High-throughput sequencing of microdissected chromosomal regions.
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.
Weise
Anja
A
Institute of Human Genetics and Anthropology, Jena, Germany.
Timmermann
Bernd
B
Grabherr
Manfred
M
Werber
Martin
M
Heyn
Patricia
P
Kosyakova
Nadezda
N
Liehr
Thomas
T
Neitzel
Heidemarie
H
Konrat
Kateryna
K
Bommer
Christiane
C
Dietrich
Carola
C
Rajab
Anna
A
Reinhardt
Richard
R
Mundlos
Stefan
S
Lindner
Tom H
TH
Hoffmann
Katrin
K
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