ARRAY-BASED TRANSLOCATION AND REARRANGEMENT ASSAYS

• US 20090239764A1
• Filed: 03/11/2009
• Published: 09/24/2009
• Est. Priority Date: 03/11/2008
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Sparks, Andrew, Jones, Keith W., Fu, Glenn K., Shapero, Michael H.
• Granted Patent: US 9,074,244 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2539/101 Subtraction analysis
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

Methods for detecting genomic rearrangements are provided. In one embodiment, methods are provided for the use of paired end tags from restriction fragments to detect genomic rearrangements. Sequences from the ends of the fragments are brought together to form ditags and the ditags are detected. Combinations of ditags are detected by an on-chip sequencing strategy that is described herein, using inosine for de novo sequencing of short segments of DNA. In another aspect, translocations are identified by using target specific capture and analysis of the captured products on a tiling array.

Claims

1. A method for detecting a chromosomal translocation in a sample from an individual comprising:

(a) obtaining a pool of capture probes consisting of a plurality of DNA fragments complementary to a first chromosome of interest;

(b) obtaining a tester sample preparation comprising DNA fragments from the sample flanked by common priming sequences;

(c) combining the tester sample preparation with the pool of capture probes to allow specific hybridization of the capture probes to complementary fragments in the tester sample preparation and thereby forming complexes;

(d) capturing complexes formed between capture probes and tester fragments in (c);

(e) recovering the tester fragments captured in (d) and amplifying the recovered tester fragments;

(f) amplifying the recovered tester fragments using primers to the common priming sequences;

(g) hybridizing the amplified fragments to a tiling array comprising a plurality of probes to said first chromosome of interest and a plurality of probes to one or more second chromosomes to obtain a hybridization pattern; and

(h) analyzing the hybridization pattern wherein the presence of hybridization to probes to one of said second chromosomes is indicative of a chromosomal translocation between the first chromosome and one of said second chromosomes.

2. The method of claim 1 wherein the individual is a human.

3. The method of claim 2 wherein the array comprises a plurality of probes to each human chromosome.

4. The method of claim 3 where said probes to each human chromosome are spaced over the entirety of each human chromosome, excluding the centromere region, at an average spacing of about 5,000 basepairs.

5. The method of claim 1 wherein the pool of capture probes further consists of a plurality of probes from at least one additional chromosome.

6. The method of claim 1 wherein the tester sample preparation is obtained by fragmenting the sample with one or more restriction enzymes, ligating an adapter sequence to the ends generated by fragmentation and amplification of the adapter-ligated fragments by PCR using a primer complementary to the adapter sequence.

7. The method of claim 1 wherein the tester sample preparation is obtained by fragmenting the sample by a method selected from shearing, chemical fragmentation or sonication.

8. A method for analysis of genomic rearrangements in a sample comprising:

(a) digesting the sample with a selected restriction enzyme to obtain restriction fragments that have a first end tag and a second end tag flanking a central portion, wherein the sequences of the end tags in the genome can be determined by using a computer to identify the sequence surrounding each restriction site for said selected restriction enzyme;

(b) ligating the fragments to a common backbone fragment to form first circular molecules comprising restriction fragments and the backbone, wherein the common backbone fragment has a first type IIS restriction enzyme recognition site at its first end and a second type IIS restriction enzyme recognition site at its second end;

(c) cleaving the first circular molecule using a type IIS restriction enzyme to form a first fragment comprising the backbone fragment flanked by the first end tag and the second end tag and a second fragment containing the central portion of the restriction fragment;

(d) ligating the ends of the first fragment to form second circular molecules, wherein the ends of said first end tag and said second end tag are ligated together to form a ditag;

(e) amplifying at least a portion of the second circular molecule using a pair of primers complementary to said backbone to obtain amplification target comprising said ditag flanked by priming sites;

(f) hybridizing the amplification target to an array comprising a plurality of ditag sequencing probes, wherein said plurality comprises probes for each end tag sequence, wherein ditags hybridize to probes that are complementary to a first end tag in the ditag; and

,

(g) determining at least a partial sequence of the second end tag in a plurality of the ditags, wherein the presence in a ditag of two non-neighboring end tags indicates a genomic rearrangement.

9. The method of claim 8 wherein the partial sequence is at least 5 bases.

10. The method of claim 8 wherein the partial sequence is 6 bases.

11. The method of claim 8 wherein the partial sequence is at least 6 bases.

12. The method of claim 8 wherein the first type IIS restriction enzyme and the second type IIS restriction enzyme are the same type IIS restriction enzyme.

13. The method of claim 12 wherein the type IIS restriction enzyme is MmeI.

14. The method of claim 8 wherein the first end tag and the second end tag are each between 10 and 20 bases in length.

15. The method of claim 8 wherein the first end tag and the second end tag are each between 18 and 27 bases in length.

16. The method of claim 8 wherein for each base to be sequenced in the second end tag the array comprises a probe comprising the complement of the first end tag and between 0 and 5 inosines.

17. A ditag sequencing array, said array comprising:

(a) a plurality of ditag sequencing probe sets, wherein each probe in a ditag sequencing probe set is complementary to the same end tag in a collection of end tags, wherein said collection of end tags is determined by selecting a restriction enzyme, using a computer to identify the to 30 bases immediately adjacent to each recognition site for said restriction enzyme in a selected sequence or collection of sequences, wherein the probe set comprises at least 4 different probes, present at different features of the array, wherein the probes have different numbers of inosines at the ends of the probes.

18. The array of claim 17 wherein the array comprises at least 100,000 different ditag sequencing probe sets.

19. The array of claim 18 wherein each probe set comprises a first probe with 0 inosines, a second probe with 1 inosine, a third probe with 2 inosines, a fourth probe with 3 inosines, a fifth probe with 4 inosines and a sixth probe with 5 inosines.

20. A method for detecting genomic rearrangements comprising:

(a) fragmenting a genomic sample with a restriction enzyme to obtain restriction fragments each having a first terminal sequence and a second terminal sequence, said first and second terminal sequences being the sequences immediately adjacent to the cleavage site of the restriction enzyme;

(b) ligating the restriction fragments each to a common backbone fragment, wherein said common backbone fragment includes a first and a second recognition site for a type IIs restriction enzyme, thereby forming first circular molecules each comprising said backbone fragment and one of said restriction fragments;

(c) cutting said first circular fragments with said type IIs restriction enzyme to obtain ditag fragments comprising at least 10 bases of said first terminal sequence, at least 10 bases of said second terminal sequence and said common backbone fragment;

(d) ligating the ends of said ditag fragments to form second circular molecules;

(e) amplifying a portion of the second circular molecules wherein said portion includes the at least 10 bases of said first terminal sequence and at least 10 bases of said second terminal sequence, to obtain an amplification product;

(f) analyzing said amplification product to identify a plurality of combinations of first and second terminal sequences present in the same fragment;

(g) comparing the results of (f) to expected combinations of first and second terminal sequences, to identify combinations that are not expected, wherein the combinations that are not expected are indicative of genomic rearrangements.

21. The method of claim 20 wherein the expected combinations used in step (g) are from a database of ditag combinations predicted from in silico digestion of a genome.