Compositions and methods for intramolecular nucleic acid rearrangement

• US 9,410,149 B2
• Filed: 11/06/2015
• Issued: 08/09/2016
• Est. Priority Date: 08/20/2009
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Brenner, Sydney, Mikawa, Gi, Osborne, Robert, Slatter, Andrew
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/935,255
• Publication Number:
  US 20160060693A1
• Time in Patent Office: 277 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.2, 435/91.53, 435/287.2, 422/430, 536/23.1, 506/2, 506/16
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• 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 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Aspects of the present invention are drawn to processes for moving a region of interest in a polynucleotide from a first position to a second position with regard to a domain within the polynucleotide, also referred to as a “reflex method”. In certain embodiments, the reflex method results in moving a region of interest into functional proximity to specific domain elements present in the polynucleotide (e.g., primer sites and/or multiplex identifier). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.

Claims

1. A method of analyzing genomic DNA, comprising:

a) generating a first plurality of different segments of a first nucleic acid molecule of a contiguous region in the genomic DNA, wherein said contiguous region in the genomic DNA is represented by a plurality of nucleic acid molecules, and wherein each of said first plurality of different segments is appended to a first multiplex identifier (MID) sequence;

b) generating a second plurality of different segments of a second nucleic acid molecule of said contiguous region, wherein each of said second plurality of different segments is appended to a second MID sequence and said first MID sequence and said second MID sequence are different; and

c) sequencing the first and second plurality of different segments and the appended first and second MID sequences, respectively, to analyze the genomic DNA.

2. The method of claim 1, wherein said genomic DNA is mammalian genomic DNA.

3. The method of claim 2, wherein said mammalian genomic DNA is human genomic DNA.

4. The method of claim 1, wherein the segments generated in steps a) and b) are in the range of 100 to 5,000 bp in length.

5. The method of claim 1, wherein the segments generated in steps a) and b) are 400 bp or less in length.

6. The method of claim 1, wherein in step a):

the different segments of said first nucleic acid molecule are distributed along said first nucleic acid molecule; and

the different segments of said second nucleic acid molecule are distributed along said second nucleic acid molecule.

7. The method of claim 6, wherein in step b):

the different segments of said first nucleic acid molecule are distributed along said first nucleic acid molecule at intervals in the range of 250 bp to 5 kb; and

the different segments of said second nucleic acid molecule are distributed along said second nucleic acid molecule at intervals in the range of 250 bp to 5 kb.

8. The method of claim 1, wherein following steps a) and b):

substantially all of said first nucleic acid molecule of said contiguous region is represented in said first plurality of segments; and

substantially all of said second nucleic acid molecule of said contiguous region is represented in said second plurality of segments.

9. The method of claim 1, wherein said contiguous region is greater than 2 kb in length.

10. The method of claim 1, wherein said genomic DNA is genomic DNA from a virus or a bacterium.

11. The method of claim 1, further comprising providing a third plurality of different segments of a third nucleic acid molecule of said contiguous region, wherein each of said third plurality of segments is appended to a third MID sequence and said third MID sequence is different from said first and second MID sequences.

12. A method of analyzing nucleic acids, comprising:

a) generating a first plurality of different segments of a first nucleic acid molecule from a contiguous region of genomic DNA, and wherein each of said first plurality of different segments is appended to a first multiplex identifier (MID) sequence;

b) generating a second plurality of different segments of a second nucleic acid molecule from said contiguous region, wherein each of said second plurality of different segments is appended to a second MID sequence and said first MID sequence and said second MID sequence are different,

c) sequencing the first plurality of different segments and appended first MID sequences, and the second plurality of different segments and appended second MID sequences; and

d) identifying the first plurality of different segments as segments of the first nucleic acid molecule from the first MID sequence, and identifying the second plurality of different segments as segments of the second nucleic acid molecule from the second MID sequence.

13. A method of analyzing a plurality of contiguous genomic sequences, comprising:

a) providing a sample comprising the plurality of contiguous genomic sequences;

b) tagging a plurality of fragments of the contiguous genomic sequences with a multiplex identifier (MID) sequence such that fragments of a first contiguous nucleic acid sequence share a common MID sequence that is different from an MID sequence of fragments of a second contiguous nucleic acid sequence; and

c) sequencing the plurality of fragments and MID sequences to identify fragments of the contiguous sequence.