Design and selection of genetic targets for sequence resolved organism detection and identification

• US 7,668,664 B2
• Filed: 08/22/2007
• Issued: 02/23/2010
• Est. Priority Date: 07/02/2004
• Status: Expired due to Fees

• Current Assignee: The Government Of The United States As Represented By The Secretary Of The Navy
• Original Assignee: the united states of america as represented by the secretary of the navy
• Inventors: Wang, Zheng, Malanoski, Anthony P., Lin, Baochuan, Stenger, David A, Schnur, Joel M
• Primary Examiner(s): Smith; Carolyn L.
• Application Number:
  US11/843,126
• Publication Number:
  US 20080033706A1
• Time in Patent Office: 916 Days
• Field of Search: None
• US Class Current: 702/20
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

A computer-implemented method as follows. Providing a list of target sequences associated with one or more organisms. Providing a list of candidate prototype sequences suspected of hybridizing to one or more of the target sequences. Generating a collection of probes corresponding to each candidate prototype sequence, each collection of probes having a set of probes for every subsequence. The sets consist of the corresponding subsequence and every variation of the corresponding subsequence formed by varying a center nucleotide of the corresponding subsequence. Generating a set of fragments corresponding to each target sequence. Calculating the binding free energy of each fragment with a perfect complimentary sequence of the fragment. Determining which extended fragments are perfect matches to any of the probes. Assembling a base call sequence corresponding to each candidate prototype sequence.

Claims

1. A computer-implemented method comprising:

providing a list of target sequences associated with one or more organisms in a list of organisms;

providing a list of candidate prototype sequences suspected of hybridizing to one or more of the target sequences;

generating a collection of probes corresponding to each candidate prototype sequence, each collection of probes comprising a set of probes for every subsequence having a predetermined, fixed subsequence length of the corresponding candidate prototype sequence, the set consisting of the corresponding subsequence and every variation of the corresponding subsequence formed by varying a center nucleotide of the corresponding subsequence;

generating a set of fragments corresponding to each target sequence, each set of fragments comprising every fragment having a predetermined, fixed fragment length of the corresponding target sequence;

calculating the binding free energy of each fragment with a perfect complementary sequence of the fragment, and if any binding free energy is above a predetermined, fixed threshold, the fragment is extended one nucleotide at a time until the binding free energy is below the threshold or the fragment is the same length as the probes corresponding to the corresponding target sequence, generating a set of extended fragments; and

determining which extended fragments are perfect matches to any of the probes; and

assembling a base call sequence corresponding to each candidate prototype sequence comprising;

a base call corresponding to the center nucleotide of each probe of the corresponding prototype sequence that is a perfect match to any extended fragment, but for which the other members of the set of probes containing the perfect match probe are not perfect matches to any extended fragment; and

a non-base call in all other circumstances;

wherein the method is at least partly performed using a suitably programmed computer.

2. The method of claim 1, wherein a subset of the target sequences corresponds to a single gene in common to a subset of the organisms.

3. The method of claim 1, wherein the list of organisms comprises a plurality of strains of a single species.

4. The method of claim 1, further comprising:

selecting a nominal target sequence known to be found in at least one of the organisms; and

performing a similarity search against a database of known sequences to add additional target sequences having at least a predetermined degree of similarity to the nominal target sequence;

wherein the list of candidate prototype sequences is identical to the list of target sequences.

5. The method of claim 1, wherein the subsequence length is 25.

6. The method of claim 1, wherein the fragment length is 13.

7. The method of claim 1, wherein the binding free energy is calculated according to an oligonucleotide nearest neighbor model.

8. The method of claim 1, wherein the binding free energy threshold is about −

14.5 kcal/mol.

9. The method of claim 1, further comprising:

generating for each candidate prototype sequence a list of matching organisms containing that candidate prototype sequence for which the base call sequence corresponding to that candidate prototype sequence comprises a fixed minimum number of base calls;

moving the candidate prototype sequence corresponding to the longest list of matching organisms to a list of final prototype sequences;

removing the matching organisms corresponding to the moved prototype sequence from the list of organisms; and

repeating the moving and removing until the list of organisms is empty.

10. The method of claim 9, wherein the minimum number of base calls is 50.

11. The method of claim 9, further comprising:

fabricating a resequencing microarray containing each set of probes corresponding to each final prototype sequence.

12. The method of claim 11, wherein the microarray further comprises a sequence complementary to each probe on the microarray.