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          Institute: MPI für molekulare Genetik     Collection: Sequencing Group     Display Documents



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ID: 460797.0, MPI für molekulare Genetik / Sequencing Group
High-throughput sequencing of microdissected chromosomal regions.
Authors:Weise, Anja; Timmermann, Bernd; Grabherr, Manfred; Werber, Martin; Heyn, Patricia; Kosyakova, Nadezdaa; Liehr, Thomas; Neitzel, Heidemarie; Konrat, Kateryna; Bommer, Christiane; Dietrich, Carola; Rajab, Anna; Reinhardt, Richard; Mundlos, Stefan; Lindner, Tom H.; Hoffmann, Katrin
Language:English
Date of Publication (YYYY-MM-DD):2009-11-04
Title of Journal:European Journal of Human Genetics
Journal Abbrev.:Euro. J. Hum. Genet.
Sequence Number of Article:ejhg.2009.196
Copyright:2009 Macmillan Publishers Limited All rights reserved
Review Status:not specified
Audience:Experts Only
Abstract / Description:The linkage of disease gene mapping with DNA sequencing is an essential strategy for defining the genetic basis of a disease. New massively parallel sequencing procedures will greatly facilitate this process, although enrichment for the target region before sequencing remains necessary. For this step, various DNA capture approaches have been described that rely on sequence-defined probe sets. To avoid making assumptions on the sequences present in the targeted region, we accessed specific cytogenetic regions in preparation for next-generation sequencing. We directly microdissected the target region in metaphase chromosomes, amplified it by degenerate oligonucleotide-primed PCR, and obtained sufficient material of high quality for high-throughput sequencing. Sequence reads could be obtained from as few as six chromosomal fragments. The power of cytogenetic enrichment followed by next-generation sequencing is that it does not depend on earlier knowledge of sequences in the region being studied. Accordingly, this method is uniquely suited for situations in which the sequence of a reference region of the genome is not available, including population-specific or tumor rearrangements, as well as previously unsequenced genomic regions such as centromeres.
Free Keywords:genomic selection;
enrichment;
microdissection;
next-generation sequencing
Comment of the Author/Creator:Correspondence: Dr K Hoffmann, Institute of Medical Genetics, Charité University Medicine, Augustenburgerplatz 1, Berlin 13353, Germany. Tel: +49 30 450 569 129/122; Fax: +49 30 450 569 915; E-mail: khoffma@gmx.de
External Publication Status:published
Document Type:Article
Communicated by:Richard Reinhardt
Affiliations:MPI für molekulare Genetik
External Affiliations:1.Institute of Human Genetics and Anthropology, Jena, Germany;
2.Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA;
3.Institute of Medical Genetics, Charité University Medicine, Berlin, Germany;
4.Institute for Human Genetics, Charité University Medicine, Berlin, Germany;
5.Genetic Unit, Ministry of Health, Muscat, Sultanate of Oman;
6.Division of Nephrology, Department of Medicine, Neurology, and Dermatology, University Clinic Leipzig, Leipzig, Germany.
Identifiers:URL:http://www.nature.com/ejhg/journal/vaop/ncurrent/a...
DOI:10.1038/ejhg.2009.196
ISSN:1018-4813
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