Transcriptome sequencing through random ligation

Patent metadata

Field	Value
Publication number	US-2025179556-A1
Application number	US-202519048745-A
Country	US
Kind code	A1
Filing date	Feb 7, 2025
Priority date	Feb 28, 2018
Publication date	Jun 5, 2025
Grant date	—

Abstract

The present disclosure provides methods and systems for producing full-length sequencing information of transcriptomes from single cells or from the bulk. Random ligation and circularization of barcoded or non-barcoded complementary deoxyribonucleic molecules can be used to provide a circular template for amplification and subsequent sequencing.

First claim

1 .- 8 . (canceled)

9 . A method, comprising:
(a) ligating a plurality of different nucleic acid analytes to generate a plurality of templates comprising different combinations of nucleic acid analytes; (b) partitioning, into a partition among a plurality of partitions: (i) a template of the plurality of templates or a product thereof, and (ii) a plurality of barcode molecules comprising a partition-specific barcode sequence; and (c) generating a plurality of amplicons within the partition using the template and the plurality of barcode molecules, wherein amplicons of the plurality of amplicons comprise (i) the partition-specific barcode sequence, and (ii) an overlapping sequence of the template or complement thereof.

10 . The method of claim 9 , wherein the method further comprises sequencing the plurality of amplicons or derivatives thereof to obtain a plurality of sequence reads, wherein sequence reads of the plurality of sequence reads comprise the partition-specific barcode sequence or complement thereof and the overlapping sequence of the template or complement thereof.

11 . The method of claim 10 , wherein the method further comprises assembling the sequence reads of the plurality of sequence reads based on the overlapping sequence of the template or complement thereof to generate an assembled template sequence.

12 . The method of claim 9 , wherein the ligating the plurality of different nucleic acid analytes comprises random ligation.

13 . The method of claim 11 , wherein the assembled template sequence comprises sequences of nucleic acid analytes comprised by the template.

14 . The method of claim 11 , wherein the assembled template sequence comprises a contiguous sequence comprising sequences of nucleic acid analytes comprised by the template.

15 . The method of claim 13 , wherein an individual nucleic acid analyte molecule comprised by the template is identified by a combination of the nucleic acid analytes comprised by the template.

16 . The method of claim 14 , wherein an individual nucleic acid analyte molecule comprised by the template is identified by a combination and an arrangement of the nucleic acid analytes comprised by the template.

17 . The method of claim 9 , wherein the plurality of different nucleic acid analytes comprises single-stranded nucleic acids.

18 . The method of claim 9 , wherein the plurality of different nucleic acid analytes comprises double-stranded nucleic acids.

19 . The method of claim 11 , wherein the assembled template sequence comprises a full-length sequence of a nucleic acid analyte comprised by the template.

20 . The method of claim 19 , wherein the full-length sequence of the nucleic acid analyte is a full-length cDNA sequence.

21 . The method of claim 11 , wherein the plurality of different nucleic acid analytes comprises cDNA molecules.

22 . The method of claim 21 , wherein the assembled template sequence comprises a plurality of full-length sequences of the cDNA molecules.

23 . The method of claim 22 , wherein the cDNA molecules are generated from RNA molecules from a plurality of cells, and wherein the cDNA molecules comprise cell-specific barcode sequences associated with cells from which the cDNAs are derived.

24 . The method of claim 23 , wherein the cDNA molecules do not comprise a unique molecular identifier.

25 . The method of claim 24 , wherein an individual cDNA molecule comprised by the template is identified by a combination of cDNA molecules comprised by the template or by the arrangement of the combination of cDNA molecules comprised by the template.

26 . The method of claim 9 , wherein the plurality of different nucleic acid analytes are derived from one or more cells.

27 . The method of claim 9 , wherein the plurality of different nucleic acid analytes comprise cell-free DNA.

28 . The method of claim 27 , wherein the cell-free DNA is circulating cell-free DNA.

29 . The method of claim 9 , wherein the plurality of barcode molecules is a plurality of barcoded amplification primers.

30 . The method of claim 29 , wherein the barcoded amplification primers comprise random N-mer priming sequences.

31 . The method of claim 29 , wherein generating the partition comprises co-partitioning into the partition: i) the template, and ii) a particle coupled to the plurality of barcoded amplification primers; wherein the barcoded amplification primers are released from the particle in the partition.

32 . The method of claim 9 , wherein the partition is a droplet or a well.

33 . The method of claim 9 , wherein the template is a circular template.

34 . The method of claim 9 , wherein the template is a ligation product of at least 5 different nucleic acid analyte molecules.

35 . The method of claim 9 , wherein the template is a circular template, wherein the method comprises performing rolling circle amplification of the circular template to generate a rolling circle amplification product (RCP), and wherein the partition generated in (b) comprises the RCP or a fragment of the RCP.

36 . The method of claim 35 , wherein the method comprises fragmenting the RCP, and wherein the partition generated in (b) comprises a fragment of the RCP.

37 . A method, comprising:
(a) ligating a plurality of different nucleic acid analytes to generate a plurality of templates comprising different combinations of nucleic acid analytes; (b) generating a partition among a plurality of partitions, wherein the partition comprises (i) a template of the plurality of templates or a product thereof, and (ii) a plurality of barcoded amplification primers comprising a partition-specific barcode sequence; (c) generating a plurality of amplicons within the partition using the template and the plurality of barcoded amplification primers, wherein amplicons of the plurality of amplicons comprise (i) the partition-specific barcode sequence, and (ii) an overlapping sequence of the template or complement thereof.

38 . The method of claim 37 , further comprising:
(d) obtaining sequence reads of the amplicons or derivatives thereof, wherein the sequence reads comprise the partition-specific barcode sequence or complement thereof and the overlapping sequence of the template or complement thereof; and (e) assembling the sequence reads based on the overlapping sequence of the template or complement thereof to generate an assembled sequence.

Assignees

• 10X Genomics Inc

Inventors

• Gopalan Vijay Kumar Sreenivasa
• Ryvkin Paul
• Bent Zachary
• Terry Jessica Michele
• Jaffe David
• Marks Patrick
• Mikkelsen Tarjei Sigurd

Classifications

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · 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

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title