Quantitative detection and analysis of moleculesip

Patent metadata

Field	Value
Publication number	US-12534721-B2
Application number	US-202418651990-A
Country	US
Kind code	B2
Filing date	May 1, 2024
Priority date	Oct 6, 2022
Publication date	Jan 27, 2026
Grant date	Jan 27, 2026

Abstract

The invention provides systems and methods for making sequencing libraries that are useful for quantitatively analyzing nucleic acids in a sample. Sample nucleic acids are randomly cleaved at, and PCR handled are attached to, a random cut site. The nucleic acid is amplified into a sequencing library in which a sequencing primer generates a sequence read from adjacent the random cut site. The sequence reads can be mapped to a reference, but they will also include a unique identifier sequence that comes from within the nucleic acid molecule being analyzed, i.e., an intrinsic molecular identifier (IMI). The IMI is unique for each molecule and can thus be used to deduplicate sequence reads originating from the same molecule.

First claim

What is claimed is:

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.

Assignees

• Illumina Inc

Inventors

• Meltzer Robert

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1096 Primary

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title