Overview and Background of the Call for Expressions of Interest

The COVID-19 pandemic and the emergence of SARS-CoV-2 variants whose transmissibility characteristics are likely to alter the dynamics of the epidemic in France have highlighted the need to strengthen SARS-CoV-2 genomic surveillance capabilities. The objective of this surveillance is to detect the emergence and track the spatiotemporal distribution of viruses with mutations that may have functional consequences, such as increased infectivity, transmissibility, virulence, or immune evasion.

In France, genomic surveillance falls under the purview of the National Reference Center (CNR) for Respiratory Infections (Institut Pasteur) and its associated laboratories (Hospices Civils de Lyon and Institut Pasteur de la Guyane); it is conducted in close collaboration with Santé publique France. The emergence of new SARS-CoV-2 variants has made it necessary to supplement the sequencing capabilities already in place at the CNR with additional resources, in order to enable more precise monitoring of variant circulation across the country.

To this end, the Ministries of Health and Research have decided to implement a large-scale project to increase these sequencing capacities in France. Called EMERGEN, this project was launched in January 2021 and includes several work packages dedicated to the collection of biological samples, sequencing platforms, and the bioinformatics infrastructure enabling the centralization, analysis, and sharing of genomic data and related metadata, and the use of this data for public health or research purposes.

The call for expressions of interest aims to supplement SARS-CoV-2 genomic sequencing capacity for surveillance purposes (random selection) with an additional cumulative volume of up to 5,000 sequences per week, depending on the level of the epidemic.

Selection of Shortlisted Candidates

The selection of candidates will take place in two stages.

1. An initial selection based on a review of the application materials to determine the list of candidates eligible to submit a series of 10 sequences with associated data/metadata for verification, under real-world conditions, by the CNR and the IFB, of their compliance with the specifications.

2. The final selection of successful candidates will be based on the results of the real-world analysis of the compliance of the submitted sequences and data/metadata with the requirements of the specifications.

Tentative Schedule

• Publication of the Call for Expressions of Interest: 05/26/21

• Submission of applications: 06/11/21

• Selection of applications for the field testing phase: 06/30/21

• Request for submission of samples: starting June 30, 2021

• Analysis of submitted samples: approximately 3 weeks

• Notification of results to applicants: around 07/27/21

Frequently Asked Questions

Can a laboratory based abroad apply for the AMI-EMERGEN?

For a foreign laboratory to apply for this call for expressions of interest, it must meet the requirements specified in the text of the AMI and the associated specifications.

In particular, regarding the origin of RT-PCR samples testing positive for SARS-CoV-2, AMI-EMERGEN aims to increase sequencing capacity in France to ensure monitoring of variants within French national territory. Thus, samples sequenced by the applicant laboratory’s technical platform, on the one hand, and submitted to one of the public platforms, on the other, must originate solely from French national territory. Additionally, payment to selected laboratories will be made via fee-for-service billing through the National Health Insurance system; therefore, the applicant laboratory must first ensure that it is eligible for this payment method.

What are the expected sequencing volumes per additional platform selected under the AMI-EMERGEN call for proposals?

Under the AMI EMERGEN initiative, medical laboratories are sought that, for a given technical platform, have the human and material resources to perform at least 1,000 sequences per week from RT-PCR samples that are positive for SARS-CoV-2.

Can a private company apply for the AMI?

To be eligible to apply for this AMI, the applicant must be a medical biology laboratory and must meet the requirements specified in the AMI consultation rules and the associated specifications.

Selected laboratories will be compensated through fee-for-service billing by the Health Insurance system; therefore, the applicant must first ensure that they are eligible for this compensation.

Can an industrial company make its NGS technical platform available to an LBM?

It is up to the applicant LBM to describe in its application the technical and organizational arrangements that will enable it to meet the requirements of the specifications.

To do so, they must refer in particular to section 2.2—content of the application package—in the text of the Call for Expressions of Interest (the package must include, in particular, a description of the methodologies used, the workflow used for sequencing analyses, the human and material resources of the technical platform, the laboratory’s experience and that of its technical platform in terms of sequencing infectious pathogens using the NGS method, etc.).

How should consensus sequences and FastQ files be submitted via EMERGEN-DB?

All files (raw reads, consensus genome, variants, metadata) will be transferred to a trusted third-party service provider, which will ensure the encryption of all sensitive information (pseudonymization keys) and upload to the server of the French Institute of Bioinformatics, which will handle the subsequent steps (validation of compliance and quality, integration into the EMERGEN-DB database, submission to international repositories).

What is the volume of samples and sequences to be submitted?

Santé publique France will specify to each selected platform both the volume of sequences to be processed and the volume of samples to be submitted.

These volumes will be defined by Santé publique France no later than the 25th of each month, and Santé publique France will provide notification of the volume to be sequenced.

What types of samples will be sequenced under this Call for Expressions of Interest for the selected laboratories?

This call for expressions of interest (AMI) describes the framework within which laboratories expressing interest in supplementing SARS-CoV-2 sequencing capacity must respond. Selected laboratories will be required to sequence samples randomly selected by the laboratory for surveillance purposes. In parallel with this call for expressions of interest, the Regional Health Agencies (ARS) may request that laboratories perform sequencing for intervention purposes (e.g., clusters).

What type of laboratory is eligible to apply?

