Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans.

Mitochondrial diseases are a clinically heterogeneous group of diseases caused by mutations in either nuclear or mitochondrial DNA (mtDNA). The diagnosis is challenging and has frequently required a tissue biopsy to obtain a sufficient quantity of mtDNA. Less-invasive sources mtDNA, such as peripheral blood leukocytes, urine sediment, or buccal swab, contain a lower quantity of mtDNA compared to tissue sources which may reduce sensitivity. Cellular apoptosis of tissues and hematopoetic cells releases fragments of DNA and mtDNA into the circulation and these molecules can be extracted from plasma as cell-free DNA (cfDNA). However, entire mtDNA has not been successfully identified from the cell free fraction previously. We hypothesized that the circular nature of mtDNA would prevent its degradation and a higher sensitivity method, such as next generation sequencing, could identify intact cf-mtDNA from human plasma. Plasma was obtained from patients with mitochondrial disease diagnosed from skeletal muscle biopsy (n = 7) and healthy controls (n = 7) using a specially cfDNA collection tube (Streck Inc.; La Vista, NE). To demonstrate the presence of mtDNA within these samples, we amplified the isolated DNA using custom PCR primers specific to overlapping fragments of mtDNA. cfDNA samples were then sequenced using the Illumina MiSeq sequencing platform. We confirmed the presence of mtDNA, demonstrating that the full mitochondrial genome is in fact present within the cell-free plasma fraction of human blood. Sequencing identified the mitochondrial haplogroup matching with the tissue specimen for all patients. We report the existence of full length mtDNA in cell-free human plasma that was successfully used to perform haplogroup matching. Clinical applications for this work include patient monitoring for heteroplasmy status after mitochondrially-targeted therapies or haplogroup monitoring as a measure of stem cell transplantation.

Newell C ，Hume S ，Greenway SC ，Podemski L ，Shearer J ，Khan A ... -  《-》

Very Short Mitochondrial DNA Fragments and Heteroplasmy in Human Plasma.

Zhang R ，Nakahira K ，Guo X ，Choi AM ，Gu Z ... -  《Scientific Reports》

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

Morshneva A ，Kozyulina P ，Vashukova E ，Tarasenko O ，Dvoynova N ，Chentsova A ，Talantova O ，Koroteev A ，Ivanov D ，Serebryakova E ，Ivashchenko T ，Sukhomyasova A ，Maksimova N ，Bespalova O ，Kogan I ，Baranov V ，Glotov A ... -  《-》

Mitochondrial and nuclear disease panel (Mito-aND-Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost-effective and sensitive NGS-based method.

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a robust high-throughput platform for nuclear and mitochondrial DNA (mtDNA) analyses. We developed a custom Agilent SureSelect Mitochondrial and Nuclear Disease Panel (Mito-aND-Panel) capture kit that allows parallel enrichment for subsequent NGS-based sequence analysis of nuclear mitochondrial disease-related genes and the complete mtDNA genome. Sequencing of enriched mtDNA simultaneously with nuclear genes was compared with the separated sequencing of the mitochondrial genome and whole exome sequencing (WES). The Mito-aND-Panel permits accurate detection of low-level mtDNA heteroplasmy due to a very high sequencing depth compared to standard diagnostic procedures using Sanger sequencing/SNaPshot and WES which is crucial to identify maternally inherited mitochondrial disorders. We established a NGS-based method with combined sequencing of the complete mtDNA and nuclear genes which enables a more sensitive heteroplasmy detection of mtDNA mutations compared to traditional methods. Because the method promotes the analysis of mtDNA variants in large cohorts, it is cost-effective and simple to setup, we anticipate this is a highly relevant method for sequence-based genetic diagnosis in clinical diagnostic applications.

Abicht A ，Scharf F ，Kleinle S ，Schön U ，Holinski-Feder E ，Horvath R ，Benet-Pagès A ，Diebold I ... -  《-》

An Effective Strategy to Eliminate Inherent Cross-Contamination in mtDNA Next-Generation Sequencing of Multiple Samples.

Heteroplasmic mutations in mitochondrial DNA (mtDNA) play critical roles in mitochondrial disease, aging, and cancer. Recently, next-generation sequencing (NGS) has been widely used to detect mtDNA mutations for diagnosis and monitoring of the above-mentioned diseases. However, little attention is paid on inherent cross-contamination generated during mtDNA capture and sequencing of mixed samples, which may seriously reduce the detection accuracy of mtDNA heteroplasmic mutations. In this study, a novel sequencing strategy based on a unique double-barcode design was established. The results showed that when single barcode-based analysis strategy was used, cross-contamination level of 20 DNA samples ranged from 0.27% to 11.90% on HiSeq 2500 and from 0.93% to 17.70% on HiSeq X ten, whereas double barcode-based strategy could effectively eliminate cross-contamination. Moreover, the data indicated that cross-contamination was mainly derived from capture process and was significantly affected by different NGS platforms. In addition, contamination level was negatively related to sequencing depth. Moreover, cross-contamination significantly increased the false-positive calling of mtDNA heteroplasmic mutations and remarkably affected the heteroplasmy level of mtDNA mutations. In contrast, cross-contamination had no notable effect on classification of mtDNA haplogroup. Taken together, our novel double barcode-based sequencing strategy is effective in eliminating cross-contamination, enhancing the detection accuracy of mtDNA NGS, and improving its application in diagnosis or monitoring of diseases associated with mtDNA mutations.

Yin C ，Liu Y ，Guo X ，Li D ，Fang W ，Yang J ，Zhou F ，Niu W ，Jia Y ，Yang H ，Xing J ... -  《-》