Related Experiment Video
Updated: Aug 9, 2025
07:24
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
1.6K
Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing
Andrea Legati1, Daniele Ghezzi1,2,3, Carlo Viscomi4
1Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.
Methods in Molecular Biology (Clifton, N.J.)
|February 22, 2023
Summary
Next-generation sequencing (NGS) is key for diagnosing mitochondrial disorders. This study presents a protocol for whole mitochondrial DNA (mtDNA) sequencing and heteroplasmy quantification, addressing unique challenges in mitochondrial genetics.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Next-generation sequencing (NGS) is crucial for diagnosing genetic disorders.
- Mitochondrial encephalomyopathies are heterogeneous disorders often linked to mitochondrial DNA (mtDNA) mutations.
- Sequencing mtDNA presents unique challenges due to mitochondrial genetics and data analysis requirements.
Purpose of the Study:
- To present a clinically relevant protocol for whole mtDNA sequencing.
- To detail a method for quantifying mtDNA variant heteroplasmy levels.
- To address the specific challenges of applying NGS to mtDNA analysis.
Main Methods:
- Utilized a single PCR amplicon approach for whole mtDNA sequencing.
- Developed a protocol starting from total DNA extraction.
- Focused on accurate heteroplasmy level quantification of mtDNA variants.
Main Results:
- Successfully generated a single PCR amplicon covering the entire mtDNA.
- Established a method for quantifying heteroplasmy levels of mtDNA variants.
- Provided a detailed and clinically relevant protocol for mtDNA analysis.
Conclusions:
- The described protocol enables comprehensive mtDNA sequencing and heteroplasmy quantification.
- This method overcomes specific challenges associated with mitochondrial genetics and NGS data analysis.
- The protocol is suitable for both diagnosis and discovery of disease-related mtDNA variants.
Keywords:
HeteroplasmyMitochondrial DNAMitochondrial diseaseMitochondrial haplogroupsNext generation sequencingSingle ampliconMore Related Videos
Related Concept Videos
03:00Next-generation Sequencing
91.9K
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
91.9K
02:59Animal Mitochondrial Genetics
7.7K
Among all the organelles in an animal cell, only mitochondria have their own independent genomes. Animal mitochondrial DNA is a double-stranded, closed-circular molecule with around 20,000 base pairs. Mitochondrial DNA is unique in that one of its two strands, the heavy, or H, -strand is guanine rich, whereas the complementary strand is cytosine rich and called the light, or L, -strand. Compared to nuclear DNA, mitochondrial DNA has a very low percentage of non-coding regions and is marked by...
7.7K
01:57Sanger Sequencing
755.6K
DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
755.6K
03:21RNA-seq
10.2K
RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
10.2K
02:16Comparing Mitochondrial, Chloroplast, and Prokaryotic Genomes
12.6K
The present-day mitochondrial and chloroplast genomes have retained some of the characteristics of their ancestral prokaryotes and also have acquired new attributes during their evolution within eukaryotic cells. Like prokaryotic genomes, mitochondrial and chloroplast genomes neither bind with histone-like proteins nor show complex packaging into chromosome-like structures, as observed in eukaryotes. Unlike mitotic cell divisions observed in eukaryotic cells, mitochondria and chloroplasts...
12.6K
01:29Modern Molecular Taxonomy
69
Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...
69