What type of sample will the laboratory receive for sequencing?
If the laboratory applies for Option 1 (Retrospective with samples provided by CDC) CDC will be providing frozen cultures and the receiving laboratory will be responsible for reviving and growing the culture and subsequent DNA extraction and processing for sequencing. If the laboratory applies for Option 2 (Prospective testing of isolates within your jurisdiction) the laboratory will likely be using a liquid culture such as the MGIT culture as the source of material for DNA extraction.
Is there a protocol for DNA extraction?
Yes, CDC has provided an example DNA extraction protocol. Unfortunately it was not initially included in the RFP due to an oversight on our part. The protocol has been added as Appendix D to the updated RFP and is directly linked above. Please note that the exact protocol does not have to be used and only serves as an example.
Are there heat-killing studies that accompany the DNA extraction protocol that would limit the scope or size validations that would need to happen in a receiving laboratory?
No. The CDC performs the CTAB (cetyl trimethyl ammonium bromide) method which does not involve a heat kill step. Instead they have demonstrated that the CTAB method meets their institutional requirements for removing the DNA extract from the BSL-3 laboratory for sequencing.
Is there any concern with combining MTBC and other bacterial pathogens on the same run?
In CDC’s limited experience combining MTBC with other bacterial pathogens has been difficult due to the GC richness of MTBC and the need to add increased DNA quantity to overcome this. Therefore, in mixed runs there have been some issues with not generating enough data from the MTBC isolates on a mixed run. CDC currently runs a MiSeq at 2x250 with 16 MTBC isolates. However, we are encouraging applicants to propose protocols that aim to optimize having mixed runs of MTBC and other bacterial pathogens.
In the letter of support should the applicant define a jurisdiction that fits the minimum and maximum (Option 2)?
Yes, the intent was to have laboratories work with their TB control program to ensure they are aware and to define a jurisdiction or a restricted population that would enable the laboratory to have sufficient numbers of samples as outlined in the RFP.
If a laboratory wants to apply for Option 3-the combination of prospective and retrospective sequencing, do threshold of 192-768 isolates/year?
No, the selected site would define the number of samples to be prospectively sequenced from their jurisdiction with the remainder of samples being sent from CDC to meet the 16 sequences per month minimum.
What is the expected turnaround time?
In the RFP we have allotted for a 2-month* turnaround time for the retrospective studies (Option 1-samples sent from CDC to laboratory). It will likely take 2-4 weeks of growth after reviving the frozen culture to have sufficient quantities for a successful DNA extraction. A 3-month turnaround time has been allotted for the prospective studies (Option 2). The longer turnaround time for Option 2 was to ensure that laboratories are able to 1) get sufficient samples to have a full run (16 sequences of TB), and or 2) to provide additional time for optimizing mixed runs.
*Note to those on the informational teleconference this answer was misstated. The answer above and the RFP are correct, 2 month turnaround time for Option 1, and a 3 month turnaround for Option 2.
How will failed runs count against the turnaround time?
We are aware that there is a significant amount of work that goes into preparing MTBC samples before they are sequenced and that runs can fail for multiple reasons. This is an exploratory project, particularly as it relates to the potential for having mixed runs (MTBC with other bacterial pathogens) and we hope to learn from this pilot phase. We expect applicants to have solid plans in place to minimize failures but no one will be penalized harshly for failed runs.
What is the preferred mechanism for data transfers for the FasQ files?
Currently the plan is to use a secure ftp site for data transfer. APHL and CDC will also explore different mechanisms for data transfer that would work best with the selected sites.
Is there a reason that CDC is using FTP rather than a cloud based solution such as BaseSpace?
Yes, unfortunately they are not yet approved for use by CDC.
Who is going to be analyzing or performing the bioinformatics?
Once the FasQ files are received by CDC, the files will be run them through the scripted pipeline developed at CDC for data analysis.
Will the CDC or APHL be providing technical assistance or troubleshooting?
CDC will only be providing minimal technical assistance and troubleshooting which will occur by phone or email for the selected laboratories. Laboratories should expect to handle the majority of technical issues internally.
If we are applying for Option 2, do the isolates need to be newly identified?
The RFP states on page 2 for option 2: "Applicants may propose any jurisdiction – city, county, region, or state—for which they will sequence one sample from culture positive TB cases as they are identified." The goal is to see how WGS would work in a real-time setting so the focus should be on newly identified isolates. However, you may propose to include recently acquired isolates (within a month or two of initiation of the project) with a justification for their inclusion and it will be considered as part of the review process.
How would CDC share the WGS data with the awarded grantees? Would the information go to the program or the laboratory?
All laboratories performing testing will receive feedback regarding the quality of the sequence data.
- For those applicants that will be performing sequencing of retrospective samples from the CDC (Option 1), communications on the analysis of the sequencing will include the relevant TB control program (and jurisdictional PHL as deemed appropriate). The communication will be provided in a joint meeting including representatives from CDC/DTBE Molecular Epidemiology Activity (MEA) and the Laboratory Branch, as applicable. If the PHL performing the retrospective testing is not located within the jurisdiction of interest, they will not be included as part of the results communication without the consent of the relevant jurisdictional TB Program.
- For those applicants that will be performing prospective analysis (Option 2), there will not be any real-time analysis for genetic relatedness performed by CDC. WGS data transmitted to CDC will be archived for anticipated analysis through pipelines, including a wgMLST pipeline, currently in development. The timeframe for the finalization of these pipelines is unknown but anticipated by the end of the project period (i.e., June 2016). The PHL performing the testing retains ownership of the data and is welcome to perform their own genetic analysis if they wish.