en) * | 2011-05-20 | 2015-07-07 | Fluidigm Corporation | Nucleic acid encoding reactions |
CN103547362B (en) | 2011-05-23 | 2016-05-25 | 哈佛学院院长等 | Emulsion, comprise the control of multiple emulsion |
DE202012013668U1 (en) | 2011-06-02 | 2019-04-18 | Raindance Technologies, Inc. | enzyme quantification |
CN106268389A (en) | 2011-07-06 | 2017-01-04 | 哈佛学院院长等 | Multiple Emulsion and for preparing the technology of multiple Emulsion |
US8658430B2 (en) | 2011-07-20 | 2014-02-25 | Raindance Technologies, Inc. | Manipulating droplet size |
US10385475B2 (en) | 2011-09-12 | 2019-08-20 | Adaptive Biotechnologies Corp. | Random array sequencing of low-complexity libraries |
US9279159B2 (en) | 2011-10-21 | 2016-03-08 | Adaptive Biotechnologies Corporation | Quantification of adaptive immune cell genomes in a complex mixture of cells |
AU2012347460B2 (en) | 2011-12-09 | 2017-05-25 | Adaptive Biotechnologies Corporation | Diagnosis of lymphoid malignancies and minimal residual disease detection |
US9499865B2 (en) | 2011-12-13 | 2016-11-22 | Adaptive Biotechnologies Corp. | Detection and measurement of tissue-infiltrating lymphocytes |
EP2791358B1 (en) * | 2011-12-13 | 2017-06-14 | Single Cell Technology, Inc. | Method of screening a plurality of single secreting cells for functional activity |
EP2817418B1 (en) * | 2012-02-24 | 2017-10-11 | Raindance Technologies, Inc. | Labeling and sample preparation for sequencing |
ES2663234T3 (en) | 2012-02-27 | 2018-04-11 | Cellular Research, Inc | Compositions and kits for molecular counting |
EP2820174B1 (en) | 2012-02-27 | 2019-12-25 | The University of North Carolina at Chapel Hill | Methods and uses for molecular tags |
US9670529B2 (en) | 2012-02-28 | 2017-06-06 | Population Genetics Technologies Ltd. | Method for attaching a counter sequence to a nucleic acid sample |
EP3495503A1 (en) | 2012-03-05 | 2019-06-12 | President and Fellows of Harvard College | Systems and methods for epigenetic sequencing |
JP6302847B2 (en) | 2012-03-05 | 2018-03-28 | アダプティヴ バイオテクノロジーズ コーポレーション | Determination of paired immunoreceptor chains from frequency matched subunits |
CN107586832B (en) | 2012-05-08 | 2021-03-30 | 适应生物技术公司 | Compositions and methods for measuring and calibrating amplification bias in multiplex PCR reactions |
AU2013293240A1 (en) * | 2012-07-24 | 2015-03-05 | Adaptive Biotechnologies Corp. | Single cell analysis using sequence tags |
JP6525872B2 (en) * | 2012-08-08 | 2019-06-05 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Increasing dynamic range to identify multiple epitopes in cells |
JP6514105B2 (en) | 2012-08-13 | 2019-05-15 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Methods and systems for detecting biological components |
US10323279B2 (en) | 2012-08-14 | 2019-06-18 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10752949B2 (en) | 2012-08-14 | 2020-08-25 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US9951386B2 (en) | 2014-06-26 | 2018-04-24 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10221442B2 (en) | 2012-08-14 | 2019-03-05 | 10X Genomics, Inc. | Compositions and methods for sample processing |
US10400280B2 (en) | 2012-08-14 | 2019-09-03 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
CN111748607B (en) * | 2012-08-14 | 2024-04-30 | 10X基因组学有限公司 | Microcapsule compositions and methods |
US10273541B2 (en) | 2012-08-14 | 2019-04-30 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US11591637B2 (en) * | 2012-08-14 | 2023-02-28 | 10X Genomics, Inc. | Compositions and methods for sample processing |
US9701998B2 (en) | 2012-12-14 | 2017-07-11 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US9567631B2 (en) | 2012-12-14 | 2017-02-14 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US20160040229A1 (en) | 2013-08-16 | 2016-02-11 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US11913065B2 (en) | 2012-09-04 | 2024-02-27 | Guardent Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
CN104781421B (en) | 2012-09-04 | 2020-06-05 | 夸登特健康公司 | System and method for detecting rare mutations and copy number variations |
US10876152B2 (en) | 2012-09-04 | 2020-12-29 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
EP2898096B1 (en) * | 2012-09-21 | 2024-02-14 | The Broad Institute, Inc. | Methods for labeling of rnas |
DE112013004650T5 (en) * | 2012-09-24 | 2015-06-18 | Cb Biotechnologies, Inc. | Multiplex pyrosequencing using non-interfering, noise-blocking polynucleotide identification tags |
CA2886647A1 (en) | 2012-10-01 | 2014-04-10 | Adaptive Biotechnologies Corporation | Immunocompetence assessment by adaptive immune receptor diversity and clonality characterization |
US10533221B2 (en) | 2012-12-14 | 2020-01-14 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
CN108753766A (en) | 2013-02-08 | 2018-11-06 | 10X基因组学有限公司 | Polynucleotides bar code generating at |
