The invention is directed to a method for detecting RNA, DNA or protein target sequences by a) Hybridizing a library of probes having the general formula (I)

P—(CL-D).sub.x  (I)  With P: probes having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X: integer between 1 and 5  to RNA, DNA or protein target sequences wherein the library comprises probes P having different sequences of nucleotides or amino acids and cleavable linkers CL of different groups which are cleavable with different means b) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first image c) Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes; removing the thus cleaved fluorophores D and detecting the remaining hybridized probes via their fluorophores D by a second image d) Detecting the removed fluorophores D by comparing the first and second image. e) Obtaining a part of the sequence information of the target sequences via the sequence information of the probes P associated with the removed fluorophores D f) Repeating step c) until all groups of chemical linkers CL are cleaved.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises: A-(B-L).sub.J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L).sub.J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises: A-(B-L).sub.J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L).sub.J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

The present disclosure provides a method for sequencing target polynucleotide molecules. In some embodiments, the present disclosure provides a method of sequencing by synthesis where different subsets of nucleotide-conjugate complexes are sequentially formed and detected during each iterative extension of a plurality of nascent nucleic acid copy strands, where each nascent nucleic acid copy strand is complementary to one of a plurality of target polynucleotide molecules. In some embodiments, the plurality of target polynucleotide molecules are arrayed on a solid support.

The present disclosure provides a method for sequencing target polynucleotide molecules. In some embodiments, the present disclosure provides a method of sequencing by synthesis where different subsets of nucleotide-conjugate complexes are sequentially formed and detected during each iterative extension of a plurality of nascent nucleic acid copy strands, where each nascent nucleic acid copy strand is complementary to one of a plurality of target polynucleotide molecules. In some embodiments, the plurality of target polynucleotide molecules are arrayed on a solid support.

Single-cell analysis of a population of cells reveals cellular genotypes (e.g., single nucleotide variants and copy number variations) and phenotypes (e.g., protein expression) of individual cells. In one scenario, individual cells can be classified according to their respective genotypes and phenotypes. In one scenario, genotypes and phenotypes of all cells in the population are informative for identifying subpopulations of cells, thereby revealing intra-population heterogeneity. The identification of subpopulations of cells is informative for improving the understanding of cellular biology, especially in the context of diseases such as cancer, and is further informative for the better design of diagnostics and therapies.

Single-cell analysis of a population of cells reveals cellular genotypes (e.g., single nucleotide variants and copy number variations) and phenotypes (e.g., protein expression) of individual cells. In one scenario, individual cells can be classified according to their respective genotypes and phenotypes. In one scenario, genotypes and phenotypes of all cells in the population are informative for identifying subpopulations of cells, thereby revealing intra-population heterogeneity. The identification of subpopulations of cells is informative for improving the understanding of cellular biology, especially in the context of diseases such as cancer, and is further informative for the better design of diagnostics and therapies.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.