Questions and Answers Update 4_ 2.13.24.docx

Q1: When referring to: Eleven independent sequencing runs shall be performed with the Illumina NovaSeq X Plus platform using the 10B flowcell: I would kindly ask to specify if you mean that each library must be run on a single Flow Cell or we can fit the 8 libraries on the 8 lanes of the Flow Cell?

A1: Each library must be run on a single Flow Cell. We will not be sending all the prepared libraries at once and so we would expect that they would each be run on a single flow cell as they are received.

Q2. The bid shall include the cost for pick-up and transport of 14 separate shipments from the ERDC-CERL laboratory – correct?

A2. Yes. Over the total period of this award the government will provide a total of fourteen (14) libraries to be sequenced that were constructed for previously collected environmental/biological samples, utilizing the contractor provided shipping methodology and supplies.

Q3. Is FEDEX or UPS overnight air (next day) shipment acceptable? Or is a door-to-door courier service that replenishes dry ice if needed (such as World Courier) required?

A3. FEDEX or UPS overnight is acceptable if overnight shipping is available from Champaign to their location.

Q4. Will the US Army ERDC-CERL lab personnel prepare the sample(s) for overnight shipment, i.e. provide the packaging, dry ice or blue ice, etc.?

A4. The amendment includes edits to Section C.4 and C.6 of the solicitation to address this question. The government personnel will perform the packing activities, but the supplies and costs are the responsibility of the contractor and are to be included in the proposed firm fixed price, along with detail of the shipment methodology which is to be included in the proposal detail in accordance with Section L text.

Q5. Does the ERDC-CERL lab require the return of any leftover DNA library materials as part of the project scope/cost?

A5. No, we do not need leftover library materials returned.

Q6. We are performing independent research on the Combined Synopsis/Solicitation (W9132T24Q0007).

We would like to know if this is a brand new contract OR if there is (was) an incumbent performing these services.

If not brand new, could you please provide the current / previous contract number?

A6. This is considered a new contract.

Q7. Would it be possible to know which kit will be used for library prep?

A7. We do not use a specific kit for our library prep but, rather, we utilize a 2-step PCR prep protocol to add primers and adaptors with the first PCR and indexes with the second PCR. In between we do cleanups with SpriSelect beads for the size selection and the ratio varies based on the primer we are using. The end point is eventually, a pooled normalized library, with concentrations checked via QBIT. Details are provided below for an invertebrate library although methods would be similar for all primers:

A two-step PCR protocol is used to construct metabarcoding libraries following the Illumina 16S metagenomic sequencing library preparation guidelines, modified to target a 181–187 bp fragment of the invertebrate cytochrome c oxidase (COI) mitochondrial gene. For the first PCR (PCR1), we use the invertebrate-specific primers (ANML: LCO1490/COI-CFMRa) developed by Jusino et al., (Jusino et al., 2019). PCR1 reactions are performed in triplicate for all samples, negative controls (sterile, molecular grade water), and positive controls (genomic DNA; 0.05 ng/μL). Each 25 μL PCR1 reaction consists of 2 μL of DNA template, 12.5 μL of Q5® High-Fidelity 2X Master Mix (NEB), 0.5 μL of bovine serum albumin (BSA), 7 μL of sterile, molecular grade water, and 1.5 μL of each 10 μM primer. Thermocycling conditions for PCR1 included a 95°C incubation step for 5 min, followed by 35 cycles of 95°C for 30 s, 50°C for 1 min, and 72°C for 45 s, with a final extension at 72°C for 10 min. Triplicate reactions for each sample are pooled and amplification verified via gel electrophoresis. Pooled PCR1 products are size-selected and cleaned with SpriSelect using a 0.6× bead: sample ratio (right side), followed by a 0.9× bead: sample ratio (left side), and confirmed via gel electrophoresis. Unique dual indexes (10 bp) and Illumina sequencing adapters are then added to the amplified products using IDT for Illumina UD Indexes with a subsequent PCR step (PCR2).

PCR2 is performed in duplicate for each sample, including negative and positive controls. Each 25 μL PCR2 reaction consists of 2 μL of PCR1 template, 12.5 μL of Q5® High-Fidelity 2X Master Mix (NEB), 6.5 μL of sterile, molecular grade water, and 2 μL of each 10 μM IDT-Illumina UD index primer. Thermocycling conditions for PCR2 includ a 95°C incubation step for 3 min, followed by 10 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. Duplicate reactions for each sample are pooled and amplification verified via gel electrophoresis. Pooled PCR2 products are size-selected and cleaned with Ampure XP beads (Beckman Coulter) using a 0.8× bead: sample ratio and confirmed via gel electrophoresis. PCR2 samples are normalized and pooled into sub-libraries by PCR plate based on DNA concentrations determined using the Qubit dsDNA HS kit (Invitrogen). Ampure XP bead cleanups (0.7× bead: sample ratio) are performed again on each sub-library to concentrate DNA and ensure primer removal prior to sequencing. Sub-libraries are then normalized, pooled, and submitted for sequencing.

Q8. Task 3 now states “independent sequencing runs” which usually means a dedicated full flow cell. Later in the paragraph it refers to “each library must be run on independent lanes of a flow cell”. We assume that the ERDC now wants individuals and not a full flow cell per library. However “lanes” is plural and there is no definition of the number of dedicated lanes required per library submitted. For the 11 (2 x 150 bp) libraries, Is it one lane per library (1-1.25B reads per 10B lane per library) or are multiple lanes required per library? Please quantify the total number of reads or data (Gb) required per library.

Same question for the 3 SP (2 x 250bp) libraries. Since longer read flow cells are less common, we typically see dedicated full-flow cells per run. Is the ERDC also requesting just one lane per library on an SP flow cell, or are multiple lanes required per library, or a full SP flow cell per library? Please quantify the number of reads or data (Gb) required per library on the SP flow cell.

Task 3. DNA sequencing and demultiplexing of validated sequence libraries. After quality check validation (Task 2), the Contractor shall perform DNA sequencing on validated libraries. Eleven independent sequencing runs shall be performed with the Illumina NovaSeq X Plus platform using the 10B flowcell with paired-reads, 2x150 bp reagent chemistry. An additional three independent sequencing runs shall be performed with the Illumina NovaSeq 6000 using the SP flowcell with paired-reads, 2x250 bp reagent chemistry or the equivalent approach on an Illumina NovaSeq X Plus platform with flowcell and chemistry for paired-end sequencing 250 bp fragments. Each library must be run on independent lanes of a Flow Cell, although, because prepared libraries will be sent over the period of performance, it would not be expected that they would all be included on a single run but, rather, that they are run on independent lanes as they are received. ERDC-CERL prepared libraries may be included on runs with other projects/samples on a flow cell, although it is requested that individual lanes be devoted to ERDC-CERL libraries. All resulting sequencing reads shall be demultiplexed by the Contractor and provided to ERDC-CERL along with a report detailing sequencing results (general statistics, read counts, quality scores, duplication, read lengths, adaptor content, etc.). Results for each library should be received within one month of library shipment.

A8. Yes, we request that each library be run on its own individual lane NOT on its own individual flow cell.

For each 2 x 150bp library submitted, we would request one lane of a flow cell. We would expect 1-1.25B reads per library.

For each 2 X 250bp library, we would request one lane of a flow cell. We would expect approximately 750M reads per library.