AU2013382098B2 (en) | 2013-03-13 | 2019-02-07 | Illumina, Inc. | Methods and compositions for nucleic acid sequencing |
GB2584364A (en) | 2013-03-15 | 2020-12-02 | Abvitro Llc | Single cell bar-coding for antibody discovery |
US9868979B2 (en) | 2013-06-25 | 2018-01-16 | Prognosys Biosciences, Inc. | Spatially encoded biological assays using a microfluidic device |
CN110592182B (en) * | 2013-06-27 | 2023-11-28 | 10X基因组学有限公司 | Compositions and methods for sample processing |
US9708657B2 (en) | 2013-07-01 | 2017-07-18 | Adaptive Biotechnologies Corp. | Method for generating clonotype profiles using sequence tags |
EP3842542A1 (en) * | 2013-08-28 | 2021-06-30 | Becton, Dickinson and Company | Massively parallel single cell analysis |
US10395758B2 (en) | 2013-08-30 | 2019-08-27 | 10X Genomics, Inc. | Sequencing methods |
CN105745334B (en) | 2013-09-30 | 2019-12-10 | 吴迪 | Method for profiling molecular complexes by using proximity barcoding |
GB201317301D0 (en) * | 2013-09-30 | 2013-11-13 | Linnarsson Sten | Method for capturing and encoding nucleic acid from a plurality of single cells |
US11901041B2 (en) | 2013-10-04 | 2024-02-13 | Bio-Rad Laboratories, Inc. | Digital analysis of nucleic acid modification |
JP2017504307A (en) | 2013-10-07 | 2017-02-09 | セルラー リサーチ, インコーポレイテッド | Method and system for digitally counting features on an array |
US9709479B2 (en) * | 2013-10-25 | 2017-07-18 | Massachusetts Institute Of Technology | Method and apparatus for tracking cell identity |
US20160279068A1 (en) | 2013-11-08 | 2016-09-29 | President And Fellows Of Harvard College | Microparticles, methods for their preparation and use |
US9944977B2 (en) | 2013-12-12 | 2018-04-17 | Raindance Technologies, Inc. | Distinguishing rare variations in a nucleic acid sequence from a sample |
US9824068B2 (en) | 2013-12-16 | 2017-11-21 | 10X Genomics, Inc. | Methods and apparatus for sorting data |
JP6571665B2 (en) | 2013-12-28 | 2019-09-04 | ガーダント ヘルス, インコーポレイテッド | Methods and systems for detecting genetic variants |
EP4094834A1 (en) | 2013-12-30 | 2022-11-30 | Atreca, Inc. | Analysis of nucleic acids associated with single cells using nucleic acid barcodes |
CN106164658B (en) | 2014-02-10 | 2020-06-09 | 泰克年研究发展基金会公司 | Methods and apparatus for cell isolation, growth, replication, manipulation and analysis |
CA2938910A1 (en) | 2014-02-11 | 2015-08-20 | F. Hoffmann-La Roche Ag | Targeted sequencing and uid filtering |
EP3114240B1 (en) | 2014-03-05 | 2019-07-24 | Adaptive Biotechnologies Corporation | Methods using randomer-containing synthetic molecules |
US10066265B2 (en) | 2014-04-01 | 2018-09-04 | Adaptive Biotechnologies Corp. | Determining antigen-specific t-cells |
KR102596508B1 (en) | 2014-04-10 | 2023-10-30 | 10엑스 제노믹스, 인크. | Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same |
ES2777529T3 (en) | 2014-04-17 | 2020-08-05 | Adaptive Biotechnologies Corp | Quantification of adaptive immune cell genomes in a complex mixture of cells |
EP3456846B1 (en) * | 2014-04-21 | 2022-06-22 | President and Fellows of Harvard College | Systems and methods for barcoding nucleic acid |
US20150298091A1 (en) | 2014-04-21 | 2015-10-22 | President And Fellows Of Harvard College | Systems and methods for barcoding nucleic acids |
SG11201609053YA (en) | 2014-04-29 | 2016-11-29 | Illumina Inc | Multiplexed single cell gene expression analysis using template switch and tagmentation |
US10738352B2 (en) | 2014-05-02 | 2020-08-11 | Idac Theranostics, Inc. | Method for analyzing nucleic acid derived from single cell |
EP3155426B1 (en) | 2014-06-13 | 2023-07-19 | Immudex ApS | General detection and isolation of specific cells by binding of labeled molecules |
EP3161157B1 (en) * | 2014-06-24 | 2024-03-27 | Bio-Rad Laboratories, Inc. | Digital pcr barcoding |
CN106795553B (en) | 2014-06-26 | 2021-06-04 | 10X基因组学有限公司 | Methods of analyzing nucleic acids from individual cells or cell populations |
AU2015279546B2 (en) | 2014-06-26 | 2021-04-08 | 10X Genomics, Inc. | Processes and systems for nucleic acid sequence assembly |
EP3160654A4 (en) | 2014-06-27 | 2017-11-15 | The Regents of The University of California | Pcr-activated sorting (pas) |
JP2017532024A (en) * | 2014-09-09 | 2017-11-02 | ザ・ブロード・インスティテュート・インコーポレイテッド | Droplet-based methods and instruments for composite single cell nucleic acid analysis |
EP3194593B1 (en) * | 2014-09-15 | 2019-02-06 | AbVitro LLC | High-throughput nucleotide library sequencing |
LT3207134T (en) * | 2014-10-17 | 2019-09-10 | Illumina Cambridge Limited | Contiguity preserving transposition |
CA2964799A1 (en) | 2014-10-17 | 2016-04-21 | Illumina Cambridge Limited | Contiguity preserving transposition |
CA3001986C (en) * | 2014-10-22 | 2023-02-21 | The Regents Of The University Of California | High definition microdroplet printer |
