Nuevas técnicas de secuenciacion de adn

New Biotechnology  Volume 25, Number 4  April 2009

REVIEW

Wilhelm J. Ansorge
Ecole Polytechnique Federal Lausanne, EPFL, Switzerland

Next-generation high-throughput DNA sequencing techniques are opening fascinating opportunities in the life sciences. Novel fields and applications in biology and medicine are becoming a reality, beyond the genomic sequencing which was originaldevelopment goal and application. Serving as examples are: personal genomics with detailed analysis of individual genome stretches; precise analysis of RNA transcripts for gene expression, surpassing and replacing in several respects analysis by various microarray platforms, for instance in reliable and precise quantification of transcripts and as a tool for identification and analysis of DNA regionsinteracting with regulatory proteins in functional regulation of gene expression. The next-generation sequencing technologies offer novel and rapid ways for genome-wide characterisation and profiling of mRNAs, small RNAs, transcription factor regions, structure of chromatin and DNA methylation patterns, microbiology and metagenomics. In this article, development of commercial sequencing devices is reviewedand some European contributions to the field are mentioned. Presently commercially available very high-throughput DNA sequencing platforms, as well as techniques under development, are described and their applications in bio-medical fields discussed.

Introduction
Next-generation high-throughput DNA sequencing techniques, which are opening fascinating new opportunities in biomedicine, wereselected by Nature Methods as the method of the year in 2007 [1]. However, the path to gaining acceptance of the novel technology was not an easy one. Until a few years ago the methods used for the sequencing were the Sanger enzymatic dideoxy technique first described in 1977 [2] and the Maxam and Gilbert chemical degradation method described in the same year [3], which was used in sequence cases whichcould not easily be resolved with the Sanger technique. The two laboratories where the first automated DNA sequencers were produced, simultaneously, were those of Leroy Hood at Caltech [4], commercialised by Applied Biosystems, and Wilhelm Ansorge at the European Molecular Biology Laboratory EMBL [5,6] and commercialised by Pharmacia-Amersham, later General Electric (GE) Healthcare. The
E-mailaddress: wilhelm.ansorge@epfl.ch.
1871-6784/$ - see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.nbt.2008.12.009

Sanger method was used in the first automated fluorescent project for sequencing of a genome region, in which sequence determination of the complete gene locus for the HPRT gene was performed using the EMBL technique; in that project the important concept of paired-endsequencing was also introduced for the first time [7]. The achievement of successful and unambiguous sequencing of a real genomic DNA region, loaded with many sequence pitfalls like Alu sequences in both directions of the HPRT gene locus, demonstrated the feasibility of using an automated fluorescence-based technique for the sequencing of entire genomes, and in principle the feasibility of thetechnical sequencing part of the Human Genome project. When the international community decided on determination of the whole human genome sequence, the goal triggered the development of techniques allowing higher sequencing throughput. In Japan, the work on fluorescent DNA sequencing technology by the team of H. Kambara (http://www.hitachi.com/rd/fellow_kambara.html) in the Hitachi laboratories resultedin the development after 1996 of a high-throughput capillary array DNA sequencer. Two
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Review

Next-generation DNA sequencing techniques

REVIEW

New Biotechnology  Volume 25, Number 4  April 2009

companies, ABI (commercialising the Kambara system) and Amersham (taking over and developing further the system set up in the US by the Molecular...