The Association of Public Health Laboratories (APHL), in cooperation with the US Centers for Disease Control and Prevention's (CDC) Influenza Division (ID), is seeking to identify up to five (5) state or local public health laboratories (PHLs) to serve as Influenza Sequencing Centers (ISCs) in support of national influenza surveillance initiatives. The sequencing centers will perform influenza virus genomic sequencing on specimens from their jurisdictions using next generation sequencing (NGS) based methods and a data assembly pipeline approved by APHL and CDC.
Eligible laboratories include all US public health laboratories with the following capabilities, resources and facilities in place. Specific expectations regarding the methodologies to be used by the reference center are outlined in Appendix A: Expectations for Influenza Sequencing Center. All applicants are required to agree to the following minimum requirements (as outlined in Appendix B: Minimum Requirements for Influenza Sequencing Center):
Surveillance networks are in place to receive specimens required to sequence 500 specimens per year;
Sufficient equipment, laboratory space and workforce capacity for the proposed work;
Established capacity to perform influenza NGS while meeting expected quality metrics;
Willingness to alter or amend existing sequencing protocols;
Willingness to increase frequency of performing certain methods (if required) to meet expected turnaround times;
Ability to collect all required metadata;
Willingness to share copies of QA or biosafety documentation associated with relevant procedures to APHL and CDC upon request;
Informatics capabilities to stream NGS read-level data to APHL Informatics Messaging Services (AIMS) cloud-based environment in near real-time using the Amazon Simple Storage Service (S3) Utility synchronization tools with IT support;
Ability to upload consensus genomic data to public database with prescribed naming convention and required metadata;
Ability to retain residual clinical specimens (properly stored, not inactivated) for up to 12 months post-selection for sequencing and provide CDC with specimens, upon request, for additional characterization/use at CDC;
Ability to contract with APHL have an existing relationship with a third party that can contract directly with APHL on behalf of the laboratory.
Anticipated RFP Schedule
November 11, 2022 – RFP Issued
November 29, 2022 – Informational teleconference (optional)
December 2, 2022 – Letter of Intent Due to APHL (see below)
December 12, 2022 – RFP Responses Due
January 6, 2023 – Proposal review completed
January 11, 2022 – As needed, follow-up interviews/proposals due
January 12, 2022 – Final review completed and awardees selected
January - June 2023 – Contract year one (training and proficiency testing)
APHL will communicate any modification to this anticipated schedule on APHL's procurement website (www.aphl.org/rfp) and via an email blast to public health laboratories (PHLs).
Confirmation of Intent to Respond
APHL requires that prospective applicants submit a brief email statement indicating an intent to submit a proposal. APHL must receive this email by no later than
5:00 pm EST on Friday, December 2, 2022. To allow for appropriate review process planning, a letter of intent is required for consideration.
APHL must receive complete responses by
5:00 pm EST on Monday, December 12, 2022. Please see Proposal-Required Submissions section for items that must be included in the completed proposal. Applicants may send proposals via email to
APHL will send an email acknowledging the receipt of your application; if you do not receive an acknowledgement within 2 business days, please email the RFP point of contact above to confirm receipt.
The official RFP document will provide detailed information in regards to this request, please read it in its entirety.
Questions and Answers
Is there an expected turnaround time for influenza sequencing results to be shared with the CDC analysis pipelines?
A:The intent is to have a regular production of influenza viruses throughout the season. It depends less on turnaround time and more on a regular, weekly run of sequencing (primarily during the influenza season).
What is the weekly or monthly testing expectation for funded sites?
A:Ideally, a maximum of 90 viruses per week (96 well plate with controls) per week. However, regular production of sequences thorough different time points in the season is important.
Does CDC have a pre-screening PCR assay and Ct value cutoff for positive samples to determine whether they are appropriate for sequencing?
A: CDC does not have a required pre-screening assay. PCR results with a Ct <28 for influenzas A or influenza B will work well for sequencing.
Are there specific extraction platforms that are required for the amplicon sequencing method that will be shared by the CDC? If so, what are the required platforms?
A: There is not a requirement for the extraction method. CDC has found that most extraction methods used by public health labs are sufficient at providing good quality RNA.
Can the CDC share their sequencing procedures and quality assessment steps with labs prior to the RFP response due date?
A:Yes; CDC can share wet-lab protocols prior to the RFP due date. CDC will provide a summary of quality metric thresholds. Please reach out to Melissa Warren (email@example.com) if you would like this information.
Please share the metadata elements that need to be submitted to Clarity LIMS.