EP3212790B1 (en) | 2014-10-29 | 2020-03-25 | Adaptive Biotechnologies Corp. | Highly-multiplexed simultaneous detection of nucleic acids encoding paired adaptive immune receptor heterodimers from many samples |
JP2017532042A (en) | 2014-10-29 | 2017-11-02 | 10エックス ゲノミクス,インコーポレイテッド | Methods and compositions for targeted nucleic acid sequencing |
US9975122B2 (en) | 2014-11-05 | 2018-05-22 | 10X Genomics, Inc. | Instrument systems for integrated sample processing |
US10246701B2 (en) | 2014-11-14 | 2019-04-02 | Adaptive Biotechnologies Corp. | Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture |
US10900065B2 (en) | 2014-11-14 | 2021-01-26 | University Of Washington | Methods and kits for labeling cellular molecules |
EP3498866A1 (en) | 2014-11-25 | 2019-06-19 | Adaptive Biotechnologies Corp. | Characterization of adaptive immune response to vaccination or infection using immune repertoire sequencing |
EP3227684B1 (en) | 2014-12-03 | 2019-10-02 | Isoplexis Corporation | Analysis and screening of cell secretion profiles |
KR102321863B1 (en) | 2015-01-12 | 2021-11-08 | 10엑스 제노믹스, 인크. | Method and system for preparing nucleic acid sequencing library and library prepared using same |
EP4092681A1 (en) | 2015-01-13 | 2022-11-23 | 10X Genomics, Inc. | Systems and methods for visualizing structural variation and phasing information |
EP4112744A1 (en) | 2015-02-04 | 2023-01-04 | The Regents of the University of California | Sequencing of nucleic acids via barcoding in discrete entities |
GB201501907D0 (en) * | 2015-02-05 | 2015-03-25 | Technion Res & Dev Foundation | System and method for single cell genetic analysis |
MX2017010142A (en) | 2015-02-09 | 2017-12-11 | 10X Genomics Inc | Systems and methods for determining structural variation and phasing using variant call data. |
DK3256604T3 (en) * | 2015-02-10 | 2020-05-25 | Illumina Inc | Methods and compositions for analyzing cellular components |
EP3256624A4 (en) * | 2015-02-13 | 2018-07-25 | Vaccine Research Institute of San Diego | Materials and methods to analyze rna isoforms in transcriptomes |
CN107250379B (en) | 2015-02-19 | 2021-12-28 | 贝克顿迪金森公司 | High throughput single cell analysis combining proteomic and genomic information |
US10697000B2 (en) | 2015-02-24 | 2020-06-30 | 10X Genomics, Inc. | Partition processing methods and systems |
US11047008B2 (en) | 2015-02-24 | 2021-06-29 | Adaptive Biotechnologies Corporation | Methods for diagnosing infectious disease and determining HLA status using immune repertoire sequencing |
BR112017018054A2 (en) | 2015-02-24 | 2018-07-24 | 10X Genomics Inc | Methods for Covering Targeted Nucleic Acid Sequences |
WO2016138496A1 (en) * | 2015-02-27 | 2016-09-01 | Cellular Research, Inc. | Spatially addressable molecular barcoding |
US11873483B2 (en) | 2015-03-11 | 2024-01-16 | The Broad Institute, Inc. | Proteomic analysis with nucleic acid identifiers |
US10876156B2 (en) * | 2015-03-13 | 2020-12-29 | President And Fellows Of Harvard College | Determination of cells using amplification |
WO2016160844A2 (en) | 2015-03-30 | 2016-10-06 | Cellular Research, Inc. | Methods and compositions for combinatorial barcoding |
WO2016161273A1 (en) | 2015-04-01 | 2016-10-06 | Adaptive Biotechnologies Corp. | Method of identifying human compatible t cell receptors specific for an antigenic target |
ES2955916T3 (en) | 2015-04-10 | 2023-12-11 | Spatial Transcriptomics Ab | Multiplex analysis of biological specimens of spatially distinguished nucleic acids |
CN107636169A (en) * | 2015-04-17 | 2018-01-26 | 生捷科技控股公司 | The method that profile space analysis is carried out to biomolecule |
AU2016248995B2 (en) | 2015-04-17 | 2022-04-28 | President And Fellows Of Harvard College | Barcoding systems and methods for gene sequencing and other applications |
EP3286326A1 (en) | 2015-04-23 | 2018-02-28 | Cellular Research, Inc. | Methods and compositions for whole transcriptome amplification |
US11124823B2 (en) | 2015-06-01 | 2021-09-21 | Becton, Dickinson And Company | Methods for RNA quantification |
SG10201912283RA (en) | 2015-08-28 | 2020-02-27 | Illumina Inc | Nucleic acid sequence analysis from single cells |
US10647981B1 (en) | 2015-09-08 | 2020-05-12 | Bio-Rad Laboratories, Inc. | Nucleic acid library generation methods and compositions |
CN108026524A (en) | 2015-09-11 | 2018-05-11 | 赛卢拉研究公司 | Method and composition for nucleic acid library standardization |
KR20180085717A (en) | 2015-09-24 | 2018-07-27 | 에이비비트로, 엘엘씨 | Affinity-oligonucleotide conjugates and their use |
MX2018003533A (en) | 2015-09-24 | 2019-04-25 | Abvitro Llc | Hiv antibody compositions and methods of use. |
JP2018537414A (en) | 2015-10-13 | 2018-12-20 | プレジデント アンド フェローズ オブ ハーバード カレッジ | System and method for making and using gel microspheres |
