This response will be provided in 3 parts. Part 1 of 3. Before answering specific questions, the Government would like to note that two protocols were provided as supplemental information for the request for proposals, and out of those two, the saliva extraction protocol is the most desirable candidate to transition to an automated platform. However, the protocols provided were intended only as examples of what the Government may automate on a technically acceptable liquid handling platform. The intent of this RFP is to procure a liquid handler that can support automation of a variety of extraction protocols with a common set of steps, such as pipetting, agitation, and incubation, with other, less common steps to be performed offline. The list of Salient Characteristics included in the RFP lists the minimum requirements for a bid to be considered technically acceptable; potential bidders should consider the requirements listed in the Salient Characteristics over the steps of the sample protocols when the two appear to be in conflict. 1. Protocol 1 & 2 – Do incubation steps need to be automated? - Automated incubation is a requirement listed in the Salient Characteristics. 2. Protocol 1 & 2 – specifically, what steps will be done offline? - Steps that are not covered by the capabilities listed in the Salient Characteristics, such as centrifugation and evaporation, do not need to be automated. a. Does the evaporation step need to be automated? - See above

This is part 2 of 3. Note that this response required a third part. 3. Could we get specific vendor and part number for all tube types, including Oakridge, Hungate and unspecified sample tubes? - We use sample tubes from a variety of vendors while staying within the same tube sizes (2 mL and 5 mL cryo-vials, 1.5 mL or 2 mL microcentrifuge tubes). As such, we don't have specific vendor or part numbers for these tubes. Specifications for Hungate and Oak Ridge tubes are in the answer below; however, we can utilize alternative tubes as long as they meet the requirements of the protocol. a. If flexible, what are the requirements of your protocols for the steps dictated by the Oakridge and the Hungate tubes? - Hungate tubes used in the manual protocol are 16x100 mm and are capped with a screw top with septum. These tubes are made of glass and are suitable for long-term storage of extracts in diethyl ether. Oak Ridge tubes are 30 mL with round bottoms. These fit into our existing refrigerated centrifuge and are resistant to solvents, but are not suitable for long-term storage of diethyl ether. b. Are there specific materials that are unacceptable for specific steps (i.e. plastic vs glass)? - Diethyl ether is incompatible with most plastic pipets. High-density plastic containers such as Oak Ridge tubes are compatible for short periods but not for long-term storage. 4. Please list the specific (vendor/ part number) labware types you expect the temperature-controlled compartment capable of reaching 4°C and able to hold? - We do not have specific vendors or part numbers in mind, but we envision a compartment capable of incubating a full- or half- rack with tubes. Specifically within the protocols provided as examples, a rack of 8 Oak Ridge tubes or a rack of 1.5 mL or 2 mL microcentrifuge tubes would be incubated within the compartment. Other protocols may have other incubation requirements, but we still anticipate incubating racks of tubes within the size range listed in the answer to questions 3 and 3a. 5. What is the labware for the saliva samples (vendor/ part number)? - The samples will be stored in 2 mL cryo0vials prior to and after extraction, but microcentrifuge tubes and/or glass vials will be used for intermediate steps in the protocol. a. will that be the microcentrifuge tubes? or 2ml cryo-tubes or something else? - See above

Part 3 of 3 6. Protocol 2, step II: what labware will be used for this step? (vendor/part number) - Assuming this refers to the saliva extraction protocol, we intend to use polypropylene microcentrifuge tubes at this time. 7. Protocol 1 & 2—What labware (what vessels) needs to be incubated and at what temperature? Which of these incubations need to happen online versus offline? - Automated incubation and temperature requirements are listed in the Salient Characteristics.

Below are the responses. They are grouped between responses that apply to both protocols, responses for Analytes from Saliva, and finally Answers for extraction of short chain fatty acids. The following answers apply to both protocols: • Are all centrifugation steps performed offline? - Yes • Barcodes - Our current labware doesn't have barcodes, but we want barcodes on labware for the automated system sufficient to at least track whether a sample has completed the protocol. • Can pipette mixing with instrument channels be performed instead of vortex mixing? - The purpose of vortex mixing is to create a temporary emulsion between aqueous and organic solvents. Pipet mixing is insufficient to create those conditions. Answers for Extraction of Analytes from Saliva: • What is the source labware size/type? - 1.5-2 mL microcentrifuge tubes and cryovials • What are the target labware size/type? - primarily 2 mL cryovials. Microcentrifuge tubes may be utilized for intermediate steps. • Is the evaporation step performed offline? - Yes Answers for Extraction of Short Chain Fatty Acids: • What is the source labware size/type? - 30 mL HDPE centrifuge tubes (Oakridge tubes), 5 mL glass screw-top tubes (Hungate tubes), glass serological pipets • Are the source fecal samples pre-homogenized and in a slurry state? - samples are in a watery slurry state, and solids will settle after a few minutes. This protocol utilizes the supernatants of the samples, so samples are not homogenized as the time of extraction. • Should all incubations on ice be performed online or offline? - Refrigerated incubation should be performed online. See requirement 1.v of the salient characteristics for specific temperature requirements. • Are the liquid transfer volumes (i.e. 1mL – 5mL) typical for this protocol? - Yes

The solicitation deadline has been extended until Friday January 22nd, 2021 at 1700 Eastern Time.

1. Stepwise protocol to be run on the system - Our intention is to have a system that can support a variety of extraction protocols. We intend to perform certain steps manually (such as centrifugation), but we intend to automate capabilities listed in the salient characteristics. Sample Protocols will be provided in two separate responses. 2. Total sample throughput per day - Sample throughput is variable, but a minimum of 60-80 samples per day is preferred. 3. Consumables (microplates) that are used - These protocols utilize 1-5 mL sample tubes, not microplates. Anticipated consumables include sample tubes (centrifuge tubes and cryo-vials) and pipet tips. We are flexible regarding consumable brands and dimensions as long as they fit the requirements of our protocols.

No, per the equipment condition requirements, only NEW equipment will be acceptable.

No, well plates are not an acceptable alternative.