A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency

Eriko Adachi, Ryuichi Nakagawa, Atsumi Tsuji-Hosokawa, Maki Gau, Shizuka Kirino, Analia Yogi, Hisae Nakatani, Kei Takasawa, Tomomi Yamaguchi, Tomoki Kosho, Masanori Murakami, Toshihiro Tajima, Tomonobu Hasegawa, Tetsuya Yamada, Tomohiro Morio, Osamu Ohara, Kenichi Kashimada

研究成果: Article査読


CONTEXT: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. OBJECTIVE: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. METHODS: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. RESULTS: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS "duplex" analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, "CH2a," in the region from the latter part of intron 5 to exon 6. CONCLUSION: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.

ジャーナルThe Journal of clinical endocrinology and metabolism
出版ステータスPublished - 2024 2月 20

ASJC Scopus subject areas

  • 内分泌学、糖尿病および代謝内科学
  • 生化学
  • 内分泌学
  • 臨床生化学
  • 生化学、医学


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