For a clinical laboratory to apply for this call for expressions of interest, it must meet the requirements specified in the text of the call and the associated specifications.

Additionally, compensation for selected laboratories will be based on the French National Health Insurance’s fee-for-service pricing; therefore, applicant laboratories must first ensure they are eligible for this payment method.

Do the data to be submitted to EMERGEN include the data that the IFB will need to submit the sequences to GISAID?

The document used to submit data to EMERGEN-DB will be updated to combine all the information required for both GISAID and EMERGEN into a single document.

How was the list of variants to be detected in the document available for download on EMERGEN DB established?

The subclades were selected based on risk analyses conducted by Santé publique France and the National Reference Center for Respiratory Infectious Diseases (including influenza), as well as the list posted on covariants.org and the detections made to date by the various laboratories that performed the sequencing. This list is regularly updated, resulting in a new version of the document available for download on EMERGEN-DB.

What should be done if variants with an atypical mutation profile are detected?

When a variant with newly identified or rarely detected mutations is identified, it is necessary to notify the CNR immediately to report this new detection and to determine the nomenclature to be used.

Does the June 11 deadline apply to electronic submission of the application or to both electronic and postal submission?

Applications must be received no later than Friday, June 11, at 12:00 p.m. (French time). Any application received after this deadline will be rejected.

• By mail to the following address: Santé publique France – Direction des Maladies Infectieuses, c/o the EMERGEN team, 12 rue du Val d’Osne, 94410 Saint-Maurice;

• By email to the following address: ami-emergen@santepubliquefrance.fr (a confirmation of receipt will then be sent to the sender).

How is sequencing distributed across the country?

The applicant’s geographic location and the extent of its territorial coverage in terms of the origin of SARS-CoV-2 RT-PCR samples are criteria for evaluating applications. Indeed, the selection of laboratories should provide insight into the dynamics of the spread of different variants across the entire country.

Is a public-private partnership feasible within the framework of the AMI?

There is no a priori objection to such a partnership. It is up to the applicant laboratory to describe in its application the technical and organizational arrangements that will enable it to meet the requirements specified in the AMI consultation rules and the associated specifications. The selected laboratories will be compensated through fee-for-service billing by the Health Insurance system; therefore, the applicant must first ensure that it is eligible for this payment method.

Can a laboratory partner with other LBMs (private and/or public) to meet the requirement of performing 1,000 sequences per week?

There is no a priori objection to such a partnership. It is up to the applicant laboratory to describe in its application the technical and organizational arrangements that will enable it to meet the requirements specified in the AMI consultation rules and the associated specifications. The selected laboratories will be compensated through fee-for-service billing by the Health Insurance system; therefore, the applicant must first ensure that it is eligible for this payment method.

Can the sequencing of the 10 samples be done in advance?

The procedures for selecting samples to be sent to the CNR and for submitting sequences and data/metadata will be specified by Santé publique France to each selected candidate following the review of the application files, starting June 30, 2021.

What are the thresholds for determining whether an allelic variant is considered (VAF, depth)?

If an allelic variant is observed for the first time in the laboratory, the mutation combinations should be noted and searched for in GISAID. If these combinations have been identified elsewhere and if the obtained sequence meets the expected specifications (minimum coverage to call a base: >10X for Illumina and Ion Torrent; >20X for MinION; Below this threshold, the base is not determined (i.e., called N); the sequence is validated if >99% of the sequence is determined (i.e., without ambiguous N bases) with an average coverage depth of 1000x and differentiation between a deletion and a coverage gap), then the result must be taken into account. Furthermore, upon detection of a variant possessing a combination of newly identified mutations, it is necessary to immediately notify the CNR to report this new detection and to determine the nomenclature to be adopted.

Regarding sequencing data, what types of data should we retain and for how long?

The choice of file type and data retention period are currently under discussion, and this information will be communicated to the selected laboratories.

Can you specify the expected structure of FASTA sequences?

For the structure of FASTA sequences, the following is expected:

• a zip archive for each sample, containing all of its individual files. A sample’s zip archive will contain:

  • the raw sequences (fastq.gz or bam)

  • the consensus genome for that sample (single-file FASTQ)

  • the variant file (VCF format)

• another archive (zip) for the files describing all samples in the batch as a whole. The zip archive for the sample batch will contain:

  • the metadata table, compliant with an upcoming version that will contain the fields required for submission to GISAID and ENA.

  • the multi-fasta consensus genomes file

  • the Nextclade assignment file generated by the platform

  • the Pango assignment file generated by the platform

How are sequences transferred to the IFB?

All files (raw reads, consensus genomes, variants, metadata) will be transferred to a trusted third-party service provider, which will ensure the encryption of all sensitive information (pseudonymization keys) and upload to the French Institute of Bioinformatics’ server, which will handle the subsequent steps (validation of compliance and quality, integration into the EMERGEN-DB database, submission to international repositories).

Since consensus sequences depend on the workflow and bioinformatics pipeline, what will be the consequence of higher localized coverage?

The specifications state that the expected requirements for sequencing are as follows:

• Minimum coverage to call a base: >10X for Illumina and Ion Torrent; >20X for MinION. Below this threshold, the base is not determined (i.e., called N);

• Sequence validated if >99% of the sequence is determined (i.e., without ambiguous N bases) with an average coverage depth of 1000x;

• If the obtained sequence is validated according to the above criteria, then there will be no consequences for higher localized coverage.