SEMI-RANDOM BARCODES FOR NUCLEIC ACID ANALYSIS

• US 20160017320A1
• Filed: 07/10/2015
• Published: 01/21/2016
• Est. Priority Date: 07/15/2014
• Status: Active Application

• Current Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Original Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Inventors: Wang, Yexun, Wu, Zhong
• Application Number:
  US14/796,279
• Publication Number:
  US 20160017320A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present disclosure provides oligonucleotides that comprise semi-random barcode sequences. Such oligonucleotides may be incorporated into reverse transcription primers, PCR primers, or portions of sequencing adapters in preparing sequencing libraries. The resulting sequencing libraries can be used for accurate sequencing, including DNA or RNA counting and mutation detection. Methods and kits for preparing sequencing adapters and sequencing libraries are also provided.

Claims

1. A plurality of single-stranded (ss) oligonucleotides, wherein each oligonucleotide comprises from the 5′

to 3′

direction a 1^st sequence and a 2^nd sequence;

(a) the 1^st sequence is a semi-random sequence consisting of (Xmer)n, wherein Xmer is 3-mer, 4-mer, 5-mer, or 6-mer, and n is an integer from 2 to 8, and

(b) the 2^nd sequence is

(i) at least 10 nucleotides in length,

(ii) fully or substantially complementary to a target sequence, and

(iii) the same among the plurality of oligonucleotides.

2. The plurality of ss oligonucleotides of claim 1, wherein Xmer is 3-mer, and n is 3, 4, 5, 6, or 7.

3. The plurality of ss oligonucleotides of claim 1, wherein Xmer is 4-mer, and n is 3, 4, 5, or 6.

4. The plurality of ss oligonucleotides of claim 1, wherein the total number of different Xmers in the 1^st sequences of the oligonucleotides is at least 5.

5. The plurality of ss oligonucleotides of claim 1, wherein Xmer is 3-mer, and the total number of different Xmers in the 1^st sequences of the oligonucleotides is at least 8.

6. The plurality of ss oligonucleotides of claim 1, wherein each oligonucleotide further comprises

(c) a 3^rd sequence that

(i) has a defined sequence that is 3 to 8 nucleotides in length,

(ii) is located 5′

to the 2^nd sequence, and

(iii) is the same among the oligonucleotides.

7. The plurality of ss oligonucleotides of claim 6, wherein the 3^rd sequence is located 5′

to the 1^st sequence.

8. The plurality of ss oligonucleotides of claim 6, wherein each of the oligonucleotides further comprises

(d) a 4^th sequence that is

(i) located 3′

to the 2^nd sequence, and

(ii) the same among the oligonucleotides.

9. The plurality of ss oligonucleotides of claim 1, wherein the 5′

terminus of the oligonucleotide is phosphorylated.

10. The plurality of ss oligonucleotides of claim 1, wherein the number of different oligonucleotides is at least 100.

11. The plurality of ss oligonucleotides of claim 1, wherein different Xmers have 2 or more nucleotide differences from each other.

12. The plurality of ss oligonucleotides of claim 1, wherein the target sequence is a portion at least 10 nucleotides long of one strand of an at least partially double-stranded sequencing adaptor.

13. The plurality of ss oligonucleotides of claim 1, wherein each oligonucleotide is a reverse transcription primer, and wherein the 2^nd sequence comprises poly(T).

14. The plurality of ss oligonucleotides of claim 13, wherein each of the oligonucleotides further comprises:

(d) a 4^th sequence that is

(i) located 5′

to the 1^st sequence, the 2^nd sequence, and the 3^rd sequence if present, and

(ii) the same among the oligonucleotides.

15. The plurality of ss oligonucleotides of claim 1, wherein each oligonucleotide is a PCR primer, and wherein the target sequence comprises a conserved sequence of a region of interest.

16. The plurality of ss oligonucleotides of claim 15, wherein each of the oligonucleotides further comprises:

(d) a 4^th sequence that is

(i) located 5′

to the 1^st sequence, the 2^nd sequence, and the 3^rd sequence if present, and

(ii) the same among the oligonucleotides.

17. The plurality of ss oligonucleotides of claim 15, wherein the region of interest is a 16S RNA gene.

18. A plurality of sets of single-stranded (ss) oligonucleotides, wherein each set comprises the plurality of ss oligonucleotides of claim 6, and wherein the oligonucleotides in different sets are identical to each other except in the 3^rd sequences thereof.

19. A kit for preparing a plurality of double-stranded (ds) sequencing adapters, comprising:

(i) (a) the plurality of ss oligonucleotides of claim 1, or

(b) the plurality of sets of oligonucleotides, wherein each set comprises a plurality of ss oligonucleotides, wherein each oligonucleotide comprises the 1^st sequence and the 2^nd sequence of claim 1, and further comprises a 3^rd sequence that (1) has a defined sequence that is 3 to 8 nucleotides in length, (2) is located 5′

to the 2^nd sequence, and (3) is the same among the oligonucleotides, and wherein the oligonucleotides in different sets are identical to each other except in the 3^rd sequences thereof, and

(ii) a 2^nd ss oligonucleotide that comprises the target sequence (“

Sequence B”

).

