Dataset Information

A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing.

ABSTRACT: Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25?ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.

SUBMITTER: Yao Y

PROVIDER: S-EPMC6874554 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing.

Yao Yue Y Nishimura Motoi M Murayama Kei K Kuranobu Naomi N Tojo Satomi S Beppu Minako M Ishige Takayuki T Itoga Sakae S Tsuchida Sachio S Mori Masato M Takayanagi Masaki M Yokoyama Masataka M Yamagata Kazuyuki K Kishita Yoshihito Y Okazaki Yasushi Y Nomura Fumio F Matsushita Kazuyuki K Tanaka Tomoaki T

Scientific reports 20191122 1

Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities o ...[more]

PMID: 31757988

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Dataset Information

A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing.

Publications

A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing.

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