Granted Patent B2

US 12,534,721 · App. 18/651,990 · Granted Jan 27, 2026

Inventor: Robert Meltzer (Belmont, MA)

Assignee: Illumina, Inc.

C12N15/1096C12Q1/6806C12Q1/6834C12Q1/686C12Q1/6874

Abstract

Claims (31)

1 . A method for generating nucleic acid library, the method comprising the steps of:

providing a sample comprising a plurality of cells, each comprising sample nucleic acids;

hybridizing sample nucleic acids to a construct comprising a solid support to which is attached, via a linker, a cellular barcode sequence, and a capture sequence;

extending said construct from said capture sequence to form a duplex comprising an extended construct;

exposing said duplex to a transposase, thereby to generate a unique identifier sequence at a 3′ end of said construct; and

amplifying said extended construct; thereby to create a nucleic acid library.

2 . The method of claim 1 , wherein the extending step reverse transcribes a sample nucleic acid into a cDNA in the construct.

3 . The method of claim 2 , wherein the transposase cuts the cDNA at a random cut site.

4 . The method of claim 3 , wherein the unique identifier sequence is provided by a segment of the cDNA adjacent the random cut site.

5 . The method of claim 1 , further comprising sequencing the library to generate sequence reads, mapping the sequence reads to genes in a reference, and collapsing reads that include the same unique identifier sequence.

6 . The method of claim 1 , further comprising isolating the cells into partitions and creating sequencing libraries from single cells in the partitions.

7 . The method of claim 1 , wherein the solid support is a bead comprising a plurality of copies of the cellular barcode sequence.

8 . A method for generating a nucleic acid library, the method comprising:

capturing RNA molecules from a single cell with capture oligos that include a first PCR handle;

extending the capture oligos to form duplexes comprising the RNA molecules and cDNA; and

cleaving the duplexes at, and attaching second PCR handles to, random cut sites to thereby form constructs that each include a label defined by intrinsic sequence of a cDNA segment adjacent the random cut site.

9 . The method of claim 8 , further comprising:

amplifying the constructs to form amplicons;

sequencing the amplicons to produce sequence reads; and

counting sequence reads with duplicate intrinsic sequences as one RNA molecule from the single cell.

10 . The method of claim 8 , wherein the capture oligos are linked to a solid support in an aqueous partition that includes the single cell.

11 . The method of claim 10 , wherein the solid support comprises a bead and the aqueous partition is a droplet.

12 . The method of claim 8 , further comprising forming a plurality of droplets that each include, on average, one bead decorated with capture oligos and zero or one single cell.

13 . The method of claim 12 , wherein the plurality of droplets are formed substantially simultaneously by shearing or vortexing a vessel comprising an aqueous phase, an immiscible phase, oligo-linked beads, and cells.

14 . The method of claim 9 , wherein the capture oligos include cell barcodes.

15 . The method of claim 14 , wherein the constructs include at least the first PCR handles, the cell barcodes, cDNAs, and the second PCR handles.

16 . The method of claim 15 , wherein the amplicons includes copies of the first and second PCR handles and wherein the copies of the first and second PCR handles anneal to sequencing adaptors.

17 . The method of claim 9 , further comprising mapping the sequence reads to a reference to identify genes from which one or more of the RNA molecules were transcribed.

18 . The method of claim 17 , further comprising providing a report with transcription levels of the genes in the single cells based on the counted sequence reads and identified genes.

19 . The method of claim 8 , wherein the cleaving and/or the attaching steps are performed by an enzyme that creates the random cut sites, wherein the enzyme comprises a transpose.

20 . The method of claim 8 , wherein the intrinsic sequence of the cDNA is copied from genetic material of the single cell and wherein, due to the random cut site, each duplex includes a label useful to uniquely identify the cDNA is sequencing data.

Assignments (2)

Continuity (3)

References Cited (322)