EP3362074B1 (en) | 2015-10-16 | 2023-08-09 | President and Fellows of Harvard College | Regulatory t cell pd-1 modulation for regulating t cell effector immune responses |
US11092607B2 (en) | 2015-10-28 | 2021-08-17 | The Board Institute, Inc. | Multiplex analysis of single cell constituents |
WO2017075297A1 (en) | 2015-10-28 | 2017-05-04 | The Broad Institute Inc. | High-throughput dynamic reagent delivery system |
KR20180097536A (en) * | 2015-11-04 | 2018-08-31 | 아트레카, 인크. | A combination set of nucleic acid barcodes for the analysis of nucleic acids associated with single cells |
US11371094B2 (en) | 2015-11-19 | 2022-06-28 | 10X Genomics, Inc. | Systems and methods for nucleic acid processing using degenerate nucleotides |
SG11201804086VA (en) | 2015-12-04 | 2018-06-28 | 10X Genomics Inc | Methods and compositions for nucleic acid analysis |
WO2017106777A1 (en) | 2015-12-16 | 2017-06-22 | Fluidigm Corporation | High-level multiplex amplification |
EP3390668A4 (en) | 2015-12-17 | 2020-04-01 | Guardant Health, Inc. | Methods to determine tumor gene copy number by analysis of cell-free dna |
EP3400298B1 (en) | 2016-01-08 | 2024-03-06 | Bio-Rad Laboratories, Inc. | Multiple beads per droplet resolution |
WO2017138984A1 (en) | 2016-02-11 | 2017-08-17 | 10X Genomics, Inc. | Systems, methods, and media for de novo assembly of whole genome sequence data |
US10633648B2 (en) | 2016-02-12 | 2020-04-28 | University Of Washington | Combinatorial photo-controlled spatial sequencing and labeling |
AU2017238054B2 (en) | 2016-03-21 | 2023-10-19 | Dana-Farber Cancer Institute, Inc. | T-cell exhaustion state-specific gene expression regulators and uses thereof |
CN109072288A (en) | 2016-05-02 | 2018-12-21 | 赛卢拉研究公司 | Accurate molecule bar coding |
WO2017197338A1 (en) | 2016-05-13 | 2017-11-16 | 10X Genomics, Inc. | Microfluidic systems and methods of use |
US10301677B2 (en) | 2016-05-25 | 2019-05-28 | Cellular Research, Inc. | Normalization of nucleic acid libraries |
EP3465502B1 (en) | 2016-05-26 | 2024-04-10 | Becton, Dickinson and Company | Molecular label counting adjustment methods |
US10640763B2 (en) | 2016-05-31 | 2020-05-05 | Cellular Research, Inc. | Molecular indexing of internal sequences |
US10202641B2 (en) | 2016-05-31 | 2019-02-12 | Cellular Research, Inc. | Error correction in amplification of samples |
ES2898088T3 (en) | 2016-06-01 | 2022-03-03 | Hoffmann La Roche | Immuno-PETE |
US10465242B2 (en) | 2016-07-14 | 2019-11-05 | University Of Utah Research Foundation | Multi-sequence capture system |
WO2018031691A1 (en) | 2016-08-10 | 2018-02-15 | The Regents Of The University Of California | Combined multiple-displacement amplification and pcr in an emulsion microdroplet |
US10428325B1 (en) | 2016-09-21 | 2019-10-01 | Adaptive Biotechnologies Corporation | Identification of antigen-specific B cell receptors |
SG11201901733PA (en) | 2016-09-26 | 2019-04-29 | Cellular Res Inc | Measurement of protein expression using reagents with barcoded oligonucleotide sequences |
EP3519433A1 (en) | 2016-10-03 | 2019-08-07 | Juno Therapeutics, Inc. | Hpv-specific binding molecules |
ES2870639T3 (en) | 2016-10-24 | 2021-10-27 | Geneinfosec Inc | Hiding information present in nucleic acids |
EP3538672A1 (en) | 2016-11-08 | 2019-09-18 | Cellular Research, Inc. | Methods for cell label classification |
JP7232180B2 (en) | 2016-11-08 | 2023-03-02 | ベクトン・ディキンソン・アンド・カンパニー | Methods of expression profile classification |
JP7348066B2 (en) | 2016-11-11 | 2023-09-20 | アイソプレキシス コーポレイション | Compositions and methods for simultaneous analysis of single cell genome, transcriptome and proteome |
SG10202100951SA (en) | 2016-11-21 | 2021-03-30 | Nanostring Technologies Inc | Chemical compositions and methods of using same |
WO2018098372A1 (en) | 2016-11-22 | 2018-05-31 | IsoPlexis Corporation | Systems, devices and methods for cell capture and methods of manufacture thereof |
AU2017382905A1 (en) | 2016-12-21 | 2019-07-04 | The Regents Of The University Of California | Single cell genomic sequencing using hydrogel based droplets |
US10011872B1 (en) | 2016-12-22 | 2018-07-03 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10815525B2 (en) | 2016-12-22 | 2020-10-27 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10550429B2 (en) | 2016-12-22 | 2020-02-04 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
WO2018132610A1 (en) | 2017-01-13 | 2018-07-19 | Cellular Research, Inc. | Hydrophilic coating of fluidic channels |
CN117512066A (en) | 2017-01-30 | 2024-02-06 | 10X基因组学有限公司 | Method and system for droplet-based single cell bar coding |
EP3577232A1 (en) | 2017-02-01 | 2019-12-11 | Cellular Research, Inc. | Selective amplification using blocking oligonucleotides |
US10995333B2 (en) | 2017-02-06 | 2021-05-04 | 10X Genomics, Inc. | Systems and methods for nucleic acid preparation |
