High throughput detection of molecular markers based on aflp and high through-put sequencing

Abstract

The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter-ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3′ end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular marker.

Description

[0001]This application is a Continuation of U.S. patent application Ser. No. 12 / 296,009, filed Feb. 6, 2009, which is a U.S. National Stage Application of PCT / NL2007 / 000094, filed Apr. 4, 2007, which claims priority to U.S. Provisional Patent Applications 60 / 788,706, filed Apr. 4, 2006 and 60 / 880,052, filed Jan. 12, 2007, all of which are incorporated herewith by reference in entirety.FIELD OF THE INVENTION[0002]The present invention relates to the field of molecular biology and biotechnology. In particular, the invention relates to the field of nucleic acid detection identification. More in particular the invention relates to methods for the detection and identification of markers, in particular molecular markers. The invention is concerned with the provision of high throughput methods for the detection and identification of molecular markers. The invention further relates to the application of the method in the identification of and / or detection of nucleotide sequences that are re...

AI Technical Summary

Benefits of technology

[0043]Thus, the present inventors have found that by incorporation of a sample-specific identifier in the adaptor-ligated restriction fragment and / or the determination of only part of the sequence of the restriction fragment provides for a very efficient and reliable improvement of the existing technologies. It was found that by incorporation of a sample-specific identifier, multiple samples can be sequenced in a single run and by sequencing only part of the restriction fragment, adequate identification of the restriction fragment can be achieved.

Problems solved by technology

The identification of polymorphisms in large samples such as genomes is at present a laborious and time-consuming task.

Although the throughput of AFLP is very high due to high multiplexing levels in the amplification and detection steps, the rate limiting step is the resolving power of electrophoresis.

These limitations also hamper throughput.

However, most high throughput sequencing technologies cannot yet provide sequencing reads that encompass entire AFLP fragments, which are typically 100-500 bp in length.

So far, detection of AFLP markers / sequences by sequencing has not been economically feasible due to, among other limitations, cost limitations of Sanger dideoxy sequencing technology and other conventional sequencing technologies.

However, detection by sequencing of the entire restriction fragment is still relatively uneconomical.

Furthermore, the current state of the art sequencing technology such as disclosed herein elsewhere (from 454 Life Sciences, www.454.com and Solexa, www.solexa.com), despite their overwhelming sequencing power, can only provide sequencing fragments of limited length.

Also the current methods do not allow for the simultaneous processing of many samples in one run.

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