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Improved strategies for transcript profiling using high throughput sequencing technologies

A high-throughput, sequencing technology, applied in the fields of molecular biology and genetics, can solve problems that are difficult to monitor/exclude and affect the accuracy of results, and achieve the effect of rapid analysis and fast methods

Inactive Publication Date: 2009-02-11
KEYGENE NV
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

However, while multiplexing capability, throughput, and robustness are very important advantages of DNA chips, chip-based transcript profiling has two very important limitations, namely, the need for sequence information to enable the creation of chips, highly homologous sequence (such as member sequences derived from repeated gene families) may affect the accuracy of the results
The latter limitation is difficult to monitor / exclude as it is an intrinsic feature of hybridization-based assays

Method used

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  • Improved strategies for transcript profiling using high throughput sequencing technologies
  • Improved strategies for transcript profiling using high throughput sequencing technologies
  • Improved strategies for transcript profiling using high throughput sequencing technologies

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Embodiment Construction

[0035] The invention provides a method for determining cDNA nucleotide sequence, said method comprising the steps of:

[0036] (a) provide cDNA;

[0037] (b) applying a complexity reduction method to at least a portion of said cDNA to obtain a first cDNA library comprising cDNA fragments;

[0038] (c) determining at least part of the nucleotide sequences of the cDNA fragments in the first library using high-throughput sequencing;

[0039] (d) aligning the nucleotide sequences of the cDNA fragments of said first library in step d) to generate a contig of said first library; and

[0040] (e) Determining the nucleotide sequence of the cDNA.

[0041] To date, the use of high-throughput sequencing of cDNA coupled with this complexity reduction method to characterize transcripts has not been disclosed or suggested in the field of sequencing technology.

[0042] In step (a) of the method, cDNA is provided. It is well known in the art how to prepare cDNA. A method of preparation is...

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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.

Description

technical field [0001] The present invention relates to the fields of molecular biology and genetics. The present invention relates to improved strategies based on the determination of the sequence of transcripts using high-throughput sequencing techniques. The present invention also relates to improved strategies for unbiased transcript profiling. Background technique [0002] Transcript characterization is one of the basic techniques used in modern biotechnology research. A major application area of ​​transcriptional profiling is the discovery of genes involved in complex traits. This includes biological phenomena such as the discovery of genes involved in (human) disease to identify targets for the development of treatments for disease (target discovery), elucidation of biochemical pathways that control the synthesis of biomolecules (fermentation industry), dissection of plants or animals Complex traits in breeding (gene discovery), and many other phenomena. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6874C12Q1/6869Y02A90/10C12Q2525/191C12Q2537/143C12Q2537/159C12Q2539/107
Inventor M·J·T·范艾杰克
Owner KEYGENE NV
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