Search

EP-4741511-A2 - METHODS AND COMPOSITIONS FOR ANALYZING CELLULAR COMPONENTS

EP4741511A2EP 4741511 A2EP4741511 A2EP 4741511A2EP-4741511-A2

Abstract

Embodiments of the present invention relate to analyzing components of a cell. In some embodiments, the present invention relate to analyzing components of a single cell. In some embodiments, the methods and compositions relate to sequencing nucleic acids. In some embodiments, the methods and compositions relate to identifying and/or quantitating nucleic acid, proteins, organelles, and/or cellular metabolites.

Inventors

  • FISHER, JEFFREY S.
  • GUNDERSON, KEVIN L.
  • RIGATTI, ROBERTO
  • STEEMERS, FRANK J.

Assignees

  • ILLUMINA, INC.

Dates

Publication Date
20260513
Application Date
20160210

Claims (15)

  1. A method of barcoding, the method comprising: (a) encapsulating cells or organelles in permeable particles, wherein at least some of the permeable particles comprise a single cell or organelle; (b) lysing the cells or organelles in the permeable particles while maintaining nucleic acids within the permeable particles; and (c) amplifying the nucleic acids, or copies thereof, in the permeable particles to produce amplification products; and (d) attaching a unique combination of nucleic acid indices to the amplification products in the permeable particles using combinatorial indexing.
  2. The method of claim 1, wherein the attaching in step (d) is by ligation, polymerase extension, tagmentation and/or hybridization.
  3. The method of claim 1, wherein the combinatorial indexing of step (d) comprises one or more rounds of: (i) splitting the permeable particles into a plurality of compartments, wherein at least some of the compartments receive multiple permeable particles; (ii) adding nucleic acid indices to the permeable particles in the plurality of compartments, wherein each compartment receives a different index and each index diffuses into multiple particles; and (iii) pooling the permeable particles from the compartments.
  4. The method of claim 1, wherein the combinatorial indexing of step (d) comprises: (i) splitting the permeable particles into multiple first compartments, wherein at least some of the first compartments receive multiple permeable particles; (ii) attaching nucleic acid indices to the amplified nucleic acids in the permeable particles of the first compartments, wherein each first compartment receives a different nucleic acid index and each index diffuses into multiple permeable particles; (iii) combining the permeable particles from the first compartments into a pool; (iv) splitting the pool of (iii) into multiple second compartments, wherein at least some of the second compartments receive multiple permeable particles; and (v) attaching nucleic acid indices to the amplified nucleic acids in the permeable particles of the second compartments, wherein: each second compartment each receives a different nucleic acid index, and the indices of (v) are attached to products made in step (ii).
  5. The method of claim 1, wherein the combinatorial indexing of step (d) comprises: (i) splitting the permeable particles into multiple first compartments, wherein at least some of the first compartments receive multiple permeable particles; (ii) adding first nucleic acid indices to the permeable particles in the plurality of the first compartments, wherein each compartment receives a different index; and (iii) combining the permeable particles from the first compartments into a pool; and (iv) splitting the pool of (iii) into multiple second compartments, wherein at least some of the second compartments receive multiple permeable particles; and (v) adding second nucleic acid indices to the permeable particles in the plurality of second compartments, wherein each compartment receives a different index.
  6. The method of claim 1, wherein: (i) the permeable particles are in the range of 20 microns to 200 microns in diameter; or (ii) the permeable particles are made of a polymer; or (iii) the permeable particles are made of a cross-linked polymer.
  7. The method of claim 1, wherein the encapsulating of step (a) is done using microfluidics.
  8. The method of claim 1, wherein the nucleic acids and amplification products are encapsulated in the permeable particles and the permeable particles are permeable to nucleotides, oligonucleotide adapters primers, and/or enzymes.
  9. The method of claim 1, further comprising sequencing the product of step (d), or an amplification product thereof, to produce sequence reads; optionally wherein the method further comprises analyzing the sequence reads to detect a mutation, a copy number alteration, a methylation state, open chromatin, or a transcriptome.
  10. The method of claim 1, wherein the nucleic acids amplified in step (c) comprise cDNA.
  11. The method of claim 1, wherein the nucleic acids amplified in step (c) comprise genomic DNA; optionally wherein the genomic DNA is amplified by whole genome amplification (WGA).
  12. The method of claim 1, wherein the amplification is done by PCR.
  13. The method of claim 1, wherein: (i) the encapsulating cells or organelles are mammalian; or (ii) the encapsulating cells or organelles are bacterial.
  14. The method of claim 1, wherein the cells or organelles are fixed.
  15. The method of claim 1, wherein the lysis is done using a protease.

