# US 8,771,958 B2 · Nucleotide sequence from amplicon subfragments

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- Number: US 8,771,958 B2
- Document ID: US8771958B2
- Status: Granted
- Kind: B2
- Application number: 13/975,234
- Filed: 2013-08-23
- Published: 2013-12-26
- Granted: 2014-07-08
- Earliest claimed: 2005-06-15
- Assignees: Callida Genomics, Inc.
- Applicants: Callida Genomics, Inc.
- Inventors: Radoje Drmanac
- Web page: https://patentgrep.com/patent/US8771958B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07K 1/047: {Simultaneous synthesis of different peptide species; Peptide libraries}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/682: Signal amplification
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6869: Methods for sequencing
- G01N 15/1404: Handling flow, e.g. hydrodynamic focusing
- G01N 15/1434: Optical arrangements
- C12Q 2525/151: repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
- C12Q 2525/313: Branched oligonucleotides
- C12Q 2531/125: Rolling circle
- C12Q 2565/513: characterised by the pattern of the arrayed oligonucleotides
- Y10S 977/778: within specified host or matrix material, e.g. nanocomposite films
- Y10S 977/789: in array format
- Y10S 977/792: Nucleic acid array, e.g. human genome array
- Y10S 977/88: with arrangement, process, or apparatus for testing
- Y10S 977/882: Assembling of separate components, e.g. by attaching
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.

## Claims

### Claim 1

A method of characterizing nucleotide sequences of one or more target polynucleotides, the method comprising the steps of:
providing a plurality of aliquots, at least some of which comprise one or more fragments of the target polynucleotide(s);
amplifying the fragments in the aliquots to produce first amplicons;
randomly fragmenting the amplicons in the aliquots to produce subfragments;
incorporating oliqonucleotide tags of known sequence into at least some of the subfragments in at least some of the aliquots, whereby the tag identifies the aliquot;
obtaining sequence reads of the target polynucleotides and the oliqonucleotide tag of at least some of the subfragments; and
assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s), wherein the assembling comprises grouping the sequence reads according to the sequence of the oliqonucleotide tags.

### Claim 2 (depends on 1)

The method of claim 1, wherein any given portion of the target polynucleotide(s) is represented by a single non-overlapping fragment in sixty percent or more of the aliquots containing such portion.

### Claim 3 (depends on 1)

The method of claim 1, wherein at least sixty percent of the aliquots containing more than one fragment contain only non-overlapping fragments.

### Claim 4 (depends on 1)

The method of claim 1, wherein at least ninety percent of the aliquots containing more than one fragment contain only non-overlapping fragments.

### Claim 5 (depends on 1)

The method of claim 1, comprising pooling tagged subfragments from a plurality of the aliquots before obtaining the sequence reads.

### Claim 6 (depends on 1)

The method of claim 1, wherein the step of obtaining nucleic acid sequence reads comprises:
amplifying the subfragments to produce second amplicons;
forming a regular array in which the second amplicons are fixed to a surface; and
obtaining sequence reads from the second amplicons on the array.

### Claim 7 (depends on 6)

The method of claim 6, wherein the second amplicons are fixed to the surface in a rectilinear or hexagonal pattern.

### Claim 8 (depends on 1)

The method of claim 1, wherein providing the plurality of aliquots comprises fragmenting and amplifying genomic DNA from a sample, and then separating the fragmented and amplified genomic DNA into the aliquots.