Musselwatch_RNA_seq_SOW_Final.docx

STATEMENT OF WORK

TITLE: Acquisition of services for RNA-sequencing

I. BACKGROUND INFORMATION

The National Center for Coastal Ocean Science (NCCOS) Key Species and Bioinformatics Branch serves as a leader in near ocean and estuarine research to protect and understand the coastal waters of the U.S. in an effort to enhance the nations blue economy, improve public health and safety, and to conserve our natural resources and environment.

The Key Species and Bioinformatics Branch is part of the Stressor Detection and Impacts Division within NCCOS and is responsible for characterization of environmental stressors (both natural and anthropogenic) and accessing the impact of those stressors on the near ocean ecosystem. Current areas of research within SDI include toxicological impacts on key ecological species of contaminants of concern (both novel and legacy).

II. SCOPE OF WORK

RNA Sequencing

The NCCOS Key Species and Bioinformatics Branch in Charleston SC, is seeking quotes for library preparation and sequencing for samples. We estimate the need for processing approximately 200 individual RNA samples. Library preparation should include polyA purification, cDNA synthesis, shearing/fragmentation, size selection, barcoding using a unique multiplexing indexing (UMI) strategy to reduce barcode hopping, pooling, and library validation prior to sequencing. Targeted sequencing depth for this project approximately 50 million read pairs per sample from the Illumina NovaSeq platform with 150 x 150 bp paired end reads. Deliverables should be raw fastq files. Currently no bioinformatics support for read cleaning, read mapping, assembly, or analysis is required. However, it is desirable that the contractor offer those services. The 200 samples will be submitted for library preparation and sequencing in a minimum of two separate batches : 1) a batch of 150 total RNA samples of the quagga mussel (Dreissena bugensis) and 2) a batch of 50 total RNA samples of adult grass shrimp (Palaemon pugio). Fastq files containing sequencing data should be provided to the end user within 2 months of receiving each batch of samples and sequencing providers should allow a timeline of up to one year after the initiation of the contract for the submission of sample batches.

SPECIFICATIONS

The Contractor shall provide the Government Contracting Officer Representative the following:

· Sequence data in the form of FASTQ files. This must consist of a minimum of 50 million read pairs (i.e., 50 million 2x150 bp paired-end reads) per sample of nucleic acid sequence, that pass initial quality assessment in the Illumina instrument quality assessment pipeline from stranded mRNA RNA-Seq performed as specified in this contract, and is of acceptable quality level.

· Clear association of reads with a sample ID provided by the Key Species and Bioinformatics Branch.

· Clear descriptions of all methods used must be disclosed to the Key Species and Bioinformatics Branch as would be expected for inclusion of sequencing data in the peer-reviewed literature (e.g., instrument/kit used).

The Contractor shall generate the sequence read data in compliance with the following performance requirements.

· Stranded RNA-Seq sequencing must generate paired end reads of at least 150 bp (2 x 150 bp).

· All libraries must be constructed using the same techniques/kits, and all libraries must be sequenced on the same platform, unless negotiated with and approved by the Government Contracting Officer Representative.

· Prior to or as part of library preparation, total RNA samples must be ribosomal RNA depleted or polyA selected. That is, the Contractor will perform mRNA RNA-Seq.

· Library preparation should follow standard industry-accepted practices; any custom approaches of the Contractor that could impact sequence quantity or quality, or that could result in sequencing bias, must be discussed with and agreed to by the Government Contracting Officer Representative.

· Standard techniques should be used to assess library quality, and shall be provided to the Key Species and Bioinformatics Branch if requested (e.g., bioanalyzer traces).

· Standard quality assessment/control approaches associated with library construction and sequencing must be performed by the Contractor.

· Sequence data must be collected on Illumina HiSeq, NovaSeq, or NextSeq instruments.

· Multiplexing is allowed within the parameters of the individual instrument being used to yield the minimum number of acceptable quality reads per sample. If multiplexing is performed, the Contractor must demultiplex clean reads, and ensure clear association of reads with the sample ID provided by the Government Contracting Officer Representative.

· Sequencing data must be returned to the Branch in FastQ format in clean (i.e., passing Illumina’s instrument quality assessment pipeline) de-multiplexed form. The above requirement for no less than 50 million paired-end (2x150 bp) reads applies to the clean data.

· Appropriate levels of randomization to mitigate potential batch effects and use of spike-in controls, are to be negotiated between the Contractor and Government Contracting Officer Representative.

The Contractor must provide a description of laboratory facilities and key equipment that will be used to complete the work. The Contractor must possess Illumina sequencing equipment (HiSeq, NovaSeq, and/or NextSeq and any necessary supporting equipment – i.e., the platform the Contractor will use for sequencing), equipment for quality and quantity assessment as appropriate for library preparation from total RNA and sequencing, and equipment for library construction.

The Contractor must provide evidence that it has performed research similar or greater in scope to that described in this Statement of Work, including preparation of mRNA RNA-Seq paired end read libraries and Illumina sequencing of mRNA samples.

The Contractor must be able to process samples (including all steps for conversion of total RNA sample into sequence data) at an average rate of 100 samples/month or faster.

III. PERIOD OF PERFORMANCE

The period of performance shall be 6/30/2021.

IV. PLACE OF PERFORMANCE

All work shall be completed at the Contractor’s facility. Delivery of the resulting data shall be submitted electronically to NCCOS, Charleston, SC.

V. DELIVERABLES

VI. PERFORMANCE REQUIREMENT SUMMARY

Prior to final acceptance of the data and final payment by NCCOS, all of the following shall be completed successfully:

· Sequence data in the form of FASTQ files. This must consist of a minimum of 50 million read pairs (i.e., 50 million 2x150 bp paired-end reads) per sample of nucleic acid sequence, that pass initial quality assessment in the Illumina instrument quality assessment pipeline from stranded mRNA RNA-Seq performed as specified in this contract, and is of acceptable quality level.

· Clear association of reads with a sample ID provided by the Government Contracting Officer Representative.

· Clear descriptions of all methods used must be disclosed to the Government Contracting Officer Representative as would be expected for inclusion of sequencing data in the peer-reviewed literature (e.g., instrument/kit used).

VII. RISK ASSESSMENT

The HSPD-12 Security Risk Level assigned to this Task Order is: No Risk Level Required

· PERSONNEL

Technical Point of Contact (TPOC):

Name: Thomas Greig Phone: 843-460-9660 Email: thomas.greig@noaa.gov