Linking sequence reads using paired code tags

• US 9,074,251 B2
• Filed: 04/05/2011
• Issued: 07/07/2015
• Est. Priority Date: 02/10/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Steemers, Frank J., Gunderson, Kevin, Royce, Thomas, Pignatelli, Natasha, Goryshin, Igor Yu, Caruccio, Nicholas
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US13/080,345
• Publication Number:
  US 20120208724A1
• Time in Patent Office: 1,554 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

Artificial transposon sequences having code tags and target nucleic acids containing such sequences. Methods for making artificial transposons and for using their properties to analyze target nucleic acids.

Claims

1. A method of sequencing a target nucleic acid, comprising:

(a) contacting a target nucleic acid with a plurality of transposons under conditions such that a plurality of the transposons are inserted into the target nucleic acid,

wherein the transposons that are inserted into the target nucleic acid each comprise a first transposase recognition site, a second transposase recognition site, and a barcode pair,

wherein the barcode pair comprises a first barcode sequence, a second barcode sequence and a linker comprising a fragmentation site disposed between the first and second barcode sequences, wherein the first barcode sequence comprises a first strand and a second strand that is not complementary to the first strand of the first barcode sequence,

wherein the barcode pair is disposed between the first and second transposase recognition sites;

(b) fragmenting the target nucleic acid at the fragmentation sites thereby preparing a library of template nucleic acid fragments;

(c) obtaining sequence reads from the nucleic acid fragments; and

(d) assembling a representation of at least a portion of the target nucleic acid from the sequence reads, wherein the assembling comprises determining the order of the sequence reads from the identities of the first barcode sequence and the second barcode sequence of each barcode pair.

2. The method of claim 1, wherein the target nucleic acid comprises cDNAs from a single cell.

3. The method of claim 1, wherein the target nucleic acid comprises nucleic acids from a plurality of species.

4. The method of claim 1, wherein the target nucleic acid comprises nucleic acids from a plurality of haplotypes.

5. The method of claim 1, wherein the fragmentation site comprises an enzyme cleavage site selected from the group consisting of a restriction endonuclease recognition sequence, a nickase recognition sequence, and a ribonucleotide sequence.

6. The method of claim 5, wherein the fragmentation site is a restriction endonuclease site and wherein fragmentation comprises fragmenting the target nucleic acid with a restriction endonuclease enzyme that recognizes the restriction endonuclease recognition site.

7. The method of claim 5, wherein the fragmentation site is a nickase recognition site and wherein fragmentation comprises fragmenting the target nucleic acid with a nickase enzyme that recognizes the nickase recognition sequence.

8. The method of claim 1, wherein the second barcode sequence of the barcode pair is different from the first barcode sequence of the barcode pair.

9. The method of claim 1, wherein the second barcode sequence comprises a first strand and a second strand that is not complementary to the first strand of the second barcode sequence.

10. The method of claim 1, wherein at least 90% of the barcode pairs in the plurality of transposons are different from each other.

11. A method of sequencing a target nucleic acid comprising:

(a) contacting a target nucleic acid with a plurality of transposons under conditions such that a portion of the transposons are inserted into the target nucleic acid,

wherein the transposons that are inserted into the target nucleic acid each comprise a first transposase recognition site, a second transposase recognition site, and a barcode pair,

wherein the barcode pair comprises a first barcode sequence, a second barcode sequence and a linker comprising a first primer site disposed between the first and second barcode sequences, wherein the first barcode sequence comprises a first strand and a second strand that is not complementary to the first strand of the first barcode sequence,

wherein the barcode pair is disposed between the first and second transposase recognition sites;

(b) hybridizing a first primer to the first primer site;

(c) amplifying a portion of the transposon using the first primer that is hybridized to the first primer site, to create a plurality of amplified nucleic acids for sequencing;

(d) obtaining sequence reads from the amplified nucleic acids; and

(e) assembling a representation of at least a portion of the target nucleic acid from the sequence reads, wherein the assembling comprises determining the order of the sequence reads from the identities of the first barcode sequence and the second barcode sequence of each barcode pair.

12. The method of claim 11, wherein the assembling further comprises identifying the presence of a first barcode sequence in one sequencing read and the presence of a second barcode sequence in another sequencing read, thereby indicating proximity between the two sequencing reads in the target nucleic acid.

13. The method of claim 12, wherein step (d) is terminated after the first and second barcode sequences are identified.

14. The method of claim 11, wherein the linker further comprises a second primer site and the method further comprises hybridizing a second primer to the second primer site.

15. The method of claim 14, wherein the amplifying of the portion of the transposon uses both the first primer that is hybridized to the first primer site and the second primer that is hybridized to the second primer site.

16. The method of claim 11, wherein the second barcode sequence of the barcode pair is different from the first barcode sequence of the barcode pair.

17. The method of claim 11, wherein the second barcode sequence comprises a first strand and a second strand that is not complementary to the first strand of the second barcode sequence.

18. The method of claim 11, wherein at least 90% of the barcode pairs in the plurality of transposons are different from each other.

19. A method of sequencing a target nucleic acid, comprising:

(a) contacting a target nucleic acid with a plurality of transposons under conditions such that a plurality of the transposons are inserted into the target nucleic acid,

wherein the transposons that are inserted into the target nucleic acid each comprise a first transposase recognition site, a second transposase recognition site, and a barcode pair,

wherein the barcode pair comprises a first barcode sequence, a second barcode sequence and a linker comprising a fragmentation site disposed between the first and second barcode sequences, wherein the fragmentation site is a ribonucleotide sequence and wherein fragmentation comprises fragmenting the target nucleic acid with a RNAse,

wherein the barcode pair is disposed between the first and second transposase recognition sites;

(b) fragmenting the target nucleic acid at the fragmentation sites thereby preparing a library of template nucleic acid fragments;

(c) obtaining sequence reads from the nucleic acid fragments; and

(d) assembling a representation of at least a portion of the target nucleic acid from the sequence reads, wherein the assembling comprises determining the order of the sequence reads from the identities of the first barcode sequence and the second barcode sequence of each barcode pair.

20. The method of claim 19, wherein the target nucleic acid comprises cDNAs from a single cell.

21. The method of claim 19, wherein the target nucleic acid comprises nucleic acids from a plurality of species.

22. The method of claim 19, wherein the target nucleic acid comprises nucleic acids from a plurality of haplotypes.