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.
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Acknowledgements
We thank Anna Kosiura, Isabelle Kühndahl, Christina Roehr and Phillipe Schroeter for their technical expertise. We thank Mathias Meyer for his advice. Friedrich C Luft and Eddy Rubin critically read the manuscript. The Deutsche Forschungsgemeinschaft (DFG SFB 577 project A4 to KH, LI820/11-1 and LI820/17-1 to TL, Li768/6-1 and 6-2 to THL), the IZKF Jena (Start-up S16), the Stiftung Leukämie and the University Jena supported the study. Katrin Hoffmann received a Rahel Hirsch fellowship from the Charité Medical Faculty.
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Weise, A., Timmermann, B., Grabherr, M. et al. High-throughput sequencing of microdissected chromosomal regions. Eur J Hum Genet 18, 457–462 (2010). https://doi.org/10.1038/ejhg.2009.196
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DOI: https://doi.org/10.1038/ejhg.2009.196
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