STRATEGIES FOR TRRANSCRIPT PROFILING USING HIGH THROUGHPUT SEQUENCING TECHNOLOGIES

• US 20090247415A1
• Filed: 12/21/2006
• Published: 10/01/2009
• Est. Priority Date: 12/22/2005
• Status: Abandoned Application

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia
• Application Number:
  US12/158,039
• Publication Number:
  US 20090247415A1
• Time in Patent Office: Days
• US Class Current: 506/5
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2539/107 Representational Difference Analysis [RDA]
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Described is a method for determining a nucleotide sequence within cDNA, the frequency of a nucleotide sequence in a cDNA sample, as well as a method for (unbiased) determination of relative transcript levels of genes without sequence information of these genes being required, said methods using complexity reduction and (high throughput) sequencing.

Claims

1. A method for determining a nucleotide sequence of cDNA comprising the steps of:

(a) Providing cDNA;

(b) Performing a complexity reduction on at least a portion of the cDNA to obtain a first library of the cDNA comprising cDNA fragments;

(c) Determining at least part of the nucleotide sequences of the cDNA fragments of the first library by high-throughput sequencing;

(d) Aligning the nucleotide sequences of the cDNA fragments of the first library of step d) to generate contigs of the first library; and

(e) Determining the nucleotide sequence of the cDNA.

5. A method according to claim 1, wherein the complexity reduction is carried out by a method, selected from the group consisting of the Amplified Fragment Length Polymorphism technique, indexed linking, genome portioning, Serial Analysis of Gene Expression and modifications thereof, Massively Parallel Signature Sequencing, Real-Time Multiplex Ligation-dependent Probe Amplification, High Coverage Expression Profiling, a universal micro-array system, the transcriptome subtraction method, fragment display, differential display and ordered differential display.

7. A method according to claim 1, wherein the high-throughput sequencing is performed on a solid support such as a bead.

9. A method according to any of claim 7, wherein the high-throughput sequencing comprises the steps of:

(c1) ligating sequencing-adaptors to the fragments;

(c2) annealing sequencing-adaptor-ligated fragments to beads, each bead annealing with a single fragment;

(c3) emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;

(c4) performing emulsion PCR to amplify sequencing-adaptor-ligated fragments on the surface of beads;

(c5) selecting/enriching beads containing amplified sequencing-adaptor-ligated fragments;

(c6) loading the beads in wells, each well comprising a single bead; and

(c7) generating a pyrophosphate signal.

10. A method according to claim 1, wherein the complexity reduction is performed by a method comprising the steps of:

(a) Digesting the cDNA with at least one restriction endonuclease to fragment it into restriction fragments;

(b) Ligating the restriction fragments with at least one double-stranded synthetic oligonucleotide adaptor having one end compatible with one or both ends of the restriction fragments to produce adaptor-ligated restriction fragments;

(c) Contacting said adaptor-ligated restriction fragments with one or more oligonucleotide primers under hybridizing conditions, said one or more oligonucleotide primers having a primer sequence including a nucleotide sequence section complementary to part of the at least one adaptor and to part of the remaining part of the recognition sequence of the restriction endonuclease; and

(d) Amplifying said adaptor-ligated restriction fragments by elongation of the hybridized one or more oligonucleotide primers.

11. A method according to claim 10, wherein the primer further comprises a selected sequence at the 3′

end of the primer sequence, said selected sequence comprising 1-10 selective nucleotides being complementary to a section located immediately adjacent to the remaining part of the recognition sequence of the restriction endonuclease.

12. A method according to claim 11, wherein the selected sequence at the 3′

end of the primer sequence comprises 1-8 selective nucleotides, preferably 1-5, more preferably 1-3.

13. A method according to claim 10, wherein said adaptor further comprises an identifier sequence.

2. A method for determining the frequency of a nucleotide sequence comprising the steps of:

(a) Providing cDNA;

(b) Performing a complexity reduction on at least a portion of the cDNA to obtain a first library of the cDNA comprising cDNA fragments;

(c) Determining at least part of the nucleotide sequences of the cDNA fragments of the first library by sequencing; and

(d) Determining the frequency of a nucleotide sequence.

3. A method for determining relative transcription levels of a nucleotide sequence in cDNA samples comprising the steps of:

(a) Determining the frequency of a nucleotide sequence in a first cDNA sample by performing a method as defined in claim 2 on said first cDNA sample;

(b) Determining the frequency of the same nucleotide sequence in a second and/or further cDNA sample by performing a method as defined in claim 2 on said second and/or further cDNA sample; and

(c) Comparing the frequency of the nucleotide sequence in said first cDNA sample with the frequency of the same nucleotide sequence in said second and/or further cDNA sample to obtain relative transcription levels of the nucleotide sequence.

6. A method according to claims 2, 3, 4, or 5, wherein the sequencing is carried out by means of high-throughput sequencing.

8. A method according to claims 6 or 7, wherein the high-throughput sequencing is based on Sequencing-by-Synthesis, preferably Pyrosequencing.

4. A method for determining relative transcription levels of a nucleotide sequence in cDNA samples comprising the steps of:

(a) Providing a first cDNA sample;

(b) Performing a complexity reduction on the first cDNA sample to obtain a first library;

(c) Tagging the first library to obtain a first tagged library;

(d) Consecutively or simultaneously performing step (a) and (b) with a second and/or further cDNA sample, preferably using a different tag for each cDNA sample, to obtain a second and/or further tagged library;

(e) Combining the first tagged library and second and/or further tagged library to obtain a combined library;

(f) Determining at least part of the nucleotide sequences of the combined library by sequencing;

(g) Determining the frequency of the nucleotide sequence in the first cDNA sample and the second and/or further DNA sample; and

(h) Comparing the frequency of the nucleotide sequence in the first cDNA sample with the frequency of the nucleotide sequence in the second and/or further cDNA sample to obtain relative transcription levels of the nucleotide sequence in the cDNA samples.

14. A method according to claim 4, wherein the tag is an identifier sequence.