Method for the High Throughput Screening of Transposon Tagging Populations and Massive Parallel Sequence Identification of Insertion Sites

• US 20090208943A1
• Filed: 11/08/2006
• Published: 08/20/2009
• Est. Priority Date: 11/14/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Vandenbussche, Michiel Marcel Albert, Gerats, Antonius Gerardus Marie, van Tunen, Adrianus Johannes, van Eijk, Michael Josephus Theresia
• Granted Patent: US 8,071,310 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

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

Claims

1. 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) restrict the DNA using one or more, preferably two or more, most preferably two, restriction endonucleases, preferably at least one of which is a frequent cutting restriction endonuclease that does not cut in the transposon and preferably at least one is a rare cutting restriction endonuclease that cuts in the transposon, ligate adaptors to the restriction fragments to thereby prepare adaptor-ligated restriction fragments;

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

(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 thereby 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 nucleotide sequences from the database with the phenotype of interest;

(j) identifying the member(s) of the transposon population containing the fragment(s) of step (i);

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

2. Method according to claim 1, wherein the pooling is a 3D-pooling strategy.

3. Method according to claim 1 wherein the database comprises EST sequences, genomic sequences of the species of interest and/or the non-redundant sequence database of GenBank or similar sequence databases.

4. Method according to claim 1, wherein the high throughput sequencing is based on Sanger sequencing, preferably by capillary electrophoresis.

5. Method according to claim 1, wherein the high throughput sequencing is sequencing-by-synthesis, preferably pyrosequencing.

6. Method according to claim 1, wherein sequencing is performed on a solid support such as a bead.

7. Method according to claim 6, 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 micro reactors, each water-in-oil micro reactor comprising a single bead;

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

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

(6) loading the beads in wells, each well comprising a single bead; and

(7) generating a pyrophosphate signal.

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