RU-2861650-C2 - METHODS AND COMPOSITIONS FOR COMBINATORIAL INDEXING OF NUCLEIC ACIDS

Abstract

FIELD: biotechnology. SUBSTANCE: described is a method for producing a plurality of combinatorially indexed beads, comprising: (a) obtaining a population of primary indexed beads containing a first polynucleotide containing a first index, wherein the population of primary indexed beads comprises a plurality of first subpopulations of indexed beads, wherein the first subpopulations of indexed beads contain a first index different from others; (b) dividing the population of primary indexed beads into a plurality of second subpopulations of beads; (c) obtaining a population of secondary indexed beads, comprising: (i) extending the first polynucleotide from the plurality of second subpopulations of beads with a second polynucleotide containing a second index, to obtain second subpopulations of indexed beads, wherein the second subpopulations of indexed beads contain a second index different from others, and (ii) combining the second subpopulations of indexed beads to obtain a population of secondary indexed beads, resulting in the production of said plurality of combinatorially indexed beads. EFFECT: expanding methods for detecting specific nucleotide sequences present in a biological sample. 71 cl, 19 dwg, 3 tbl, 10 ex

Inventors

• MANTSO, Andrea
• BRAUN, Kolin
• NORBERG, Stiven
• KHARRINGTON, Timoti

Dates

Publication Date

20260506

Application Date

20220623

Priority Date

20210624

Claims (20)

1. 1. A method for producing a plurality of combinatorially indexed granules, comprising:
2. (a) obtaining a population of primary indexed granules comprising a first polynucleotide comprising a first index, wherein the population of primary indexed granules comprises a plurality of first subpopulations of indexed granules, wherein the first subpopulations of indexed granules comprise first indices that are different from one another;
3. (b) partitioning the population of primary indexed granules into a plurality of second subpopulations of granules;
4. (c) obtaining a population of secondary indexed granules, comprising:
5. (i) extending the first polynucleotide in a plurality of second subpopulations of granules with a second polynucleotide comprising a second index to produce second subpopulations of indexed granules, wherein the second subpopulations of indexed granules comprise second indices that are different from one another; and
6. (ii) combining the second subpopulations of indexed granules to obtain a population of secondary indexed granules, which results in obtaining the specified set of combinatorially indexed granules.
7. 2. The method according to paragraph 1, further comprising:
8. (d) dividing the population of secondary indexed granules into a plurality of third subpopulations of granules; and
9. (e) obtaining a population of tertiary indexed granules comprising:
10. (i) extending a second polynucleotide in a plurality of third subpopulations of granules with a third polynucleotide comprising a third index to produce third subpopulations of indexed granules, wherein the third subpopulations of indexed granules comprise third indices that are different from one another; and
11. (ii) combining the tertiary subpopulations of indexed granules to obtain a population of tertiary indexed granules.
12. 3. The method of claim 1 or 2, wherein (c) comprises extending the first polynucleotide with a second polynucleotide by chemical ligation.
13. 4. The method according to paragraph 3, in which:
14. the first polynucleotide contains a terminal 3'-modified deoxynucleotide (dNTP) containing a 3'-functional fragment capable of participating in a click reaction;
15. the second polynucleotide comprises a terminal 5'-modified dNTP containing a compatible 5'-functional fragment capable of participating in a click reaction with a 3'-functional fragment; and
16. The 3'-functional fragment and the 5'-functional fragment are able to react with each other to form a modified framework bond.
17. 5. The method of claim 4, wherein extending the first polynucleotide with the second polynucleotide produces a secondary indexed polynucleotide and the method further comprises modifying the secondary indexed polynucleotide to produce a modified polynucleotide containing a terminal 3'-modified deoxynucleotide (dNTP) containing a 3'-functional fragment capable of participating in a click reaction.
18. 6. The method of claim 5, wherein the modification comprises contacting the secondary indexed polynucleotide with a template-independent polymerase.
19. 7. The method of claim 6, wherein the template-independent polymerase is selected from terminal deoxynucleotidyl transferase (TdT), PolyA polymerase, or CCA-adding RNA polymerase.
20. 8. The method of claim 7, wherein the template-independent polymerase is TdT.