23. A plurality of double-stranded (ds) sequencing adapters, wherein each sequencing adapter comprises:

(a) an oligonucleotide (“

1^st oligonucleotide”

) of the plurality of single-stranded (ss) oligonucleotides of claim 1, and

(b) a 2^nd ss oligonucleotide that comprises from the 3′

to 5′

direction

(i) a sequence (“

Sequence A”

) that is fully complementary to the 1^st sequence of the 1^st oligonucleotide of the sequencing adapter, and

(ii) the target sequence (“

Sequence B”

), and

wherein the 1^st oligonucleotide anneals to the 2^nd ss oligonucleotide.

24. The plurality of ds sequencing adapters of claim 23, wherein the 1^st oligonucleotide comprises the 3^rd sequence, and wherein the 2^nd oligonucleotide further comprises

(iii) a sequence (“

Sequence C”

) that is located 3′

to Sequence B, and is fully complementary to the 3^rd sequence of the 1^st oligonucleotide.

25. The plurality of ds sequencing adapters of claim 23, wherein the 2^nd oligonucleotide further comprises

(iv) a sequence (“

Sequence D”

) located 5′

to Sequence B.

26. The plurality of ds sequencing adapters of claim 25, wherein Sequence D of the 2^nd oligonucleotide is not complementary to the 4^th sequence, if present, of the 1^st oligonucleotide.

27. The plurality of ds sequencing adapters of claim 23, wherein each ds sequencing adapter has a single nucleotide 3′

-overhang in the 2^nd oligonucleotide.

28. A plurality of sets of double-stranded (ds) sequencing adapters, wherein

(A) each set comprises a plurality of single-stranded (ss) sequencing adapters, wherein each sequencing adapter in each set comprises:

(a) an oligonucleotide (“

1^st oligonucleotide”

) of the plurality of ss oligonucleotides of claim 6, and

(b) a 2^nd ss oligonucleotide that comprises;

(i) a sequence (“

Sequence A”

) that is fully complementary to the 1^st sequence of the 1^st oligonucleotide of the sequence adapter, and

(ii) the target sequence (“

Sequence B”

) located 5′

to Sequence A, and

(iii) a sequence (“

Sequence C”

) that is located 3′

to Sequence B and is fully complementary to the 3^rd sequence of the 1^st oligonucleotide, and

wherein the 1^st oligonucleotide anneals to the 2^nd oligonucleotide; and

(B) wherein the plurality of ds sequencing adapters in different sets are identical to each other except in the 3^rd sequence of the 1^st oligonucleotide and in Sequence C of the 2^nd oligonucleotide.

32. A kit for preparing a sequencing library, comprising:

(i) the plurality of double-stranded (ds) sequencing adapters of claim 23, and

(ii) a DNA ligase.

33. A method for preparing a sequencing library, comprising:

(1) ligating the plurality of double-stranded (ds) sequencing adapters of claim 23 to ds DNA fragments of a sample, thereby generating a sequencing library.

34. The method of claim 33, wherein the ds DNA fragments are prepared by fragmenting the ds DNAs of the sample, end repairing the fragmented DNAs, and optionally performing A-addition on end-repaired DNA fragments.

35. The method of claim 33, wherein the ds DNA fragments are cDNAs.

36. The method of claim 33, further comprising:

(2) amplifying the sequencing library.

37. The method of claim 33, further comprising:

(3) sequencing the ds DNA fragments.

38. The method of claim 37, further comprising:

a) determining the copy numbers of one or more sequences of interest in the ds DNA fragments.

40. A method for preparing a plurality of sequencing libraries, comprising:

(1) ligating the sequencing adapters of each set of the plurality of sets of double-stranded (ds) sequencing adapters of claim 28 to ds DNA fragments of each of a plurality of samples, thereby generating a plurality of sequencing libraries.

47. A method for preparing a sequencing library, comprising:

(1) (a) reverse transcribing mRNAs of a sample in the presence of the plurality of single stranded (ss) oligonucleotides of claim 1 to generate cDNAss, wherein each oligonucleotide is a reverse transcription primer, and wherein the 2^nd sequence comprises poly(T), or

(b) amplifying a region of interest in a sample in the presence of the plurality of ss oligonucleotides of claim 1 to generate amplified DNAs, wherein each oligonucleotide is a PCR primer, and wherein the target sequence comprises a conserved sequence of a region of interest, and

(2) attaching the cDNAs or amplified DNA of step (1) to at least one double-stranded (ds) sequencing adapter, thereby generating a sequencing library.

49. A method for preparing a sequencing library, comprising:

(1) reverse transcribing mRNAs of a sample in the presence of the plurality of ss oligonucleotides of claim 14 to generate cDNAs, and

(2) amplifying cDNAs

(a) in the presence of a primer that comprises the 4^th sequence, wherein the 4^th sequence comprises a sequencing adapter sequence, and wherein the primer is preferably not any of the plurality of ss oligonucleotides, or

(b) in the presence of a primer comprising, from the 3′

to 5′

direction, the 4^th sequence and a sequencing adapter sequence, wherein the 4^th sequence does not comprise a sequencing adapter sequence,

thereby generating a sequencing library.

