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A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology.

Sophie Dhorne-Pollet1, Nicolas Pollet2, Eric Barrey3

  • 1Université Paris-Saclay, INRAE, AgroParisTech, Animal Genetic and Integrative Biology, Université Paris-Saclay, Jouy-en-Josas, France. sophie.pollet@inrae.fr.

Methods in Molecular Biology (Clifton, N.J.)
|June 3, 2021
PubMed
Summary
This summary is machine-generated.

We present a protocol for creating multiplexed mitochondrial DNA (mtDNA) libraries from blood for long-read sequencing. This method achieves high mtDNA enrichment, enabling accurate variant detection using portable sequencers.

Keywords:
Long read sequencingMinIONMitochondrial DNAMultiple displacement amplificationNuclear DNA depletion

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Mitochondrial DNA (mtDNA) sequencing is crucial for understanding genetic diseases and evolution.
  • Long-read sequencing technologies offer advantages for resolving complex mtDNA regions.
  • Efficient enrichment of mtDNA from blood samples is essential for successful library preparation.

Purpose of the Study:

  • To develop and describe a protocol for preparing multiplexed mtDNA libraries from blood.
  • To achieve high enrichment of mtDNA relative to nuclear DNA.
  • To enable long-read sequencing of the entire mitochondrial genome for variant calling.

Main Methods:

  • Protocol development for mtDNA library preparation from blood.
  • High enrichment of mtDNA using specific techniques.
  • Multiplexing of libraries for high-throughput sequencing.
  • Long-read sequencing using Oxford Nanopore Technologies (MinION).

Main Results:

  • Successful preparation of multiplexed mtDNA libraries from blood samples.
  • High enrichment of mtDNA achieved, minimizing nuclear DNA contamination.
  • Generation of long mtDNA sequence reads up to 16.5 kbp.
  • High-quality sequence data suitable for variant calling.

Conclusions:

  • The described protocol effectively prepares multiplexed mtDNA libraries from blood.
  • Long-read sequencing of mtDNA is feasible with high enrichment and quality.
  • This method facilitates accurate variant detection in the mitochondrial genome using portable sequencing platforms.