Methods of Nucleic Acid Sequencing

• US 20190324042A1
• Filed: 04/01/2019
• Published: 10/24/2019
• Est. Priority Date: 03/06/2012
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Fraser, Louise, Kokko-Gonzales, Paula
• Granted Patent: US 11,834,699 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Provided herein is a method of using transposition to improve methods of sequencing RNA molecules. Provided herein is a method of tagging nucleic acid duplexes, such as DNA:RNA duplexes or DNA:DNA duplexes. The method includes the steps of providing a transposase and a transposon composition, providing one or more nucleic acid duplexes immobilized on a support, and contacting the transposase and transposon composition with the one or more nucleic acid duplexes under conditions wherein the one or more nucleic acid duplexes and transposon composition undergo a transposition reaction to produce one or more tagged nucleic acid duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.

Claims

1-20. -20. (canceled)

21. A composition comprising

a DNA:

RNA duplex or a DNA;

DNA duplex immobilized on a support; and

a transposome complex, wherein the transposome complex comprises a transposase and a transposon composition,

wherein the transposon composition comprises a double-stranded nucleic acid comprising a transferred strand comprising a first transposon sequence and a non-transferred strand comprising a second transposon sequence that is complementary to the first transposon sequence.

22. The composition of claim 21, wherein the transposome complex is associated with the duplex.

23. The composition of claim 21, wherein the transposon composition comprises the double-stranded nucleic acid molecule and one or more other nucleotide sequences on one or both strands.

24. The composition of claim 21, wherein the transferred strand comprises a tag 5′

of the transposon sequence, wherein the tag comprises one or more tag domains.

25. The composition of claim 24, wherein the tag comprises a nucleotide sequence that permits identification, recognition, and/or molecular of biochemical manipulation of an oligonucleotide to which the tag is attached.

26. The composition of claim 25, wherein the tag comprises one or more of a site for annealing an oligonucleotide, a site for capture of an oligonucleotide, a site for a ligation reaction, and a site that provides identification of an oligonucleotide as originating from a particular source.

27. The composition of claim 21, wherein the transposon composition comprises a biotin, a label, or a fluorescent label.

28. The composition of claim 21, wherein the transposon composition comprises:

(i) the transferred strand comprising the first transposon sequence and one or more tag domains selected from a restriction site tag domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain; and

(ii) the non-transferred strand comprising the second transposon sequence.

29. The composition of claim 28, wherein the transferred strand comprises a sequencing tag domain, a capture tag domain, and an amplification tag domain.

30. The composition of claim 21, wherein the duplexes are immobilized on the solid support via immobilized primers on the solid support.

31. The composition of claim 30, wherein the duplex is a DNA:

RNA duplex, wherein the RNA of the RNA duplex comprises a polyA sequence and the immobilized primers comprise a polyT sequence.

32. The composition of claim 30, wherein the immobilized primers each comprise a target-specific primer and one strand of the duplex comprises a sequence complementary to each target-specific primer.

33. The composition of claim 30, wherein the RNA of the RNA:

DNA duplex or the DNA of the DNA;

DNA duplex comprises a 3′

adaptor, wherein the 3′

adaptor comprises a sequence complementary to the plurality of immobilized primers or a subset thereof.

34. The composition of claim 21, wherein the transposase is a Tn5 transposase.

35. The composition of claim 21, wherein the solid support is a flow cell reactor surface or a bead.

36. The composition of claim 21, wherein the bead is a magnetic bead, a hollow bead, or a solid bead.

37. The composition of claim 21, wherein the solid support is an array, wherein the array includes separate supports each bearing a different probe molecule.

38. The composition of claim 21, wherein the support is a plurality of beads, wherein each bead comprises a different duplex.

39. The composition of claim 21, wherein the solid support is a flow cell reactor comprising multiple microfluidic channels, optionally wherein the surface of the flow cell reactor comprises wells.

40. A composition comprising:

a DNA;

RNA duplex or a DNA;

DNA duplex immobilized on a flow cell reactor surface or a bead; and

a transposome complex associated with the duplex, wherein the transposome complex comprises

a transposase and a transposon composition,

wherein the transposon composition comprises a double-stranded nucleic acid comprising a transferred strand comprising a first transposon sequence and one or more tag domains selected from a restriction site tag domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain; and

a non-transferred strand comprising a second transposon sequence that is complementary to the first transposon sequence.