Method for the high throughput screening of transposon tagging populations and massive parallel sequence identification of insertion sites

• US 8,071,310 B2
• Filed: 11/08/2006
• Issued: 12/06/2011
• Est. Priority Date: 11/14/2005
• Status: Expired due to Fees

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: van Eijk, Michael Josephus Theresia, Gerats, Antonius Gerardus Marie, van Tunen, Adrianus Johannes, Vandenbussche, Michiel Marcel Albert
• Primary Examiner(s): Woolwine, Samuel
• Application Number:
  US12/093,574
• Publication Number:
  US 20090208943A1
• Time in Patent Office: 1,854 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/138 Amplified fragment length polymorphism [AFLP]

Abstract

A method for the identification of a gene in a transposon population is provided. The method comprises isolating genomic DNA, optionally pooling the DNA, restricting the DNA in the pools using an enzyme, ligating adaptors, amplifying the adaptor-ligated fragments with primers one of which is a primer complementary to a border of a transposon sequence, sequencing the fragments using high throughput sequencing, aligning the fragments with known sequences in a database and thereby identifying gene candidates.

Claims

1. A method for the identification of an insertion associated with a gene or sequence of interest in a member of a transposon population, comprising the steps of:

(a) isolating, individually or in pools, genomic DNA of the transposon population;

(b) optionally, pooling the DNA obtained in step (a);

(c) restricting the DNA using one or more restriction endonucleases and ligating adaptors to the restriction fragments, thereby preparing adaptor-ligated restriction fragments;

(d) amplifying the adaptor-ligated restriction fragments with a pair of primers, whereby one of the primers comprises a section that is complementary or capable of hybridizing to part of a transposon sequence and further comprises a sequencing primer binding site, wherein the other of the primers is at least complementary to the adaptor, wherein one or both primers comprise a tag;

(e) optionally, pooling the amplification products of step (d) to create a library of amplification products;

(f) optionally, fragmenting the amplification products in the library to create amplification library product fragments;

(g) determining the nucleotide sequence of the fragments of (d), (e) or (f) using high throughput sequencing;

(h) optionally, trimming the sequence of the fragments in silico to remove any adaptor and/or transposon related sequence information;

(i) identifying one or more fragments of step (g) or (h) that are capable of aligning with nucleotide sequences from a database, thereby correlating the identified fragments with a gene or phenotype of interest represented in the database;

(j) identifying members of the transposon population containing the fragment or fragments of step (i);

(k) optionally, designing a probe or PCR primer pair based on the fragments of step (i) and using said probe or PCR primer to confirm transposon insertion in the gene of interest in the genome of the member or members identified in (j).

2. The method according to claim 1, wherein step (a) or step (b) uses a 3D-pooling strategy.

3. The method according to claim 1 wherein the database comprises EST sequences or genomic sequences of the species of interest.

4. The method according to claim 1, wherein the high throughput sequencing is based on Sanger sequencing.

5. The method according to claim 4, wherein the Sanger sequencing is performed by capillary electrophoresis.

6. The method according to claim 1, wherein the high throughput sequencing is sequencing-by-synthesis.

7. The method according to claim 6, wherein the sequencing-by-synthesis is pyrosequencing.

8. The method according to claim 1, wherein sequencing is performed on a solid support.

9. The method according to claim 8, wherein the solid support is a bead.

10. The method according to claim 9 wherein sequencing comprises the steps of:

(1) annealing sequencing-adaptor-ligated fragments to beads, each bead annealing with a single fragment;

(2) emulsifying the beads in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead;

(3) performing emulsion PCR to amplify adaptor-ligated fragments on the surface of beads;

(4) selecting and/or enriching beads containing amplified adaptor-ligated fragments;

(5) loading the beads into wells, one bead per well; and

(6) generating a pyrophosphate signal.

11. The method according to claim 1, wherein at least one of the primers contains one or more nucleotides with improved binding affinity.

12. The method according to claim 1, wherein, in step (c), two or more restriction endonucleases are used.

13. The method according to claim 12, wherein, in step (c),

(i) at least one the restriction endonucleases is a frequent cutting restriction endonuclease that does not cut in the transposon, and

(ii) at least one of the restriction endonucleases is a rare cutting restriction endonuclease that cuts in the transposon.

14. The method according to claim 1, wherein in step (c) two restriction endonucleases are used.

15. The method according to claim 1, wherein, in step (d), the pair of primers is a pair of labeled primers.