US11072816B2 (en) | 2017-05-03 | 2021-07-27 | The Broad Institute, Inc. | Single-cell proteomic assay using aptamers |
CA3062248A1 (en) * | 2017-05-05 | 2018-11-08 | Scipio Bioscience | Methods for trapping and barcoding discrete biological units in hydrogel |
EP3625715A4 (en) | 2017-05-19 | 2021-03-17 | 10X Genomics, Inc. | Systems and methods for analyzing datasets |
US10844372B2 (en) | 2017-05-26 | 2020-11-24 | 10X Genomics, Inc. | Single cell analysis of transposase accessible chromatin |
SG11201901822QA (en) | 2017-05-26 | 2019-03-28 | 10X Genomics Inc | Single cell analysis of transposase accessible chromatin |
EP3631012B1 (en) | 2017-05-26 | 2022-06-08 | AbVitro LLC | High-throughput polynucleotide library sequencing and transcriptome analysis |
WO2018226546A1 (en) | 2017-06-05 | 2018-12-13 | 10X Genomics, Inc. | Gaskets for the distribution of pressures in a microfluidic system |
AU2018281745B2 (en) * | 2017-06-05 | 2022-05-19 | Becton, Dickinson And Company | Sample indexing for single cells |
BR112020001601A2 (en) | 2017-08-09 | 2020-08-11 | Juno Therapeutics Inc | methods and compositions for preparing genetically engineered cells |
WO2019032760A1 (en) | 2017-08-10 | 2019-02-14 | Rootpath Genomics, Inc. | Improved method to analyze nucleic acid contents from multiple biological particles |
WO2019032762A1 (en) | 2017-08-10 | 2019-02-14 | Rootpath Genomics, Inc. | Methods to improve the sequencing of polynucleotides with barcodes using circularisation and truncation of template |
WO2019046783A1 (en) * | 2017-08-31 | 2019-03-07 | Ohio State Innovation Foundation | Methods of making and using tandem, twin barcode molecules |
WO2019051335A1 (en) | 2017-09-07 | 2019-03-14 | Juno Therapeutics, Inc. | Methods of identifying cellular attributes related to outcomes associated with cell therapy |
CN111386351A (en) | 2017-09-22 | 2020-07-07 | 华盛顿大学 | In situ combinatorial labelling of cellular molecules |
US11084037B2 (en) * | 2017-09-25 | 2021-08-10 | Plexium, Inc. | Oligonucleotide encoded chemical libraries |
SG11202002728VA (en) | 2017-10-03 | 2020-04-29 | Juno Therapeutics Inc | Hpv-specific binding molecules |
US10837047B2 (en) | 2017-10-04 | 2020-11-17 | 10X Genomics, Inc. | Compositions, methods, and systems for bead formation using improved polymers |
US10590244B2 (en) | 2017-10-04 | 2020-03-17 | 10X Genomics, Inc. | Compositions, methods, and systems for bead formation using improved polymers |
US10501739B2 (en) | 2017-10-18 | 2019-12-10 | Mission Bio, Inc. | Method, systems and apparatus for single cell analysis |
WO2019084043A1 (en) | 2017-10-26 | 2019-05-02 | 10X Genomics, Inc. | Methods and systems for nuclecic acid preparation and chromatin analysis |
WO2019084165A1 (en) | 2017-10-27 | 2019-05-02 | 10X Genomics, Inc. | Methods and systems for sample preparation and analysis |
SG11201913654QA (en) | 2017-11-15 | 2020-01-30 | 10X Genomics Inc | Functionalized gel beads |
US10829815B2 (en) | 2017-11-17 | 2020-11-10 | 10X Genomics, Inc. | Methods and systems for associating physical and genetic properties of biological particles |
US11254980B1 (en) | 2017-11-29 | 2022-02-22 | Adaptive Biotechnologies Corporation | Methods of profiling targeted polynucleotides while mitigating sequencing depth requirements |
WO2019108851A1 (en) | 2017-11-30 | 2019-06-06 | 10X Genomics, Inc. | Systems and methods for nucleic acid preparation and analysis |
CN111699042A (en) | 2017-12-07 | 2020-09-22 | 麻省理工学院 | Single cell analysis |
CN111492068A (en) | 2017-12-19 | 2020-08-04 | 贝克顿迪金森公司 | Particles associated with oligonucleotides |
KR102653725B1 (en) | 2018-01-29 | 2024-04-01 | 세인트 쥬드 칠드런즈 리써치 호스피탈, 인코포레이티드 | Methods for Nucleic Acid Amplification |
EP3752832A1 (en) | 2018-02-12 | 2020-12-23 | 10X Genomics, Inc. | Methods characterizing multiple analytes from individual cells or cell populations |
US11639928B2 (en) | 2018-02-22 | 2023-05-02 | 10X Genomics, Inc. | Methods and systems for characterizing analytes from individual cells or cell populations |
US11525164B2 (en) | 2018-03-27 | 2022-12-13 | The Trustees Of Columbia University In The City Of New York | Spatial metagenomic characterization of microbial biogeography |
MA52193A (en) | 2018-04-05 | 2021-02-17 | Juno Therapeutics Inc | T-LYMPHOCYTE RECEPTORS AND MODIFIED CELLS EXPRESSING THEM |
CN112262218A (en) | 2018-04-06 | 2021-01-22 | 10X基因组学有限公司 | System and method for quality control in single cell processing |
US11365409B2 (en) | 2018-05-03 | 2022-06-21 | Becton, Dickinson And Company | Molecular barcoding on opposite transcript ends |
ES2945191T3 (en) | 2018-05-03 | 2023-06-29 | Becton Dickinson Co | High-throughput multi-omics sample analysis |
CA3099909A1 (en) | 2018-05-14 | 2019-11-21 | Nanostring Technologies, Inc. | Chemical compositions and methods of using same |