A:In general, the metadata needed will be the same as requested on the current CDC flu submission form.
What types of data will be transmitted through the APHL AIMS S3 bucket?
A:The raw data from the instruments to be transmitted through the APHL AIMS S3 bucket.
Are you looking for long or short read sequences?
A:The platform has not been predetermined, however based on CDC’s experience the preference is short read sequences because they yield better data quality overall.
Would Google Cloud Platform (GCP) be able to be used instead of Amazon Web Services (AWS)?
A:The plan is to utilize APHL’s current cloud-based infrastructure for initial assembly which uses an AWS framework.
Can you provide more information about Clarity LIMS? Does the lab need to have Clarity LIMS or will Clarity LIMS be hosted on the AIMS platform for us to use?
A:CDC has used Clarity LIMS as a way to understand how viruses were processed and handled in the NGS pipeline. This helps CDC with troubleshooting and understanding quality of sequence. This is not like a LIMS that contains comprehensive metadata that contains patient sample information; rather it manages process of NGS workflow. There is a cloud pipeline system that the National Influenza Reference Centers (NIRCs) use in which CDC hopes to use for this project. The initial plan is to use the cloud-hosted environment that only requires web-based access to reach this instance of Clarity LIMS. If you already have experience with that system it would be a familiar interface, if not, there would be a little bit of a learning curve. It helps with batch operations with sequencing.
Can we access a cloud-based version of Clarity hosted by APHL/CDC rather than purchasing and maintaining our own copy of Clarity?
A:Yes, this project will be utilizing the cloud hosted version from APHL that communicates with the AIMS cloud.
Will there be a standardized analysis workflow such as using IRMA?
A: Yes; CDC envisions that we will use similar standards as currently used with the NIRC – Clarity LIMS to feed into CDC’s IRMA curation pipeline.
Will there be a training session such as clarity LIMS or analysis for the sites selected?
A:Post-selection, CDC will provide training on SOPs and Clarity LIMS. CDC provides sequencing proficiency panels to NIRCs and may do the same for the ISCs.
Is the estimate of 500 for total samples tested or sequenced?
A:The estimate is 500 sequences generated from each site. There could be more or less depending on the season and the contract with APHL would stipulate the maximum.
Is there a focus on solely sequencing the most common influenza type or are all types expected to be sequenced?
A:Diversity in virus type and subtype is important. CDC will provide protocols for influenza A and influenza B and CDC hopes to get a variety of viruses.
Could you expand upon the multi-segment RT-PCR enrichment step?
A:This is the backbone of CDC’s protocols for enriching and amplifying the influenza genome using the conserved ends to amplify the entire genome. CDC will provide an SOP for influenza A and influenza B viruses – they are slightly different but use the same thermocycling parameters.
If we are a participatory site, is submission of samples to the NIRC still needed?
A:Yes; NIRCs do additional phenotypic characterization on submitted viruses which may be grown as candidate vaccine viruses. Submission to NIRCs must be maintained. ISCs will submit a subset of samples to the NIRCs and sequence additional specimens from their own jurisdiction themselves.
What needs to be included in the letter of intent?
A:You just need to notify us that you intend to apply. A one or two sentence email will suffice. APHL will respond to confirm receipt of your letter of intent.
Can we submit NextSeq data which delivers 8 files per sample instead of 2?
A:It is a possibility for this project – some changes would need to be made in the existing Clarity system first. The data transfer would be the full run output – the fastq data is still a derivative, so you need to account for full run data.
Can older specimens (collected in 11/2022 or 12/2022) be included?
A:No, CDC is interested in the most current material. Old viruses from the freezer do not have much use for this project. Representativeness and timeliness of data is important. CDC is looking at what viruses are trending and forecasting what will appear next season.
We created a budget for our department of sponsored programs this morning and realized there will be no revenue until invoicing for sequence generated, however we will spend months possibly the whole six months without any revenue, which is problematic because the DSP sets this contract up as a grant. Is there any possibility to request upfront development funds prior to sequence generation? Or would we be able to sequence frozen specimens collected during this season when we get test live to generate revenue before the end of the 6 month period?
A:APHL will set up contracts with selected sites in early 2023. Upon completion of a proficiency panel, sites would be able to invoice for reimbursement for sequencing. APHL does not have an advance contract payment plan set up at this time.
To confirm, this would be for samples in our own jurisdiction only, correct?
Would CDC be submitting sequence data to GISAID/NCBI or would the ISC be responsible for data submission to public repository? It sounds like CDC will be assembly the raw read data.
