# US 10,246,746 B2 · Preserving genomic connectivity information in fragmented genomic DNA samples

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- Number: US 10,246,746 B2
- Document ID: US10246746B2
- Status: Granted
- Kind: B2
- Application number: 14/572,556
- Filed: 2014-12-16
- Published: 2015-06-25
- Granted: 2019-04-02
- Earliest claimed: 2013-12-20
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Jeffrey S. Fisher; Frank J. Steemers; Sasan Amini; Kevin L. Gunderson
- Web page: https://patentgrep.com/patent/US10246746B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6869: Methods for sequencing
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C12Q 1/6844: Nucleic acid amplification reactions
- G16B 30/20: Sequence assembly
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

A method of sequencing a target nucleic acid polymer by (a) modifying a target nucleic acid polymer to produce a modified nucleic acid polymer; (b) producing fragments of the modified nucleic acid polymer, wherein the fragments are attached to locations on a solid support surface (c) determining nucleotide sequences from the fragments at the locations; and (d) producing a representation of the nucleotide sequence for the target nucleic acid polymer based on the nucleotide sequences from the fragments and the relative distances between the locations on the solid support surface.

## Claims

### Claim 1

A method of sequencing a target nucleic acid polymer, comprising:
(a) modifying a target nucleic acid polymer to produce a modified nucleic acid polymer, wherein the modified nucleic acid polymer comprises a plurality of sequence regions from the target nucleic acid polymer;
(b) releasing fragments of the modified nucleic acid polymer onto a solid support surface, each fragment comprising one of the sequence regions;
(c) capturing the fragments randomly at locations in an area of the solid support surface;
(d) determining nucleotide sequences of the sequence regions by detecting the fragments at the locations;
(e) determining relative physical distances between the fragments at the locations; and
(f) producing a representation of the nucleotide sequence for the target nucleic acid polymer based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between the sequence regions along a given strand of the target nucleic acid polymer.

### Claim 2 (depends on 1)

The method of claim 1, wherein the modifying comprises adding inserts into the target nucleic acid polymer to form the modified nucleic acid polymer, wherein the modified nucleic acid polymer comprises a plurality of internal inserts.

### Claim 3 (depends on 2)

The method of claim 2, wherein the fragments produced in step (b) each comprise at least a portion of an insert added in step (a).

### Claim 4 (depends on 3)

The method of claim 3, wherein the inserts further comprise cleavage sites and wherein producing of the fragments in step (b) comprises cleaving the inserts at the cleavage sites.

### Claim 5 (depends on 2)

The method of claim 2, wherein the inserts have priming sites; wherein the fragments each comprise at least a portion of the inserts having the priming sites; and wherein step (d) comprises determining nucleotide sequences from the fragments by extension of primers hybridized to the priming sites at the locations.

### Claim 6 (depends on 2)

The method of claim 2, wherein the inserts comprise ligands that attach to receptors on the solid support surface.

### Claim 7 (depends on 2)

The method of claim 2, wherein the inserts are added into the target nucleic acid polymer by transposases.

### Claim 8 (depends on 7)

The method of claim 7, wherein each of the inserts comprise a first transposon element and a second transposon element that is contiguous with the first transposon element; and wherein a transposase associated with the first transposon element and the second transposon element forms a transposome.

### Claim 9 (depends on 8)

The method of claim 8, wherein the 5′ end of the first transposon element is attached to the 5′ end of the second transposon element by a linker.

### Claim 10 (depends on 8)

The method of claim 8, wherein the first transposon element and the second transposon element each comprise a forked adapter.

### Claim 11 (depends on 8)

The method of claim 8, wherein the transposases comprise ligands and the surface comprises receptors that bind to the ligands to attach the modified nucleic acid polymer to the surface prior to the producing of the fragments.

### Claim 12 (depends on 7)

The method of claim 7, wherein step (b) comprises attaching the modified nucleic acid polymer to the solid support surface prior to the producing of the fragments.

### Claim 13 (depends on 12)

The method of claim 12, wherein the transposases are removed from the modified nucleic acid polymer prior to the attaching of the modified nucleic acid polymer to the solid support surface.

### Claim 14 (depends on 12)

The method of claim 12, wherein the transposases are removed from the modified nucleic acid polymer after the attaching of the modified nucleic acid polymer to the solid support surface.

### Claim 15 (depends on 14)

The method of claim 14, wherein the modified nucleic acid polymer is attached to the solid support surface via binding of at least one transposase to the surface.