US11254975B2 (en) * | 2018-05-24 | 2022-02-22 | National Center For Child Health And Development | Method of amplifying a polynucleotide of interest |
US11932899B2 (en) | 2018-06-07 | 2024-03-19 | 10X Genomics, Inc. | Methods and systems for characterizing nucleic acid molecules |
US11703427B2 (en) | 2018-06-25 | 2023-07-18 | 10X Genomics, Inc. | Methods and systems for cell and bead processing |
US20200032335A1 (en) | 2018-07-27 | 2020-01-30 | 10X Genomics, Inc. | Systems and methods for metabolome analysis |
EP3837381A1 (en) | 2018-08-15 | 2021-06-23 | Illumina, Inc. | Compositions and methods for improving library enrichment |
WO2020036926A1 (en) * | 2018-08-17 | 2020-02-20 | Cellecta, Inc. | Multiplex preparation of barcoded gene specific dna fragments |
CN112912513A (en) * | 2018-08-28 | 2021-06-04 | 贝克顿迪金森公司 | Sample multiplexing using carbohydrate binding reagents and membrane permeability reagents |
CN112805389A (en) | 2018-10-01 | 2021-05-14 | 贝克顿迪金森公司 | Determination of 5' transcript sequences |
WO2020086510A1 (en) * | 2018-10-22 | 2020-04-30 | The General Hospital Corporation | Multiplexed single-cell analysis using optically-encoded rna capture particles |
WO2020097315A1 (en) | 2018-11-08 | 2020-05-14 | Cellular Research, Inc. | Whole transcriptome analysis of single cells using random priming |
US11459607B1 (en) | 2018-12-10 | 2022-10-04 | 10X Genomics, Inc. | Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes |
WO2020123384A1 (en) | 2018-12-13 | 2020-06-18 | Cellular Research, Inc. | Selective extension in single cell whole transcriptome analysis |
EP3894593A2 (en) | 2018-12-13 | 2021-10-20 | DNA Script | Direct oligonucleotide synthesis on cells and biomolecules |
US11845983B1 (en) | 2019-01-09 | 2023-12-19 | 10X Genomics, Inc. | Methods and systems for multiplexing of droplet based assays |
WO2020150356A1 (en) | 2019-01-16 | 2020-07-23 | Becton, Dickinson And Company | Polymerase chain reaction normalization through primer titration |
EP3914728B1 (en) | 2019-01-23 | 2023-04-05 | Becton, Dickinson and Company | Oligonucleotides associated with antibodies |
PE20212198A1 (en) | 2019-01-29 | 2021-11-16 | Juno Therapeutics Inc | ANTIBODIES AND CHIMERIC RECEPTORS OF SPECIFIC ANTIGENS TO ORPHAN RECEPTOR 1, RECEPTOR TYROSINE KINASE TYPE (ROR1) |
US11467153B2 (en) | 2019-02-12 | 2022-10-11 | 10X Genomics, Inc. | Methods for processing nucleic acid molecules |
US11851683B1 (en) | 2019-02-12 | 2023-12-26 | 10X Genomics, Inc. | Methods and systems for selective analysis of cellular samples |
WO2020168013A1 (en) | 2019-02-12 | 2020-08-20 | 10X Genomics, Inc. | Methods for processing nucleic acid molecules |
US11655499B1 (en) | 2019-02-25 | 2023-05-23 | 10X Genomics, Inc. | Detection of sequence elements in nucleic acid molecules |
WO2020180659A1 (en) * | 2019-03-01 | 2020-09-10 | 1Cellbio Inc. | Nucleic acid labeling methods and composition |
WO2020180778A1 (en) | 2019-03-01 | 2020-09-10 | Illumina, Inc. | High-throughput single-nuclei and single-cell libraries and methods of making and of using |
EP3938537A1 (en) | 2019-03-11 | 2022-01-19 | 10X Genomics, Inc. | Systems and methods for processing optically tagged beads |
CN114302966A (en) * | 2019-03-12 | 2022-04-08 | 通用测序技术公司 | Method for capturing single cell in cell and application thereof |
WO2020214642A1 (en) | 2019-04-19 | 2020-10-22 | Becton, Dickinson And Company | Methods of associating phenotypical data and single cell sequencing data |
WO2020237222A1 (en) | 2019-05-22 | 2020-11-26 | Mission Bio, Inc. | Method and apparatus for simultaneous targeted sequencing of dna, rna and protein |
WO2020243160A1 (en) * | 2019-05-28 | 2020-12-03 | Chan Zuckerberg Biohub, Inc. | Methods and compositions for multiple-parameter single-cell analysis using spectrally encoded microbeads |
US11667954B2 (en) | 2019-07-01 | 2023-06-06 | Mission Bio, Inc. | Method and apparatus to normalize quantitative readouts in single-cell experiments |
EP4004231A1 (en) | 2019-07-22 | 2022-06-01 | Becton, Dickinson and Company | Single cell chromatin immunoprecipitation sequencing assay |
EP4049282A4 (en) * | 2019-10-25 | 2024-03-13 | Massachusetts Inst Technology | Methods and compositions for high-throughput compressed screening for therapeutics |
CN114729350A (en) | 2019-11-08 | 2022-07-08 | 贝克顿迪金森公司 | Obtaining full-length V (D) J information for immunohistorian sequencing using random priming |
EP4081657A1 (en) * | 2019-12-23 | 2022-11-02 | Illumina Inc | Nanoparticle with single site for template polynucleotide attachment |
SG11202106899SA (en) | 2019-12-23 | 2021-09-29 | 10X Genomics Inc | Methods for spatial analysis using rna-templated ligation |
WO2021146207A1 (en) | 2020-01-13 | 2021-07-22 | Becton, Dickinson And Company | Methods and compositions for quantitation of proteins and rna |
