Ditag genome scanning technology

• US 20090137402A1
• Filed: 10/11/2007
• Published: 05/28/2009
• Est. Priority Date: 10/11/2006
• Status: Abandoned Application

• Current Assignee: Northshore University Healthsystem (Advocate Aurora Health, Inc.)
• Original Assignee: Northshore University Healthsystem (Advocate Aurora Health, Inc.)
• Inventors: Chen, Jun, Wang, San Ming, Kim, Yeong Cheol
• Application Number:
  US11/907,404
• Publication Number:
  US 20090137402A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics

Abstract

The present invention provides for a method for analyzing large genomes using a process by where the genomic DNA is digested by a small base pair restriction enzyme. The fragments are then cloned and a unique ta-vector-tag is created. The tag-vector-tag fragments are purified and re-ligated to create a “ditag” library, which are then sequenced. In the final step, the sequenced ditags can be mapped back to the genome using software containing mapping algorithms and a unique ditag reference database to provide a method for scanning large portions of the genome in a reduced amount of time and cost.

Claims

1. A system for collecting genetic information using DNA sequences comprising the steps of:

1) collecting two short tags from both ends of DNA fragments to form a ditag;

2) using the 454 sequencing system for maximal collection of ditags at the genome scale;

3) identifying the DNA fragments in the human genome sequences that originated the ditags and identify the DNA fragments that are different from those in the reference human genome;

4) confirming the mapping results by using the ditag sequences directly as the sense and antisense primers in a PCR expansion to detect the original DNA fragments; and

5) performing computational and experimental analysis of DGS results.

2. A method for determining of the genome origin of a Ditag through the Ditagmap reference database comprising the steps of:

a. dividing the identified ditags into three groups, and classifying as mapped ditags, those ditags having been identified with reference ditags in a one to one correspondence, and with mismatches up to two bases, of which the p values are higher than the cutoff of 1.0e<sup>−

5</sup>;

classifying as trouble-mapped ditags, those identified ditags of which the combined p values of mapping two single tags in reference ditag database are higher than the cutoff of 1.0e<sup>−

3</sup>, or, any single tag mapping p value is larger than 1.0e<sup>−

3</sup>, which allows at most one mismatch with reference tags; and

classifying as unmapped ditags, those ditags having p values that are less than the cutoff 1.0e<sup>−

3</sup> when their two single tags are mapped to reference ditag database;

b. selecting a reference ditag having a 32-bp tag from the 5′

end and a 32-bp tag from the 3′

end of a virtual DNA fragment;

c. searching the DitagMap reference database for the experimental ditag, by comparing experimental ditags having a sequence shorter than 32 bp with reference ditags of the same length, and counting total mismatches without allowing gaps, wherein ditags having a sequence longer than 31 bp are compared with reference ditags with extra bases, such that the 16-bp in both ends of the longer ditags are aligned with the ends of each reference ditag, then the extra bases between the two 16-bp are compared with the bases in the reference ditag, and those bases with matches are assigned to the corresponding single tag;

d. identifying length of experimental ditag and the mismatches with each reference ditag;

e. calculating the probability of an experimental ditag/tag (w_ob) mapping in the reference database is calculated by using the formula;

p-score=

3. The method of claim 2, wherein the identifying the genomic origin of at least one ditag represents a genomic variation between individuals.

4. A method for producing and collecting ditag sequence information comprising the following steps:

a) obtaining a genomic DNA sample;

b) fragmenting the genomic DNA sample by restriction enzyme digestion;

c) cloning the DNA fragments generated in step b) into plasmid vectors to generate a genomic DNA library;

d) digesting the library using the restriction enzyme MmeI such that two short tags are retained on each site of the cloned DNA fragment in the same plasmid vector in a tag-vector-tag orientation;

e) religating the tag-vector tag fragments to form a ditag;

f) releasing the ditags formed in step e) from the vectors by digestion with a restriction enzyme;

g) concatemerizing the individual ditags having a suitable length for sequencing;

h) sequencing the concatemerized ditags using a 454 sequencing system;

i) extracting the ditags from the sequences based on the identification of their restriction sites;

j) mapping the ditags extracted from step i) to a reference ditag database where restriction fragments of known reference genome sequences are stored;

k) determining whether the ditag has a counterpart in the reference ditag database and identifying those ditags which have counterpart sequences to mapped; and

l) identifying the ditags which do not have a counterpart in the reference ditag database as trouble-mapped ditags.