TAGGED-FRAGMENT MAP ASSEMBLY

• US 20100261285A1
• Filed: 03/26/2010
• Published: 10/14/2010
• Est. Priority Date: 03/27/2009
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Goldstein, Peter, Preparata, Franco, Upfal, Eli
• Granted Patent: US 8,455,260 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• 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 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 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: Y10T 436/14 Heterocyclic carbon compound [i.e. , O, S, N, Se, Te, as only ring hetero atom]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for determining a sequence of a biomolecule, the method including binding a plurality of uniform probes to a biomolecule fragment, creating a collection of binding signatures for the fragment with each binding signature representing a series of distances between binding sites within the fragment, and grouping the binding signatures into a plurality of signature clusters based at least in part on distances between the binding sites in each binding signature. For each binding signature in a first cluster, a potential successor binding signature is selected from signature clusters other than the first signature cluster, and one of the potential successor binding signatures is identified as a successor binding signature. The last two steps are repeated until the successor signature represents a terminal signature, resulting in a sequence of signatures representing at least a portion of the biomolecule.

Claims

1. A method for determining a sequence of a biomolecule, the method comprising the steps of:

a) creating a biomolecule fragment from the biomolecule;

b) binding a plurality of uniform probes to the biomolecule fragment;

c) identifying a plurality of binding sites along the biomolecule fragment at which the probes bind to the biomolecule;

d) creating a collection of binding signatures for the biomolecule fragment, each binding signature representing a series of distances between binding sites within the fragment;

e) grouping the binding signatures into a plurality of signature clusters based at least in part on distances between the binding sites in each binding signature;

f) for each binding signature in a first cluster, selecting a potential successor binding signature from signature clusters other than the first signature cluster;

g) identifying one of the potential successor binding signatures as a successor binding signature;

h) repeating steps f) and g) until the successor signature represents a terminal signature, resulting in a sequence of signatures representing at least a portion of the biomolecule.

2. The method of claim 1 further comprising measuring the distances between the binding sites within the fragment.

3. The method of claim 1 further comprising assigning an error to each measured distance between binding sites.

4. The method of claim 2 wherein the distances are measured based on a number of base pairs between the binding sites.

5. The method of claim 2 wherein measuring the distances between the binding sites further comprises measuring electrical signals emitted by the fragment as the fragment passes through a nanopore.

6. The method of claim 1 further comprising ordering the signatures based on one or more characteristics of the signatures.

7. The method of claim 6 wherein the characteristics of the signatures comprise a total length of the signature.

8. The method of claim 1 wherein the biomolecule comprises a single-stranded DNA molecule.

9. The method of claim 1 wherein the biomolecule comprises a double-stranded DNA molecule.

10. The method of claim 1 wherein the signature clusters are determined based on a degree of match between the binding sites attributed to the binding signatures.

11. The method of claim 1, wherein the probes are selected from the group consisting of 6-mer probes, 7-mer probes, 8-mer probes and 9-mer probes.

12. A system for determining a sequence of a biomolecule, the system comprising:

a) an identifying module that identifies a plurality of binding sites along a fragment of the biomolecule at which a plurality of uniform probes are bound;

b) a signature-creating module that creates a collection of binding signatures for the fragment, each binding signature representing a series of distances between binding sites within the fragment;

c) a grouping module that groups the binding signatures into a plurality of signature clusters based at least in part on distances between the binding sites in each binding signature;

d) a selecting module that selects, for each binding signature in a first cluster, a potential successor binding signature from signature clusters other than the first signature cluster; and

e) a sequencing module that (i) identifies one of the potential successor binding signatures as a successor binding signature, and (ii) determines the sequence of at least a portion of the biomolecule based at least in part on a series of identified successor binding signatures.

13. An article of manufacture having computer-readable program portions embodied thereon for sequencing a biomolecule, the article comprising computer-readable instructions for:

a) creating a biomolecule fragment from the biomolecule;

b) binding a plurality of uniform probes to the biomolecule fragment;

c) identifying a plurality of binding sites along the biomolecule fragment at which the probes bind to the biomolecule;

d) creating a collection of binding signatures for the biomolecule fragment, each binding signature representing a series of distances between binding sites within the fragment;

e) grouping the binding signatures into a plurality of signature clusters based at least in part on distances between the binding sites in each binding signature;

f) for each binding signature in a first cluster, selecting a potential successor binding signature from signature clusters other than the first signature cluster;

g) identifying one of the potential successor binding signatures as a successor binding signature;

h) repeating steps f) and g) until the successor signature represents a terminal signature, resulting in a sequence of signatures representing at least a portion of the biomolecule.

14. An apparatus for sequencing a biomolecule, the apparatus comprising:

a memory for storing code that defines a set of instructions; and

a processor for executing the set of instructions to;

(i) identify a plurality of binding sites along a fragment of the biomolecule at which a plurality of uniform probes are bound;

(ii) create a collection of binding signatures for the fragment, each binding signature representing a series of distances between binding sites within the fragment;

(iii) group the binding signatures into a plurality of signature clusters based at least in part on distances between the binding sites in each binding signature;

(iv) select, for each binding signature in a first cluster, a potential successor binding signature from signature clusters other than the first signature cluster;

(v) identify one of the potential successor binding signatures as a successor binding signature; and

(vi) determine the sequence of at least a portion of the biomolecule based at least in part on a series of identified successor binding signatures.