Description

RELATED APPLICATIONS [0001] This application claims priority to Provisional Patent Application No. 63/214,693, filed June 24, 2021, entitled "METHODS AND COMPOSITIONS FOR COMBINATORIAL INDEXING OF BEAD-BASED NUCLEIC ACIDS," which is incorporated herein by reference in its entirety. LINK TO SEQUENCE LISTING [0002] This application is filed together with a sequence listing in electronic format. The sequence listing is provided as a file named ILLINC608WOSEQLIST, created on June 17, 2022, and is approximately 3.15 KB in size. The information contained in the electronic sequence listing file is incorporated herein by reference in its entirety. FIELD OF APPLICATION OF THE INVENTION [0003] Some embodiments relate to methods and compositions for producing combinatorially indexed beads. Some embodiments involve sequentially adding different indices to polynucleotides attached to the beads. In some embodiments, indices are added by chemical ligation, polymerase extension, ligation of partially double-stranded adapters, or short splint ligation. PREREQUISITES FOR THE CREATION OF THE INVENTION [0004] Detection of specific nucleotide sequences present in a biological sample is 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 predisposition to a particular disease, and assessing the response to various forms of treatment. A common method for detecting specific nucleotide sequences in a biological sample is nucleic acid sequencing. [0005] Nucleic acid sequencing technology has evolved significantly from the chemical digestion methods used by Maxam and Gilbert and the chain extension methods used by Sanger. Several sequencing techniques are currently in use, allowing for the parallel processing of thousands of nucleic acids, all on a single chip. Some platforms include bead-based and microarray-based configurations, in which silica beads are functionalized with probes, depending on the use of such configurations in applications including sequencing, genotyping, and gene expression profiling. [0006] Methods for genotyping various samples on existing bead-based arrays may require spacers to physically divide different regions of the bead chip into multiple sectors. Individual samples are then loaded into each separate section created by the spacer. However, such methods can be used with relatively small numbers of input samples, but they prove labor-intensive or difficult to control as the sample density per bead chip increases from 24 to 96, 384, 1536, or more samples per bead chip. SUMMARY OF THE INVENTION [0007] Some embodiments of the methods and compositions provided herein include methods of producing a plurality of combinatorially indexed beads, comprising: (a) obtaining a population of primary indexed beads comprising a first polynucleotide comprising a first index, wherein the population of primary indexed beads comprises a plurality of first subpopulations of indexed beads, wherein the first subpopulations of indexed beads comprise a first index that is different from others; (b) dividing the population of primary indexed beads into a plurality of second subpopulations of beads; (c) obtaining a population of secondary indexed beads, comprising: (i) extending a first polynucleotide from the plurality of second subpopulations of beads with a second polynucleotide comprising a second index to produce second subpopulations of indexed beads, wherein the second subpopulations of indexed beads comprise a second index that is different from others, and (ii) combining the second subpopulations of indexed beads to produce a population of secondary indexed beads; and optionally further comprising: (d) dividing the population of secondary indexed beads into a plurality of third subpopulations of beads; and (e) obtaining a population of tertiary indexed granules, comprising: (i) extending a second polynucleotide from the plurality of third subpopulations of granules with a third polynucleotide containing a third index, to obtain third subpopulations of indexed granules, wherein the third subpopulations of indexed granules contain a third index that is different from the others, and (ii) combining the third subpopulations of indexed granules to obtain a population of tertiary indexed granules. [0008] In some embodiments, (c) comprises extending the first polynucleotide with a second polynucleotide via chemical ligation. [0009] In some embodiments, the first polynucleotide comprises a terminal 3'-modified deoxynucleotide (dNTP) containing a 3'-functional moiety capable of participating in a click reaction; the second polynucleotide comprises a terminal 5'-modified dNTP containing a compatible 5'-functional moiety capable of participating in a click reaction with a 3'-functional moiety; and the 3'-functional moiety and the 5'-functional moiety are ca