### Claim 16 (depends on 1)

The method of claim 1, wherein the determining of nucleotide sequences comprises nanopore sequencing.

### Claim 17 (depends on 1)

The method of claim 1, wherein step (a) comprises modifying a plurality of different target nucleic acid polymers to produce a mixture of modified nucleic acid polymers, wherein each of the modified nucleic acid polymers comprises a plurality of sequence regions from an individual target nucleic acid polymer of the plurality of different target nucleic acid polymers.

### Claim 18 (depends on 17)

The method of claim 17, wherein step (e) comprises producing representations of the nucleotide sequences for the target nucleic acid polymers based on the nucleotide sequences from the fragments and the relative distances between the locations on the solid support surface.

### Claim 19 (depends on 1)

The method of claim 1, wherein the solid support surface comprises an interior surface of a flow cell.

### Claim 20 (depends on 1)

The method of claim 1, further comprising determining haplotype phase for polymorphisms occurring in the nucleotide sequences for different fragments released from the modified nucleic acid polymer.

### Claim 21

A method of sequencing a target nucleic acid polymer, comprising:
(a) modifying a target nucleic acid polymer to produce a modified nucleic acid polymer, wherein the modified nucleic acid polymer comprises a plurality of sequence regions from the target nucleic acid polymer;
(b) attaching the modified nucleic acid polymer to an area on a solid support surface;
(c) producing fragments of the modified nucleic acid polymer that is attached to the solid support surface, wherein the fragments are attached to locations at the area on the solid support surface;
(d) determining nucleotide sequences from the fragments by detecting the fragments at the locations;
(e) determining relative physical distances between the fragments at the locations; and
(f) producing a representation of the nucleotide sequence for the target nucleic acid polymers based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between the sequence regions along a given strand of the target nucleic acid polymer.

### Claim 22

A method of sequencing a target nucleic acid polymer, comprising:
(a) adding inserts into a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts;
(b) producing fragments of the modified nucleic acid polymer in a fluid that is in contact with a solid support surface, thereby releasing fragments that each comprise at least a portion of the inserts having the priming sites;
(c) capturing the fragments from the fluid randomly at locations on the solid support surface;
(d) determining nucleotide sequences from the fragments by detecting the fragments at the locations;
(e) determining relative physical distances between the fragments at the locations; and
(f) producing a representation of the nucleotide sequence for the target nucleic acid polymer based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between sequence regions along a given strand of the target nucleic acid polymer.

### Claim 23

A method of sequencing a target nucleic acid polymer, comprising:
(a) adding inserts into a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts, wherein the inserts have priming sites;
(b) producing fragments of the modified nucleic acid polymer in a fluid that is in contact with the solid support surface, thereby releasing fragments that each comprise at least a portion of the inserts having the priming sites;
(c) capturing the fragments from the fluid randomly at locations on a solid support surface;
(d) determining nucleotide sequences from the fragments by extension of primers hybridized to the priming sites at the locations;
(e) determining relative physical distances between the fragments at the locations; and
(f) producing a representation of the nucleotide sequence for the target nucleic acid polymer based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between sequence regions along a given strand of the target nucleic acid polymer.

### Claim 24

A method of sequencing a target nucleic acid polymer, comprising:
(a) adding inserts into a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts;
(b) attaching the modified nucleic acid polymer to a solid support surface;
(c) producing fragments of the modified nucleic acid polymer that is attached to the solid support surface, wherein the fragments are attached to locations on the solid support surface and wherein the fragments each comprise at least a portion of the inserts;
(d) determining nucleotide sequences from the fragments by detecting the fragments at the locations;
(e) determining relative physical distances between the fragments at the locations; and
(f) producing a representation of the nucleotide sequence for the target nucleic acid polymer based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between sequence regions along a given strand of the target nucleic acid polymer.

### Claim 25

A method of determining the source of individual sequences in a mixture of sequences from different sources, comprising:
(a) providing a mixture of target nucleic acid polymers from a plurality of different sources;
(b) modifying the mixture of target nucleic acid polymers to produce a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers comprises a plurality of sequence regions from the different sources;
(c) producing fragments of the modified nucleic acid polymers in a vessel having a solid support surface, each fragment comprising a sequence region from a single one of the different sources;
(d) capturing the fragments randomly at locations of the solid support surface, under conditions wherein fragments from a common target nucleic acid polymer preferentially localize to proximal locations on the solid support surface;
(e) determining nucleotide sequences of the fragments at the locations;
(f) determining relative physical distances between the fragments at the locations; and
(g) identifying the nucleotide sequences from the mixture of target nucleic acid polymers that are derived from a common source from the plurality of different sources based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between the sequence regions along a given strand of a target nucleic acid polymer from the mixture of target nucleic acid polymers.