US11702693B2 (en) | 2020-01-21 | 2023-07-18 | 10X Genomics, Inc. | Methods for printing cells and generating arrays of barcoded cells |
US11732299B2 (en) | 2020-01-21 | 2023-08-22 | 10X Genomics, Inc. | Spatial assays with perturbed cells |
US11835462B2 (en) | 2020-02-11 | 2023-12-05 | 10X Genomics, Inc. | Methods and compositions for partitioning a biological sample |
US11926863B1 (en) | 2020-02-27 | 2024-03-12 | 10X Genomics, Inc. | Solid state single cell method for analyzing fixed biological cells |
WO2021195277A1 (en) * | 2020-03-24 | 2021-09-30 | President And Fellows Of Harvard College | Multiplexed methods for detecting target rnas |
CN115698339A (en) * | 2020-04-07 | 2023-02-03 | 私人基因诊断公司 | Unfixed bar code |
US11851700B1 (en) | 2020-05-13 | 2023-12-26 | 10X Genomics, Inc. | Methods, kits, and compositions for processing extracellular molecules |
CN115803824A (en) | 2020-05-13 | 2023-03-14 | 朱诺治疗学股份有限公司 | Methods of identifying characteristics associated with clinical response and uses thereof |
CN115605614A (en) | 2020-05-14 | 2023-01-13 | 贝克顿迪金森公司(Us) | Primers for immune repertoire profiling |
EP4153775A1 (en) | 2020-05-22 | 2023-03-29 | 10X Genomics, Inc. | Simultaneous spatio-temporal measurement of gene expression and cellular activity |
EP4163390A1 (en) | 2020-06-03 | 2023-04-12 | Tenk Genomics, Inc. | Method for analyzing target nucleic acid from cell |
WO2021252617A1 (en) | 2020-06-09 | 2021-12-16 | Illumina, Inc. | Methods for increasing yield of sequencing libraries |
US11932901B2 (en) | 2020-07-13 | 2024-03-19 | Becton, Dickinson And Company | Target enrichment using nucleic acid probes for scRNAseq |
EP4247967A1 (en) | 2020-11-20 | 2023-09-27 | Becton, Dickinson and Company | Profiling of highly expressed and lowly expressed proteins |
AU2022227563A1 (en) | 2021-02-23 | 2023-08-24 | 10X Genomics, Inc. | Probe-based analysis of nucleic acids and proteins |
WO2022242734A1 (en) | 2021-05-21 | 2022-11-24 | 上海绾塍生物科技有限公司 | Composition and method for analyzing target molecule from sample |
WO2023023584A2 (en) | 2021-08-19 | 2023-02-23 | Eclipse Bioinnovations, Inc. | Methods for detecting rna binding protein complexes |
WO2023086847A1 (en) | 2021-11-10 | 2023-05-19 | Encodia, Inc. | Methods for barcoding macromolecules in individual cells |
WO2023150764A1 (en) * | 2022-02-07 | 2023-08-10 | Becton, Dickinson And Company | Sorting of mrna and abseq containing barcoded beads by flow |
CA3223722A1 (en) | 2022-04-07 | 2023-10-12 | Illumina, Inc. | Altered cytidine deaminases and methods of use |
WO2024073047A1 (en) | 2022-09-30 | 2024-04-04 | Illumina, Inc. | Cytidine deaminases and methods of use in mapping modified cytosine nucleotides |
WO2024073043A1 (en) | 2022-09-30 | 2024-04-04 | Illumina, Inc. | Methods of using cpg binding proteins in mapping modified cytosine nucleotides |
WO2024069581A1 (en) | 2022-09-30 | 2024-04-04 | Illumina Singapore Pte. Ltd. | Helicase-cytidine deaminase complexes and methods of use |
Family Cites Families (54)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2703908A (en) | 1954-05-12 | 1955-03-15 | Joseph J Stracker | Heat deflector for pot handle |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
JPH0672011B2 (en) | 1985-09-20 | 1994-09-14 | 東ソー株式会社 | Method for producing synthetic mordenite compact |
US4959463A (en) | 1985-10-15 | 1990-09-25 | Genentech, Inc. | Intermediates |
US4659774A (en) | 1985-11-01 | 1987-04-21 | American Hoechst Corporation | Support for solid-phase oligonucleotide synthesis |
US5602244A (en) | 1988-05-26 | 1997-02-11 | Competitive Technologies, Inc. | Polynucleotide phosphorodithioate compounds |
US5168038A (en) | 1988-06-17 | 1992-12-01 | The Board Of Trustees Of The Leland Stanford Junior University | In situ transcription in cells and tissues |
ES2093633T3 (en) | 1989-01-19 | 1997-01-01 | Behringwerke Ag | AMPLIFICATION OF NUCLEIC ACIDS USING A SINGLE PRIMER. |
US5141813A (en) | 1989-08-28 | 1992-08-25 | Clontech Laboratories, Inc. | Multifunctional controlled pore glass reagent for solid phase oligonucleotide synthesis |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US5612199A (en) | 1991-10-11 | 1997-03-18 | Behringwerke Ag | Method for producing a polynucleotide for use in single primer amplification |
US5428148A (en) | 1992-04-24 | 1995-06-27 | Beckman Instruments, Inc. | N4 - acylated cytidinyl compounds useful in oligonucleotide synthesis |
US6294323B1 (en) | 1993-04-14 | 2001-09-25 | Behringwerke Ag | Self initiating single primer amplification of nucleic acids |
US5925517A (en) | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
US5574146A (en) | 1994-08-30 | 1996-11-12 | Beckman Instruments, Inc. | Oligonucleotide synthesis with substituted aryl carboxylic acids as activators |
US5554744A (en) | 1994-09-23 | 1996-09-10 | Hybridon, Inc. | Method for loading solid supports for nucleic acid synthesis |
