High-throughput sequencing of microdissected chromosomal regions
High-throughput sequencing of microdissected chromosomal regions
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DOI：
10.1038/ejhg.2009.196
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发表时间：
2010-04-01
影响因子：
5.2
通讯作者：
Hoffmann, Katrin
中科院分区：
文献类型：
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作者：
Weise, Anja;Timmermann, Bernd;Hoffmann, Katrin
疾病基因定位与DNA测序的联系是确定疾病遗传基础的重要策略。新的大规模平行测序程序将极大地促进这一过程，尽管在测序前对目标区域进行富集仍然是必要的。对于这一步，已经描述了依赖于序列定义的探针集的各种DNA捕获方法。为了避免对目标区域中存在的序列进行假设，我们访问了特定的细胞遗传区域，为下一代测序做准备。我们直接对中期染色体的靶区进行微解剖，用退化寡核苷酸引物PCR扩增，获得了足够高质量的高通量测序材料。序列读数可以从少至6个染色体片段获得。下一代测序的细胞遗传学富集的力量在于它不依赖于被研究区域序列的早期知识。因此，该方法特别适用于基因组参考区域序列不可用的情况，包括群体特异性或肿瘤重排，以及先前未测序的基因组区域，如着丝粒。欧洲人类遗传学杂志（2010）18,457-462；doi: 10.1038 / ejhg.2009.196;2009年11月4日在线发布
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. European Journal of Human Genetics (2010) 18, 457-462; doi:10.1038/ejhg.2009.196; published online 4 November 2009