Contractor Questions and Answers FDA-CDER-25-128751_07102025.pdf

FDA-CDER-25-128751 Questions and Answers 7/10/2024

Q. Can an alternative sequencing platform to Illumina be used.

R. No.

Q. On the pricing table below – we are trying to understand why there would only be basic analysis for only 400 of the 800 samples? Or is basic analysis required for all 800 samples?

R. Basic analysis is required for all 800 samples.

Q. Please explain the “6” data handling items. 6 units of what? We assume you want FASTQ files and bioinformatic analysis data uploaded for all 800 samples, versus 6?

R. Number can be arbitrary for certain files (e.g., QC reports and basic analysis). Yes, required files for PE sequencing should include raw data files, QC reports for raw data, and mapping output for aligned data and alignment statistics.

Q. Will all 800 samples be provided in a single batch, or (perhaps?) spread equally across 6 batches over the 12 months?

R. All RNA samples will be delivered to the contractor at one time or as a single batch. The number of samples per sequencing run will be determined by the provider’s ability to generate approximately 100 total million reads per sample and across sample groups/boxes supplied.

Q. There are no specifications provided on the choice of Total RNA kit. Is a well published commercially available Total RNA kit required (i.e., Illumina), or is a homebrew kit acceptable?

R. Total RNA will be extracted in-house, and RNA samples delivered to the contractor. Receipt of high quality and sufficiently concentrated RNA should be confirmed by the provider following receipt and before further processing.

Q. Does the USDA require the use of full reaction sizes (per commercial kit supplier recommendations) or can the supplier use a smaller reaction size to reduce cost?

R. The contractor can propose a reaction size as long it meets the ~100 million total reads (~50 million paired end read pairs) per sample."

Q. Would these samples be submitted in a single batch or multiple batches?

R. While all RNA samples will be supplied in a single batch, the number of samples per sequencing run will be determined by the provider’s ability to generate approximately 100 million total reads per sample in sample groups/boxes supplied.

Q. Do you have the expected RNA yields?

R. Expect min-max RNA yields of 0.4 µg – 1µg.

Q. Can you confirm what the QTY of 6 means in the data handling in Pricing Table?

R. Number can be arbitrary for certain files (e.g., QC reports). This refers to cost for delivery of required files and basic analyses for PE sequencing including raw data files, QC reports for raw data, and mapping output for aligned data and alignment statistics.

Q. Which line item should we include the return shipping?

R. Return delivery of the RNA samples will be paid for by us.

Q. What extraction kit was used for the Total RNA extraction? Is this an optional service line you would like to see pricing for?

R. RNA extraction will be done in-house; it is not required as a service. We are currently optimizing the RNA extraction method to obtain high quality (high RIN) total RNA. Receipt of high quality and sufficiently concentrated RNA should be confirmed by the provider following receipt and before further processing.

Q. Can we deliver via AWS bucket or a self hosted sFTP, instead of Box folder?

R. Yes, a self-hosted sFTP for delivery of data can be used in place of box folder.

Q. Are samples already extracted and DNAse treated?

No. RNA samples are not extracted. Pure (260:280 ratio ~2.0) and high quality (high RIN) total RNA samples will be supplied.

Q. Is 50bp read length acceptable or is 150bp required?

R. PE 150bp sequencing is required.

Q. Is 800 samples the required number? Or can it be another number divisible by 96?

R. While all RNA samples will be supplied in a single batch, the number of samples per sequencing run will be determined by the provider’s ability to generate approximately 100 million total reads per sample across sample groups/boxes supplied.