US5624711A (en) | 1995-04-27 | 1997-04-29 | Affymax Technologies, N.V. | Derivatization of solid supports and methods for oligomer synthesis |
US6261797B1 (en) | 1996-01-29 | 2001-07-17 | Stratagene | Primer-mediated polynucleotide synthesis and manipulation techniques |
DE69733282T2 (en) | 1996-06-04 | 2006-01-19 | University Of Utah Research Foundation, Salt Lake City | Monitoring hybridization during PCR |
JPH10253701A (en) | 1997-03-10 | 1998-09-25 | Mitsubishi Electric Corp | Semiconductor tester |
EP1591541B1 (en) * | 1997-04-01 | 2012-02-15 | Illumina Cambridge Limited | Method of nucleic acid sequencing |
US6974669B2 (en) * | 2000-03-28 | 2005-12-13 | Nanosphere, Inc. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US6511803B1 (en) | 1997-10-10 | 2003-01-28 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
US6485944B1 (en) | 1997-10-10 | 2002-11-26 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
EP1028970A1 (en) | 1997-10-10 | 2000-08-23 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
DE19813317A1 (en) | 1998-03-26 | 1999-09-30 | Roche Diagnostics Gmbh | Nucleic acid amplification involving primer extension preamplification, especially for whole genome amplification |
US6391544B1 (en) | 1998-05-15 | 2002-05-21 | Abbott Laboratories | Method for using unequal primer concentrations for generating nucleic acid amplification products |
US6300070B1 (en) * | 1999-06-04 | 2001-10-09 | Mosaic Technologies, Inc. | Solid phase methods for amplifying multiple nucleic acids |
AU2001241723A1 (en) | 2000-02-25 | 2001-09-03 | Affymetrix, Inc. | Methods for multi-stage solid phase amplification of nucleic acids |
AU2003283976B2 (en) * | 2002-09-27 | 2009-12-10 | Cold Spring Harbor Laboratory | Cell-based RNA interference and related methods and compositions |
WO2004070007A2 (en) | 2003-01-29 | 2004-08-19 | 454 Corporation | Method for preparing single-stranded dna libraries |
AU2008200151B2 (en) * | 2003-01-29 | 2011-08-25 | 454 Life Sciences Corporation | Bead Emulsion Nucleic Acid Amplification |
CN1950519A (en) | 2004-02-27 | 2007-04-18 | 哈佛大学的校长及成员们 | Polony fluorescent in situ sequencing beads |
US7622281B2 (en) | 2004-05-20 | 2009-11-24 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for clonal amplification of nucleic acid |
WO2007120208A2 (en) | 2005-11-14 | 2007-10-25 | President And Fellows Of Harvard College | Nanogrid rolling circle dna sequencing |
US20080009420A1 (en) | 2006-03-17 | 2008-01-10 | Schroth Gary P | Isothermal methods for creating clonal single molecule arrays |
WO2008111990A1 (en) * | 2006-06-14 | 2008-09-18 | Cellpoint Diagnostics, Inc. | Rare cell analysis using sample splitting and dna tags |
US20080108804A1 (en) | 2006-11-02 | 2008-05-08 | Kabushiki Kaisha Dnaform | Method for modifying RNAS and preparing DNAS from RNAS |
WO2008076842A2 (en) | 2006-12-14 | 2008-06-26 | Applied Biosystems Inc. | Sequencing methods |
US20080269068A1 (en) * | 2007-02-06 | 2008-10-30 | President And Fellows Of Harvard College | Multiplex decoding of sequence tags in barcodes |
US8003312B2 (en) * | 2007-02-16 | 2011-08-23 | The Board Of Trustees Of The Leland Stanford Junior University | Multiplex cellular assays using detectable cell barcodes |
US8268564B2 (en) * | 2007-09-26 | 2012-09-18 | President And Fellows Of Harvard College | Methods and applications for stitched DNA barcodes |
EP2212434A1 (en) * | 2007-10-01 | 2010-08-04 | Applied Biosystems Inc. | Chase ligation sequencing |
US20110008775A1 (en) | 2007-12-10 | 2011-01-13 | Xiaolian Gao | Sequencing of nucleic acids |
US9184629B2 (en) | 2007-12-10 | 2015-11-10 | Clevx, Llc | Stored-power system including power management |
US20090163366A1 (en) * | 2007-12-24 | 2009-06-25 | Helicos Biosciences Corporation | Two-primer sequencing for high-throughput expression analysis |
US9328172B2 (en) * | 2008-04-05 | 2016-05-03 | Single Cell Technology, Inc. | Method of obtaining antibodies of interest and nucleotides encoding same |
EP2291533B2 (en) | 2008-07-02 | 2020-09-30 | Illumina Cambridge Limited | Using populations of beads for the fabrication of arrays on surfaces |
US20100062494A1 (en) | 2008-08-08 | 2010-03-11 | President And Fellows Of Harvard College | Enzymatic oligonucleotide pre-adenylation |
US9347092B2 (en) | 2009-02-25 | 2016-05-24 | Roche Molecular System, Inc. | Solid support for high-throughput nucleic acid analysis |
EP3998346A1 (en) * | 2009-03-30 | 2022-05-18 | Illumina, Inc. | Gene expression analysis in single cells |
EA023190B9 (en) * | 2009-04-02 | 2018-03-30 | Флуидигм Корпорейшн | Multi-primer amplification method for barcoding of target nucleic acids |
US9701998B2 (en) * | 2012-12-14 | 2017-07-11 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
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