### Claim 26

A method of determining the source for individual sequences in a mixture of sequences from different sources, comprising:
(a) providing a mixture of target nucleic acid polymers from a plurality of different sources;
(b) modifying the mixture of target nucleic acid polymers to produce a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers comprises a plurality of sequence regions from the different sources;
(c) attaching the modified nucleic acid polymers to a solid support surface;
(d) producing fragments of the modified nucleic acid polymers that are attached to the solid support surface, wherein fragments from a common source of the plurality of sources are attached to locations that are proximal on the solid support surface;
(e) determining nucleotide sequences of the fragments at the locations;
(f) determining relative physical distances between the fragments at the locations; and
(g) identifying the nucleotide sequences from the mixture of target nucleic acid polymers that are derived from a common source from the plurality of different sources based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between the sequence regions along a given strand of a target nucleic acid polymer from the mixture of target nucleic acid polymers.

### Claim 27

A method of determining the source for individual sequences in a mixture of sequences from different sources, comprising:
(a) providing a mixture of target nucleic acid polymers from a plurality of different sources;
(b) adding inserts into the target nucleic acid polymers in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts;
(c) producing fragments of the modified nucleic acid polymers in a fluid that is in contact with a solid support surface, thereby releasing fragments that each include at least a portion of each of the inserts;
(d) capturing the fragments from the fluid randomly at locations on the solid support surface;
(e) determining nucleotide sequences from the fragments by detecting the fragments at the locations;
(f) determining relative physical distances between the fragments at the locations;
(g) identifying the nucleotide sequences from the mixture of target nucleic acid polymers that are derived from a common source from the plurality of different sources based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between sequence regions along a given strand of a target nucleic acid polymer from the mixture of target nucleic acid polymers.

### Claim 28

A method of determining the source for individual sequences in a mixture of sequences from different sources, comprising:
(a) providing a mixture of target nucleic acid polymers from a plurality of different sources;
(b) adding inserts into the target nucleic acid polymers in the mixture to form a mixture of modified nucleic acid polymers, each polymer comprising a plurality of internal inserts;
(c) attaching the modified nucleic acid polymer to a solid support surface;
(d) producing fragments of the modified nucleic acid polymers that are attached to the solid support surface, wherein fragments from a common source of the plurality of sources are attached to locations that are proximal on the solid support surface;
(e) determining nucleotide sequences from the fragments by detecting the fragments at the locations;
(f) determining relative physical distances between the fragments at the locations; and
(g) identifying the nucleotide sequences from the mixture of target nucleic acid polymers that are derived from a common source from the plurality of different sources based on the nucleotide sequences from the fragments and the determined relative physical distances between the locations on the solid support surface, wherein the relative physical distances are indicative of distances between sequence regions along a given strand of a target nucleic acid polymer from the mixture of target nucleic acid polymers.

### Claim 29 (depends on 1)

The method of claim 1, wherein step (f) comprises determining that two of the fragments are derived from a common source based on the relative physical distances.

### Claim 30 (depends on 29)

The method of claim 29, wherein step (f) comprises determining that the two fragments derived from the common source have closer relative physical distances than other fragments that are not derived from the common source.

### Claim 31 (depends on 1)

The method of claim 1, wherein step (e) comprises generating a map of clusters at the locations.

### Claim 32 (depends on 1)

The method of claim 1, wherein step (f) comprises determining the distances between fragments in a plurality of overlapping fragments.

### Claim 33 (depends on 32)

The method of claim 32, wherein step (f) comprises determining that overlapping fragments are derived from different common sources based on determined relative distances between the plurality of overlapping fragments.

### Claim 34 (depends on 21)

The method of claim 21, wherein step (f) comprises determining that two of the fragments are derived from a common source based on the determined relative physical distances and comparison of the nucleotide sequences of the two fragments with a reference sequence.

### Claim 35 (depends on 1)

The method of claim 1, wherein the distances are genomic distances.

### Claim 36 (depends on 1)

The method of claim 1, wherein the fragments of the modified nucleic acid polymer are closer in relative physical distance to one another than other fragments of another modified nucleic acid polymer captured on the solid support surface.