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Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes


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.

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We thank the patients and their families for agreeing to participate in this study.


This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (21K06283 to TK, 15H04710 and 24390085 to HK) and from the Ministry of Health, Welfare and Labor (16ek0109067h0003 to HK), Japan.

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Authors and Affiliations



TM—Participated in the study design, conducted the molecular experiments, and drafted the manuscript. TK—Participated in the study design, conducted the molecular experiments, and drafted the manuscript. TO—Participated in the study design, conducted the molecular experiments. TS—Participated in the study design, conducted the molecular experiments. SM (S.Miyai)—Participated in the study design, conducted the molecular experiments. HI—Participated in the study design, conducted the molecular experiments. ES—Participated in the study design, conducted the molecular experiments. YM—Participated in the study design, conducted the molecular experiments. SM (S.Mizuno)—Participated in the study design, conducted the molecular experiments. HK—Coordination and conception of the study, critical revision of the manuscript.

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Correspondence to Hiroki Kurahashi.

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The authors declare no competing interests.

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The study was approved by the Ethics Committee for Human Genome Studies at Fujita Health University. Written informed consent to participate was obtained from the study patients and their families.

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Mariya, T., Kato, T., Sugimoto, T. et al. Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes. J Hum Genet 67, 363–368 (2022).

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