High-throughput sequencing of microdissected chromosomal regions

• Published in: European journal of human genetics : EJHG 2010-04, Vol.18 (4), p.457-462
• Main Authors: Weise, Anja, Timmermann, Bernd, Grabherr, Manfred, Werber, Martin, Heyn, Patricia, Kosyakova, Nadezda, Liehr, Thomas, Neitzel, Heidemarie, Konrat, Kateryna, Bommer, Christiane, Dietrich, Carola, Rajab, Anna, Reinhardt, Richard, Mundlos, Stefan, Lindner, Tom H, Hoffmann, Katrin
• Format: Article
• Language: English
• Subjects: Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Cancer; Cardiovascular disease; Centromeres; Chromosomes; Chromosomes, Human, Pair 1 - genetics; Chromosomes, Human, Pair 12 - genetics; Computational Biology; Cytogenetics; Deoxyribonucleic acid; DNA; DNA sequencing; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene mapping; General aspects. Genetic counseling; Genetics; Genetics of eukaryotes. Biological and molecular evolution; Genomes; Genomics; Human Genetics; Humans; Hypertension; Medical genetics; Medical research; Medical sciences; Medicine; Metaphase; Microdissection; Molecular and cellular biology; Nucleotide sequence; Polymerase Chain Reaction; RNA polymerase; Sequence Analysis, DNA
• ISSN: 1018-4813; 1476-5438; 1476-5438
• DOI: 10.1038/ejhg.2009.196

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.