Description

FIELD OF THE DISCLOSURE Embodiments of the present application relate to methods and composition for analyzing cellular components. In some embodiments, the present application relate to methods and composition for analyzing components of a single cell. In some embodiments, the present application relate to methods and composition for identifying a single cell type. In some embodiments, the methods and compositions relate to sequencing nucleic acids. Some embodiments of the methods and compositions provided are useful in deriving a composite status of such single cell. BACKGROUND The detection of specific nucleic acid sequences present in a biological sample has been used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. A common technique for detecting specific nucleic acid sequences in a biological sample is nucleic acid sequencing. Nucleic acid sequencing methodology has evolved significantly from the chemical degradation methods used by Maxam and Gilbert and the strand elongation methods used by Sanger. Today several sequencing methodologies are in use which allow for the parallel processing of nucleic acids all in a single sequencing run. As such, the information generated from a single sequencing run can be enormous. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts a schematic of a four tier combinatoric indexing of DNA contiguity preserving element (CE) created by embedding single cell contents in a polymer matrix or attaching to a bead. Compartment-specific indexes are attached at each combinatoric pooling and redistribution step (tiers). In the example shown, the four tiers result in four indexes being concatenated together (via repeated rounds of ligation, polymerase extension, tagmentation, etc.) enabling easy sequencing read out. Alternatively, the contiguity preserving element comprising DNA can be created by a compartmentalized DNA partition (i.e. a DNA dilution subsampling the original DNA sample) that has been encapsulated in a matrix or immobilized on a bead. This type of dilution is useful in phasing and assembly applications.Fig. 2 depicts a method of preparing single cell DNA or cDNA libraries using a two tier combinatorial indexing scheme wherein the first level indexes are attached via tagmentation (compartment-specific indexes in transposons) and the second tier indexes are attached by PCR (compartment-specific indexes on PCR primers). The contents of the single cell vessel (i.e. genomic DNA or cDNA) may employ an optional whole genome amplification (WGA) or whole transcriptome amplification step.Fig. 3 depicts a method of making cDNA library from the contents of a single cell in CE such as droplets. In the example shown, the indexes are being used to label different samples.Fig. 4 depicts representative contents of a single cell that can be analyzed via the combinatorial indexing scheme proposed.Fig. 5Aand5B depict exemplary schematic embodiments for creating a contiguity preserving elements (CE) from encapsulating and lysing the contents of a single cell trapped within a CE such as in polymer bead. Cell is embedded in, for example, a polymer bead. All the components from a single cell are kept in proximity to one another in the bead. Subsequently, one or more components can be amplified, modified (cDNA synthesis), and subsequently labeled with indexes or tags. Fig. 5C depicts an exemplary schematic embodiment in which sample indexing can be accomplished by spiking encoding DNA sequences (such as a plasmid) at the encapsulation, amplification/cDNA, or polymerization stage. Each sample is prepared with a different set of encoding plasmids or combination of encoding plasmid. Every combinatorially indexed CE will produce corresponding combinatorially indexed sample encoding library elements. In this way, every library element can be mapped back to its originating CE and originating sample.Fig. 6 depicts schematics for encapsulating single cell contents in CE such as polymer matrix beads.Fig. 7 depicts an exemplary schematics of high throughput analysis of cellular components by direct surface capture. "A" shows a collection of cells. "B" shows surface-bound transposomes. In "C" the cells are flowed onto the surface. In "D" cells are lysed and the cell's components are allowed to diffuse in a controlled way around the site at which the cell was captured. In "E" the nucleic acids are captured (tagmented) by the transposomes. Different cellular components are captured depending on whether the cell membrane or nuclei are lysed. By using component-specific capture moieties (i.e. antibodies, receptors, ligands), various cellular components can be captured. The analysis of the captured molecules can be carried out directly on the capturing