Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes
Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes
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DOI：
10.1038/s10038-021-01004-x
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发表时间：
2022-01-14
影响因子：
3.5
通讯作者：
Kurahashi, Hiroki
中科院分区：
文献类型：
--
作者：
Mariya, Tasuku;Kato, Takema;Kurahashi, Hiroki
小额外标记染色体（small supernumerary marker chromosomes，sSMCs）的结构分析表明，许多小额外标记染色体具有复杂的结构。然而，通过使用短读段测序仪的全基因组测序对sSMC进行结构分析是具有挑战性的，因为大多数sSMC存在低水平的嵌合现象，并且由所涉及的染色体的小区域组成。在本研究中，我们使用纳米孔长读测序技术应用自适应采样来富集目标区域，从而尝试确定两种具有先前由细胞遗传学微阵列揭示的复杂结构重排的sSMC的结构。在自适应采样中，FASTA文件中目标区域的简单规范使得能够识别测序DNA是否包含在目标中，从而促进有效的长读段测序。为了评估靶富集效率，我们平行进行了常规的对端短读段测序。自适应采样测序实现了靶富集，其覆盖率比传统的双端测序高约11.0至11.5倍。这使我们能够快速识别所有断点连接，并确定确切的sSMC结构为环状染色体。除了微同源性和微插入在交界处，我们确定了反向重复结构在两个sSMC，这表明涉及复制损伤的共同生成机制。因此，自适应采样是确定复杂染色体重排结构的简单而有益的方法。
Structural analysis of small supernumerary marker chromosomes (sSMCs) has revealed that many have complex structures. Structural analysis of sSMCs by whole genome sequencing using short-read sequencers is challenging however because most present with a low level of mosaicism and consist of a small region of the involved chromosome. In this present study, we applied adaptive sampling using nanopore long-read sequencing technology to enrich the target region and thereby attempted to determine the structure of two sSMCs with complex structural rearrangements previously revealed by cytogenetic microarray. In adaptive sampling, simple specification of the target region in the FASTA file enables to identify whether or not the sequencing DNA is included in the target, thus promoting efficient long-read sequencing. To evaluate the target enrichment efficiency, we performed conventional pair-end short-read sequencing in parallel. Sequencing with adaptive sampling achieved a target enrichment at about a 11.0- to 11.5-fold higher coverage rate than conventional pair-end sequencing. This enabled us to quickly identify all breakpoint junctions and determine the exact sSMC structure as a ring chromosome. In addition to the microhomology and microinsertion at the junctions, we identified inverted repeat structure in both sSMCs, suggesting the common generation mechanism involving replication impairment. Adaptive sampling is thus an easy and beneficial method of determining the structures of complex chromosomal rearrangements.