50. A method for preparing a sequencing library, comprising:

(a) amplifying nucleic acids of a region of interest in a sample in the presence of the plurality of ss oligonucleotides of claim 16 wherein the 4^th sequence comprises a sequencing adapter sequence, thereby generating a sequence library;

or

(b) (1) amplifying nucleic acids of a region of interest in a sample in the presence of the plurality of ss oligonucleotides of claim 16 wherein the 4^th sequence does not comprises a sequencing adapter sequence to generate amplified nucleic acids, and

(2) further amplifying the amplified nucleic acids of step (b)(1) in the presence of a primer that comprises from the 5′

to 3′

direction, the 4^th sequence and a sequencing adapter sequence, thereby generating a sequencing library.

56. A kit for preparing a sequencing library, comprising:

(1) the plurality of single-stranded (ss) oligonucleotides of claim 14, and

(2) a DNA polymerase.

89. A kit for preparing a sequencing library, comprising:

(i) the plurality of sets of double-stranded (ds) sequencing adapters of claim 28, and

(ii) a DNA ligase.

90. A kit for preparing a sequencing library, comprising:

(1) the plurality of single-stranded (ss) oligonucleotides of claim 16, and

(2) a DNA polymerase.

20-22. -22. (canceled)

29-31. -31. (canceled)

39. (canceled)

41-46. -46. (canceled)

48. (canceled)

51-55. -55. (canceled)

57. A plurality of single-stranded (ss) oligonucleotides, wherein each oligonucleotides comprises:

(a) a 1^st sequence that is a semi-random sequence consisting of (Xmer)n, wherein Xmer is 3-mer, 4-mer, 5-mer, or 6-mer, and n is an integer from 2 to 10,

(b) optionally a 2^nd sequence that

(i) has a defined sequence that is 3 to 8 nucleotides in length, and

(ii) is the same among the oligonucleotides, and

(c) a 3^rd sequence that

(i) has a defined sequence,

(ii) is the same among the oligonucleotides, and

(iii) is located 5′

to the 1^st sequence and the 2′

sequence, if present.

64. A plurality of sets of single-stranded (ss) oligonucleotides, wherein each set comprises the plurality of ss oligonucleotides of claim 57, each of the oligonucleotides comprises the 2^nd sequence, and the oligonucleotides in different sets are identical to each other except in the 2^nd sequences thereof.

65. A method for preparing a sequencing library, comprising:

(1) ligating the plurality of single-stranded (ss) oligonucleotides of claim 57 to the 5′

termini of ss nucleic acids of a sample, and

(2) amplifying the ss nucleic acids

(a) in the presence of a primer comprising the 3^rd sequence, wherein the 3^rd sequence comprises a sequencing adapter sequence, or

(b) in the presence of a primer comprising, from the 3′

to 5′

direction, the 3^rd sequence and a sequencing adapter sequence, wherein the 3^rd sequence does not comprise a sequencing adapter sequence,

thereby generating a sequencing library.

71. A kit for preparing a sequencing library, comprising:

(i) the plurality of single-stranded (ss) sequencing adapters of claim 57, and

(ii) a ligase.

58-63. -63. (canceled)

66-70. -70. (canceled)

72. (canceled)

73. A plurality of single-stranded (ss) oligonucleotides, wherein each oligonucleotide comprises:

(a) a 1^st sequence that is a semi-random sequence consisting of (Xmer)n, wherein Xmer is 3-mer, 4-mer, 5-mer, or 6-mer, and n is an integer from 2 to 10,

(b) optionally a 2^nd sequence that

(i) has a defined sequence that is 3 to 8 nucleotides in length, and

(ii) is the same among the oligonucleotides, and

(c) a 3^rd sequence that

(i) has a defined sequence,

(ii) is the same among the oligonucleotides, and

(iii) is located 3′

to the 1^st sequence and the 2^nd sequence, if present.

80. A plurality of sets of single-stranded (ss) oligonucleotides, wherein each set comprises the plurality of ss oligonucleotides of claim 73, each of the oligonucleotides comprises the 2^nd sequence, and the oligonucleotides in different sets are identical to each other except in the 2^nd sequences thereof.

81. A method for preparing a sequencing library, comprising:

(1) ligating the plurality of single-stranded (ss) oligonucleotides of claim 73 to the 3′

termini of ss nucleic acids of a sample, and

(2) amplifying the ss nucleic acids

(a) in the presence of a primer comprising a sequence complementary to the 3^rd sequence, wherein the 3^rd sequence comprises a sequencing adapter sequence, or

(b) in the presence of a primer comprising, from the 3′

to 5′

direction a sequence at least substantially complementary to the 3^rd sequence and a sequencing adapter sequence, wherein the 3^rd sequence does not comprise a sequencing adapter sequence,

thereby generating a sequencing library.

87. A kit for preparing a sequencing library, comprising:

(i) the plurality of single-stranded (ss) sequencing adapters of claim 73,

(ii) a ligase.

74-79. -79. (canceled)

82-86. -86. (canceled)

88. (canceled)