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Platform for the characterization of microbial genomes by high throughput sequencing

Dr Christophe RODRIGUEZ, Director

 "Metagenomics, at the heart of screening and characterization of microbial pathogens."

In the beginning…

The next-generation sequencing platform (NGS) was created in 2012 at the initiative of the Institute for Biomedical Research (IMRB), INSERM research center of the Faculty of Medicine of Créteil, and the Pôle of Biology and Pathology of the Henri Mondor Hospital Group (Assistance Publique-Hôpitaux de Paris), supported by INTER-DIM funding (Ile-de-France Region) and specific funding from the University Paris-Est Créteil, the Assistance Publique-Hôpitaux de Paris, and the National Agency for Research on AIDS and Viral Hepatitis (ANRS). The platform has focused on medical and microbiological research from the beginning and now occupies an important place in the development of NGS techniques for the diagnosis and characterization of microbial pathogens.

Directions of development

  • Development of diagnostic approaches to detect any viral, bacterial, or fungal pathogen in any type of diagnostic or research sample.
  • Development of pathogen characterization approaches to identify, classify, and highlight mutations or genes of interest (e.g., resistance to anti-infectives).

Blind diagnosis of infection and pathogen identification

The constant emergence of new infections, favored by globalization, travel, and global warming, sometimes requires the diagnosis of infections based on nonspecific symptoms, in poorly informative contexts, without any preliminary knowledge of the microorganism. The development of specific diagnostic tests for a given infection is expensive, time-consuming to implement, and often available too late. The laboratory has specialized in the development and application of a "universal" diagnostic test based on a metagenomic approach. These metagenomic techniques aim to establish the repertoire of all the species present in a sample. The first application was targeted metagenomics (16S or ITS) for bacteria or fungi, but this approach did not allow the search for viruses, whether DNA or RNA.

Our laboratory has developed a complete process for "shotgun metagenomics" allowing:

  • the exhaustive extraction of all pathogens from a sample
  • simultaneous and automated preparation of DNA and RNA libraries
  • massive sequencing (more than 80 million sequences per sample)
  • appropriate analysis (figure below), allowing the identification of all microorganisms in a sample

 

The "shotgun metagenomic" technique has been validated on mono- and polymicrobial samples containing bacterial, fungal, and/or viral pathogens. The figure below shows our ability to identify whole genome sequences of various viruses —including HIV, HCV, human pegivirus type 1 (HPGV), Zika virus (ZIKV), and respiratory syncytial virus (RSV)— in human samples or cell cultures (human coronavirus 229E, HCoV). The depth indicates the number of sequences generated for each region of the viral genome, allowing the identification of the pathogen by comparison with sequence libraries.

 

Pathogen characterization

The precise identification of the pathogen(s), as well as demonstration that some of their genomic characteristics with functional significance, is another advantage of the "shotgun metagenomic" technique developed on the platform. Simultaneous sequencing of the complete genome of all pathogens present in a single sample makes it possible to obtain all the genetic data, including, possibly, those for minority variants. For example, the technique allows genotyping and subtyping or the detection of resistant viral variants in samples provided by the NRC (National Reference Center) of viral hepatitis B, C, and delta.


The platform also regularly tests new equipment and/or NGS kits as part of its technology monitoring mission, particularly for diagnosis and typing.

Host-Pathogen interactions

In the context of research, NGS techniques allow the characterization of modifications affecting the host in various infection situations, with or without anti-infectious treatment. The methods developed include RNA-Seq, micro-RNA sequencing, and transcriptomics. These tools provide valuable information for the identification of antiviral targets and the understanding of antiviral mechanisms of action for the group working on the development of new broad-spectrum antiviral approaches.

Human Resources

  • Bruno COSTES, Reserch Engineer (Co-Director)
  • Vanessa DEMONTANT, Senior Technician
  • Guillaume GRICOURT, Bioinformatician
  • Corinne MARIE, Medical-Technical Services Manager
  • Natacha MARTIN, Engineer assitant
  • Mélanie MERCIER-DARTY, Senior Hospital Engineer
  • Christophe RODRIGUEZ, MCU-PH (Director)

Collaboration

  • Jean-Winoc DECOUSSER, MCU-PH, Bacterial genome
  • Paul-Louis WOERTHER, MCU-PH, Microbiota

Network

  • Platform supported by the ANRS for the realization of projects financed by this organization
  • Animation of the groupe NGS ANRS (C.Rodriguez, AG. Marcelin)
  • Ongoing creation of a NGS APHP division for diagnosis

Formations

  • Team training on the experimental or analytical part in the field of microbiology
  • Animation of an annual seminar "NGS in microbiology"
  • Supervision of Masters 2 "Microbiology" (Paris 11 and Paris 6 IMVI)
  • PhD supervision (microbiology)

    Services

    Expérimental

    • DNA, RNA extraction, all types of samples
    • Qualitative and quantitative analysis of extracted nucleic acids
    • Making of libraries (Amplicons, Nextera XT, Total RNA, mRNA, micro-RNA
    • Sequencing adapted to needs (MiSeq-NextSeq)

    Analytics

    • Screening for bacterial, fungal or viral pathogens using an analysis pipeline developed on the site by the team
    • Genomic mapping of single nucleotide polymorphism (SNP) of pathogens (not accessible for all pathogens, inquire)
    • Development of customized analysis pipelines

    Équipments

    Expérimental

    • Extractor : QiaSymphony
    • Tape Station 2000
    • 2 Hamilton StarLet (1 pré-PCR, 1 post-PCR)
    • qPCR : 1 Light cycler 480, 1 ABI 7900, 1 RotorGene
    • Sanger Sequencers : 3 ABI 3130
    • Illumina Sequencers : 2 NextSeq 500 et 1 MiSeq
    • IonTorrent Sequencer : 2 PGM, 1 S5

    Analytics

    • Servers : 4 nods (25 Co, 1 To RAM/noeud) 
    • Workstation : 4 computer MultiCore, RAM (>24Go)

    Software copyrights

    • Pyromute© (2010) (NGS data filtering and variant calling)
    • Pyrotree© (2010) (NGS data 3D phylogeny)
    • Pyrodyn© (2010) (NGS data viral evolution modeling)
    • Pyroclass© (2010) (NGS data demultiplexing)
    • Pyrotrop© (2011) (HIV-1 coreceptor usage prediction)
    • PyroMIC© (2013) (metagenomics analysis)