3-D GENOMIC REGION OF INTEREST SEQUENCING STRATEGIES

• US 20130183672A1
• Filed: 07/08/2011
• Published: 07/18/2013
• Est. Priority Date: 07/09/2010
• Status: Active Application

• Current Assignee: Cergentis B.V. (Solvias AG)
• Original Assignee: Cergentis B.V. (Solvias AG)
• Inventors: de Laat, Wouter Leonard, van Min, Max Jan
• Application Number:
  US13/807,662
• Publication Number:
  US 20130183672A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The invention relates to methods for determining the sequence of a genomic region of interest comprising a target nucleotide sequence comprising, fragmenting a crosslinked DNA, ligating the fragmented cross linked DNA, reversing the crosslinking and determining at least part of the sequences of ligated DNA fragments which comprise a target nucleotide sequence.

Claims

1. A method for determining the sequence of a genomic region of interest comprising a target nucleotide sequence the method comprising:

fragmenting a crosslinked DNA,

ligating the fragmented cross linked DNA,

reversing the crosslinking

determining at least part of the sequences of ligated DNA fragments that comprise a target nucleotide sequence, and

utilizing the determined sequences to build a sequence of the genomic region of interest.

15. The method according to claim 1, wherein in case the ploidy in a cell of a genomic region of interest is greater than 1, for each ploidy a contig is built in step i).

2. A method for determining a genomic region of interest'"'"'s sequence, said genomic region of interest comprising a target nucleotide sequence, the method comprising the steps of:

a) providing a sample of cross-linked DNA;

b) fragmenting the crosslinked DNA;

c) ligating the fragmented crosslinked DNA;

d) reversing the crosslinking;

e) optionally fragmenting the DNA of step d);

f) optionally, ligating the fragmented DNA of step d) or e) to at least one Adaptor;

g) optionally amplifying the ligated DNA fragments of step d) or e) comprising the target nucleotide sequence utilizing at least one primer that hybridises to the target nucleotide sequence, or amplifying the ligated DNA fragments of step f) using utilizing at least one primer that hybridises to the target nucleotide sequence and at least one primer that hybridises to the at least one adaptor;

h) determining at least part of the sequence of the (amplified) ligated DNA fragments of step d), e), f) or g) comprising the target nucleotide sequence;

i) building a contig of the genomic region of interest from the determined sequences.

4. The method for determining the sequence of a genomic region of interest according to claim 2, wherein the genomic region of interest comprises one or more target nucleotide sequences in addition, and wherein in the amplification step (g) a primer is provided that hybridises with the target nucleotide sequence and one or more primers are provided for the corresponding one or more additional target nucleotides, wherein the ligated DNA fragments are amplified, or circularized DNA is amplified, utilizing the primers.

5. The method according to claim 2, wherein the fragmenting step b) comprises sonication, followed by enzymatic DNA end repair.

6. The method according to claim 2, wherein the fragmenting step b) comprises fragmenting with a restriction enzyme.

7. The method according to claim 5, wherein the ligation step c) is performed in the presence of an adaptor, ligating adaptor sequences in between fragments.

8. The method according to claim 6, wherein in step b) a plurality of subsamples is processed, and for each subsample restriction enzymes with different recognition sites are used.

9. The method according to claim 8, wherein the fragmenting step e) comprises a restriction enzyme with a recognition sequence that is longer than the recognition sequence of the restriction enzyme of step b).

10. The method according to claim 2, wherein the sequences of multiple genomic regions of interests are determined.

11. The method according to claim 2, wherein an identifier is included in at least one of the oligonucleotide primers of step g).

12. The method according to claim 2, wherein prior to or after the amplification step g) a size selection step is performed.

13. The method according to claim 12, wherein the size selection step is performed utilizing gel extraction chromatography, gel electrophoresis or density gradient centrifugation.

14. The method according to claim 12, wherein DNA is selected of a size between 20-20,0000 base pairs.

16. The method according to claim 2, wherein the step i) of building a contig comprises the steps of:

identifying the fragments of step b);

assigning the fragments to a genomic region; and

building a contig for the genomic region.

17. The method according to claim 16, wherein the step of assigning the fragments to a genomic region comprises identifying the different ligation products of step f) and coupling of the different ligation products to the identified fragments.

