Q_and_A_W81XWH-17-T-0135_GOV_Response.pdf

Q&A Solicitation W81XWH-17-T-0135

1. Q: Is the subject effort considered a new requirement or a follow-on to an existing requirement?

A: This is a new requirement.

2. Q: Would you mind providing the incumbent contractor’s name and contract number?

A: N/A

3. Q: Is an incumbent contractor currently performing these services or is this a new requirement?

A: See answer to 1 and 2.

4. Q: If there is an incumbent, could you please provide the contract number?

A: See answer to question 2.

5. Q: My question relates to the proposal due date posted of 8 March 2017, listed on page 12 of the draft solicitation, Para L.1.3. If contractor questions are received by 28 Feb 2017, and a final RFP is released after that date, is a proposal response date of 8 March 2017 realistic, or will a new proposal receipt date be published in the final RFP?

A: See Solicitation Amendment 0001 for the revised proposal due date.

6. Q: Number of samples (If submitted in batches, number of samples in each batch)?

A: Number of samples: 45 for whole-metagenome sequencing and 45 samples for metatranscriptome RNA-sequencing. Due to the nature of the study, the samples (DNA/RNA extracts) will not be provided as a single big batch. Instead, samples will be provided in batches. Number of samples in each batch may vary.

7. Q: (What is the) Starting material? Purified genomic DNA, tissue, cell pellet etc.

A: The samples provided for library preparation and sequencing will be purified microbial genomic DNA and RNA extracts.

8. Q: (What is the) Required coverage per sample? Depending upon the complexity and project goal, we recommend anywhere between ~6Gb to ~20Gb per sample.

A: The complexity (microbial abundance, richness, and composition) of each sample’s microbiome will vary. Having sufficient coverage for downstream analysis is critical for the success of this project. As mentioned in 2.3.2, any modifications or adjustments to the parameters of sequencing run will be in accordance to the microbial biodiversity and species richness of each specimen.

For the Metagenome study: Considering challenges associated with host DNA contamination of microbiome, we need Paired-end reads (PE) at 150bp reads with the capacity of about 300 million reads for each sequencing run and a metagenome coverage of 50 to 100x so we can capture fungal, viral, archaeal, and bacterial genomes.

For the metatranscriptome study: Considering the size of mRNA molecules, we prefer PE at 100bp reads.

The capacity needed for proper sequence coverage will be based on the biodiversity we observe for each sample from the metagenome portion of the study.

9. Q: Is it too late to respond to this solicitation?

A: See Solicitation Amendment 0001 for the revised proposal due date.

10. Q: How many total samples will be processed under this solicitation? What is the per sample sequencing depth that is expected?

A: We will have about 45 samples for the metagenome and 45 samples for the metatranscriptome study. We want up to 100X coverage with 150bp reads to ensure sufficient sequencing of fungal, viral, archaeal, and bacterial genomes.

11. Q: Will we be allowed to have access to de-identified metadata for these samples to enable an integrated analysis of the sequence data and metadata?

A: Yes. In order to perform biologically meaningful analysis and comparison of the metagenomics and transcriptomic data, the metadata will be provided.

12. Q: Can the Government confirm acquisition timeline and vehicle?

A: See Solicitation Amendment 0001 for details, full and open competition.

13. Q: How will DNA/RNA be extracted/stored/shipped?

A: DNA will be isolated using chemical and enzymatic lysis and the DNA will be eluted in water, which should be compatible with any library preparation work flow. We are currently setting up the microbiome RNA isolation pipeline. It will likely include bead beating and the use of well-established microbiome RNA isolation kits.

14. Q: E.g. Will Trizol be used?

A: We are currently optimizing this portion of the project. If Trizol is used, RNA will be purified and eluted in PCR-grade water.

15. Q: Will there be any treatment of samples prior to shipment to site?

A: The DNA and RNA extracts will be purified and eluted in water and provided for sequencing.

16. Q: For items 2.2.2 and 2.2.3, are these separate steps for library preparation, or to both be performed on all samples?

A: The preference is to use step 2.2.2 for DNA library preparation. If the quantity of DNA is too low, use step 2.2.3 instead.

17. Q: If samples are to be treated by EITHER 2.2.2 or 2.2.3, what are expected cutoffs to use?

A: 2.2.2 technology should be used on all the DNA samples. However, if the DNA concentration is too low proceed with 2.2.2. As for the exact cut off, it would be based on the parameters stated in the library preparation kit described in 2.2.3.

18. Q: Is (the) desired analysis different for sequencing outputs from 2.2.2 and 2.2.3?

A: There ought to be no differences in the sequencing and analysis approach.

19. Q: For DNA metagenomics analysis, are there identified reference sequences for the reference based assemblies described?

A: We expect that the computational group will perform both reference-based as well as de-novo assembly as part of the metagenomics analysis tasks. There are comprehensive prokaryotic, fungal, and viral databases available that can be used for reference based analysis. We expect that the computational group will custom create a reference sequence database for each kingdom by compiling the reference data bases from various sources (e.g., NCBI) including the human microbiome project.

20. Q: Will DNA/RNA be paired (from same sample/same individual)?

A: Yes, the DNA and RNA should come from the same individuals, which then allows for overlaying of the pathway landscape (metatranscriptome) with the metagenomics data.

21. Q: Will RNA/DNA be shipped at same time? (note that deliverable dates for metatranscriptomics are all in year 2) A: Due to difference in storage and processing of DNA- and RNA-destined samples, the DNA specimens will be provided first for whole-genome sequencing; while the DNA samples are being sequenced and analyzed, the RNA extracts will be sent. We expect to devote year 1 to metagenomics sequencing and analysis and year 2 for metatranscriptomics analyses.

22. Q: Will there be longitudinal samples as part of this study?

A: No, these samples are from one-time collection.

23. Q: Are publications expected from this work?

A: Yes.

24. Q: Will contracted groups be involved in preparation of manuscripts, if so?

A: No. The contracting group will conduct the required computational analyses and provide the needed data to ISR investigators, in the appropriate publication-ready format (e.g., graphs and charts). We will also obtain the raw data (FASTA and FASTQ files) for all of the sequencing runs.