title: High-throughput sequencing of microdissected chromosomal regions.
abstract: 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.
authors.affiliation: Institute of Human Genetics and Anthropology, Jena, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Weise
authors.forename: Bernd
authors.initials: B
authors.lastname: Timmermann
authors.forename: Manfred
authors.initials: M
authors.lastname: Grabherr
authors.forename: Martin
authors.initials: M
authors.lastname: Werber
authors.forename: Patricia
authors.initials: P
authors.lastname: Heyn
authors.forename: Nadezda
authors.initials: N
authors.lastname: Kosyakova
authors.forename: Thomas
authors.initials: T
authors.lastname: Liehr
authors.forename: Heidemarie
authors.initials: H
authors.lastname: Neitzel
authors.forename: Kateryna
authors.initials: K
authors.lastname: Konrat
authors.forename: Christiane
authors.initials: C
authors.lastname: Bommer
authors.forename: Carola
authors.initials: C
authors.lastname: Dietrich
authors.forename: Anna
authors.initials: A
authors.lastname: Rajab
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.forename: Tom H
authors.initials: TH
authors.lastname: Lindner
authors.forename: Katrin
authors.initials: K
authors.lastname: Hoffmann
mesh_terms: D002878:Chromosomes, Human, Pair 1 / Q000235:genetics*; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics*; D019295:Computational Biology; D006801:Humans; D008677:Metaphase; D042282:Microdissection; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA