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.

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination

“…Within the cell, the mtDNA-gDNA content ratio is less then 1:1000 [ 75 ], which is generally in line with mtDNA content we detected in cfDNA fraction ( Figure 1 A). It must be noted that while traditional genetic and clinical tests based on mtDNA analysis are conducted on amplified or deeply sequenced mtDNA [ 75 , 76 ], conducting similar tests within NIPT places significant limits caused by low coverage of cf-mtDNA [ 77 ]. Nevertheless, our results indicate that we can perform some mtDNA tests despite low coverage.…”

Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a

“…The clinical features of mitochondrial hearing loss include (1) maternal inheritance; (2) hearing loss is sensorineural and primarily symmetrical; (3) variable penetrance and severity; and (4) childhood-onset ( Mutai et al, 2017 ). Traditionally, mitochondrial mutations were detected by restriction fragment length polymorphism assay ( Matsunaga et al, 2006 ), PCR and Sanger sequencing ( Yao et al, 2019 ), or fluorescent PCR ( Moraes et al, 2003 ).…”

An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues

“…A method using ExoV has recently been applied to study the covalently closed circular hepatitis B virus (3.2 kb) DNA (Gao, Yan, & Li, 2019). ExoV has also been used to enrich for human mitochondrial DNA (16 kb) prior to sequencing (Yao et al., 2019). The protocol outlined in this article has been used to determine the state of the HPV genome in established cell lines and organotypic raft tissues (Bienkowska‐Haba et al., 2018; Guidry et al., 2019; Myers et al., 2019).…”