18. The method for identifying the presence or absence of a genetic mutation of claim 2, wherein contigs are built for a plurality of samples, comprising the further steps of:

j) aligning the contigs of a plurality of samples; and

k) identifying the presence or absence of a genetic mutation in the genomic regions of interest from the plurality of samples.

19. The method for identifying the presence or absence of a genetic mutation of claim 2, comprising the further steps of:

j) aligning the contig to a reference sequence; and

k) identifying the presence or absence of a genetic mutation in the genomic region of interest.

25. The method according to claim 18, wherein a genetic mutation is a SNP, a deletion, an insertion, an inversion and/or a translocation.

26. The method according to claim 25, wherein a deletion and/or insertion is identified by comparing the number of fragments and/or ligation products from a sample carrying the deletion and/or insertion with a reference sample.

27. The method according to claim 25, wherein a deletion, insertion, inversion and/or translocation is identified based on the presence of chromosomal breakpoints in analyzed fragments.

28. The method according to claim 2, the presence or absence of methylated nucleotides in DNA fragments, ligated DNA fragments, and/or genomic regions of interest.

30. The method according to claim 14, wherein DNA is selected of a size between 100-3,000 base pairs.

3. A method for determining a genomic region of interest'"'"'s sequence, said genomic region of interest comprising a target nucleotide sequence, the method comprising the steps of:

a) providing a sample of cross-linked DNA;

b) fragmenting the crosslinked DNA;

c) ligating the fragmented crosslinked DNA;

d) reversing the crosslinking;

e) optionally fragmenting the DNA of step d);

f) circularising the DNA of step d) or e);

g) optionally amplifying the circularised DNA comprising the target nucleotide sequence;

h) determining at least part of the sequence of the ligated or amplified ligated DNA fragments comprising the target nucleotide utilizing high throughput sequencing;

i) building a contig of the genomic region of interest from the determined sequences.

29. A method comprising:

a) providing a sample of cross-linked DNA;

b) fragmenting the crosslinked DNA;

c) ligating the fragmented crosslinked DNA;

d) reversing the crosslinking;

e) fragmenting the DNA of step d);

f) ligating the fragmented DNA of step d) or e) to at least one adaptor;

g) amplifying the ligated DNA fragments of step d) or e) comprising the target nucleotide sequence utilizing at least one primer that hybridises to the target nucleotide sequence, or amplifying the ligated DNA fragments of step f) utilizing at least one primer that hybridises to the target nucleotide sequence and at least one primer that hybridises to the at least one adaptor; and

h) determining at least part of the sequence of the ligated or amplified ligated DNA fragments of step d), e), f) or g) comprising the target nucleotide sequence.

20. The method according to claim 29 for identifying the presence or absence of a genetic mutation, further comprising:

i) aligning the determined sequences of the ligated or amplified ligated DNA fragments to a reference sequence; and

j) identifying the presence or absence of a genetic mutation in the determined sequences.

21. The method according to claim 29 for identifying the presence or absence of a genetic mutation, further comprising:

i) aligning the determined sequences of the ligated or amplified ligated DNA fragments of a plurality of samples; and

j) identifying the presence or absence of a genetic mutation in the determined sequences.

22. The method according to claim 29 for determining the ratio of fragments carrying a genetic mutation from a cell population suspected of being heterologous, the method further comprising:

i) identifying the fragments of step b);

j) identifying the presence or absence of a genetic mutation in the fragments;

k) determine the number of fragments carrying the genetic mutation;

l) determine the number of fragments not carrying the genetic mutation; and

m) calculating the ratio of fragments carrying the genetic mutation.

23. The method according to claim 29 for determining the ratio of ligation products carrying a fragment with a genetic mutation from a cell population suspected of being heterologous, the method further comprising:

i) identifying the fragments of step b);

j) identifying the presence or absence of a genetic mutation in the fragments;

k) identifying the ligated DNA fragments carrying the fragments with or without the genetic mutation;

l) determine the number of ligated DNA fragments carrying the fragments with the genetic mutation;

m) determine the number of ligation products carrying the fragments without the genetic mutation; and

n) calculating the ratio of ligation products carrying the genetic mutation.

24. The method according to claim 22, wherein in step j) the presence or absence of a genetic mutation is identified by aligning to a reference sequence and/or by comparing fragment sequences of a plurality of samples.