A: In the initial stage of the project, CDC will be submitting consensus sequence data to public repositories. In the future, ISCs will be encouraged to submit their own data that meet a standard set of quality metrics.
What is the CDC's target turnaround time from the collection date to submission?
A: It depends on your laboratory and submitter networks; CDC does not have specific requirements but would like them to be as timely as possible. Do not sequence viruses that have been in the freezer for a long time. The emphasis is on performing timely routine sequencing throughout the influenza season.
To streamline, we could bypass normal test development and validation and just run the CDC proficiency panel and go live?
A:That’s correct. CDC will provide all the protocols you need. None of this information is going back to patients so it does not fall under CLIA.
Will APHL/CDC send us 500 flu samples to sequence or would you expect samples to come from our lab?
A:No, you would not be sent specimens for sequencing. The ISC is responsible for collecting specimens from their own jurisdiction’s submitter network for this purpose.
We have approval for a few different ways to share Influenza reads with the AIMS S3 bucket. I see there is a guidance doc for sharing via the AIMS AWS S3 bucket option on APHL from 2015, is this the most up to date guidance for preferred sharing and if not would you be able to send us updated specifics?
A:The guidance for data transfer from labs to AIMS has not changed; the 2015 guidance is still appropriate.
Would AIMs be ok with transfer from our GCP bucket storage?
A:APHL is OK with transferring data from GCP bucket to AIMS S3 bucket.
Would AIMs be ok with transfer from our GCP bucket storage?
A:APHL is OK with transferring data from GCP bucket to AIMS S3 bucket.
Is there architecture of the solution in AWS cloud?
A:Please see the below document for an overview of AIMS, the AIMS S3 Client and clarification on the steps required to install and configure the AIMS S3 Client. AIMS - S3 Transport *NIX CLI Configuration Guide - Version 2 It is also possible to use the Amazon Web Services (AWS) CLI interface or one of the AWS SDKs (Java, .NET, etc) to connect to the AIMS S3 location. This information is from the NIRC RFP in 2015, but they are still accurate.
Is there any Personal Identifiable Information (PII) in the dataset being exchanged with AIMS?
A: No PII will be transferred through AIMS for this project. Only sequencing data would be part of the dataset transfer to AIMS. Any metadata requested will be most likely limited to the fields requested as part of the WHO surveillance specimen submission forms that you currently use to submit specimens to CDC and NIRCs.
We currently have an FTP-based interface with AIMS for eCR transactions, and our lab is using this same interface to transmit/test ARLN lab results with APHL. Can this same interface be used for the influenza data, or must CrossFTP be installed per the attached PDF to connect to Amazon S3?
A: This project uses AMD specific S3 bucket. CrossFTP is not preferred. This project will use the AWS CLI tool to exchange data to the influenza S3 bucket.
My understanding is that after the primary selection, APHL ISC committee will bring the PH laboratories and their respective IT personnel to set up the AIMS S3 hosted by APHL. Will there be any formal training or instruction for this?
A: Yes, APHL will work with you to provide documentation and instruction for how to set things up.
Our IT’s major concern is that they do not have any prior experience in AWS cloud network service though we know that most of this cloud-based data transmission pipeline will be operated by our sequencing team. Would you suggest anything that we can explain them to ease their concern.
A: From what we have done for the National Influenza Reference Centers and another pilot site, the ongoing support is somewhat minimal; IT engagement is required for initial setup of a data synchronization process with AIMS cloud storage (APHL informatics technical assistance is available), but the ongoing data transmission occurs by a script or schedule job that syncs sequencing data automatically.
Will funding be provided for our IT Department as well to assist with the projects to whatever extent they would need should we be approved to become an Influenza Sequencing Center?
A: Compensation will be $150 per specimen per sequence attempted, with an anticipated maximum of 500 specimens per year. The use of funds is at the discretion of the recipient laboratory for how it is disbursed through your organization. No additional funds are provided specifically for IT department.
Is indirect an allowable use of funds?
A: The ISC contracts would be written in such a way that the ISC would invoice APHL for specimens sequenced during a set time period (e.g. monthly). The ISC contract payment would essentially be a reimbursement at the set rate per specimen, so how the funds are used once they arrive at the ISC are at the discretion of the laboratory.
Is there an "east" Amazon S3 that labs in that region would connect, or does everyone use the "west" web address?
A: AIMS has services in AWS West and East. The ISCs will receive S3 addresses based on their region.