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Master Thesis

Séquençage à haut débit par nanopores sur Oxford Nanopore Technologies MinION® : preuves de concept en pharmacogénomique

Abstract : Next generation sequencing by nanopores is based on the sequencing of long DNA fragments by passing a nucleotides single strand through a hemisynthetic nanopore. In pharmacogenomics, current methods are flawed for heterozygous composite patients, making it impossible to predict a correct phenotype. The objective of this study was to develop a targeted sequencing method for a panel of pharmacogenes, allowing in a single process the complete sequencing of genes, haplotypes phasing and the study of methylation. This method combines an amplification free enrichment mediated by CRISPR-Cas9 and sequencing on Oxford Nanopore Technologies MinION® sequencer. We first sequenced an CYP2D6 amplicon of a patient, then a panel of four pharmacogenes amplicons involved in thiopurines response by multiplexing twelve patient samples on a single flowcell. Then, we used the CRISPR-Cas9 system to enrich the CYP3A4, CYP3A5, CYP2D6, TPMT, NUDT15 and DPYD genes. Sequencing on MinION® associated with the bioinformatics pipeline we built resulted in a satisfactory quality and depth of sequencing, demonstrating the ability of this method to accurately genotype, haplotype mutations and also study DNA methylation. This proof of concept in pharmacogenomics demonstrates that MinION® sequencing represents a viable alternative to conventional next generation sequencing methods based on amplification and promises a large democratization of this approach.
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Master Thesis
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Submitted on : Thursday, September 12, 2019 - 11:17:05 AM
Last modification on : Tuesday, May 11, 2021 - 11:36:24 AM
Long-term archiving on: : Saturday, February 8, 2020 - 6:28:28 AM


  • HAL Id : dumas-02284840, version 1
  • PPN : 237839660


Théo Willeman. Séquençage à haut débit par nanopores sur Oxford Nanopore Technologies MinION® : preuves de concept en pharmacogénomique. Sciences pharmaceutiques. 2019. ⟨dumas-02284840⟩



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