Statement of Work.pdf

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STATEMENT OF WORK

1. Background

The US Geological Survey is investigating contaminant Polychlorinated biphenyl (PCB) health effects thresholds for Pacific herring (Clupea pallasii). Herring and other fishes in Puget Sound have high tissue concentrations of PCBs, yet we know little about related biological impacts. The USGS will conduct a controlled laboratory study, first exposing herring to environmentally relevant concentrations of PCBs through their food, then introducing a disease challenge to PCB-exposed fish. Sub-lethal genetic responses in herring will be assessed following PCB exposure by using an RNA-seq experiment, and immunological impacts of PCB exposure on disease challenged-fish will be determined from functional responses using RNA-seq in lymphatic tissues. To facilitate differential expression analysis, we are requesting next-generation sequencing services on fish tissues.

2. Scope of Work

A. Sample matrix and numbers

Up to sixty tissue samples of three types will be supplied to the sequencing facility preserved in RNAlater® solution, or comparable: 20 each of liver, kidney, and spleen.

B. Timeline

• Tissue samples are expected to be delivered to the chosen sequencing facility between

September 1, 2020 and December 31, 2020.

• mRNA-seq experiments and data delivery should be completed within 3 months of receipt of samples at the sequencing facility.

• The overall Period of Performance is from 09/01/2020 to 12/31/2021 with the possibility of additions, modifications, and a 1-year renewal option.

C. Methods for mRNA-seq (Specific Requirements)

RNA extraction

• RNA will be extracted from tissues and enriched using an mRNA-specific library preparation method.

• Extraction should follow kit-specific protocols for total RNA isolation. Extraction kit selection is at the discretion of the sequencing facility and should be specified with the final data.

• Extracted RNA should be checked for integrity after isolation using an Aligent Bioanalyzer, or similar, with a minimum RIN number of 7 and RNA concentration reported (target of >1µg). The USGS can supply additional tissue if RIN or RNA concentration is too low for sequencing.

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RNA Library Prep

• Library preparation is sequencing platform-specific, and must be equivalent to an Illumina

RNA TrueSeq mRNA preparation to reduce non-coding RNA sequences.

• Samples must be individually barcoded (multiplexed), so that expression is comparable among samples within a specific tissue type (liver, kidney, spleen).

Sequencing

• Sequencing for the RNA-seq experiment will follow standard protocols.

• Multiplexed samples from a particular tissue type (liver, kidney, spleen) should be sequenced in a single run to minimize inter-run variability. Within-run variability should be minimized by using a “balanced block” design, which is accomplished by sequencing all 20 multiplexed libraries for a specific tissue type across all lanes of the utilized sequencing platform, such as the Illumina NextSeq 500 platform or similar, with a minimum read depth per sample of 20 million (400 million per run) and a minimum read length of 75bp (paired).

• At least three independent sequencing runs are required, one for each tissue type.

Quality Assurance and Quality Control (QAQC)

• The chosen laboratory must have internal QAQC procedures that, at minimum, include reporting of sample-specific RIN number, total RNA concentration, and any QC samples required by platform-specific protocols, such as the use of PhiX sequencing controls.

• Extracted RNA should be checked for integrity after isolation using an Aligent Bioanalyzer, or similar, with a minimum RIN number of 7 and RNA concentration reported (target of >1µg).

Reporting requirements

• Sequencing reads must be demultiplexed and delivered in sample-specific FASTQ files to the

USGS via download or deliverable on a hard drive.

• Sample-specific RIN, total RNA concentration, barcode sequences, and run-specific QC summary files will be delivered to the USGS via download or deliverable on a hard drive.

• Extraction kit information will be reported to the USGS.

3. Period of Performance

The Period of Performance is from 09/01/2020 to 12/31/2021, with a 1-year renewal option.

4. Modifications

An option period may be exercised which would include 60 additional samples, subject to availability of funds. The extended option Period of Performance will be 1/1/2022 through 12/31/2022.