Attachment 1 SOW_NextGenSequencing.docx

Statement of Work for Next Generation Sequencing Background. The USDA ARS NCWMAC Shellfish Genetics program is conducting research on eastern oysters in order to assess transcriptomic and epigenetic dynamics during gonad development. Eastern oysters are sequential hermaphrodites and the genetic regulatory structure that controls the sex of an oyster in a given season is not understood.

Project objective. The primary objective of this work is to use next generation sequencing (NGS) to identify differentially expressed genes and differentially methylated regions between males and females during gonad development. Mantle tissue has been collected from 120 eastern oysters during gonad development and phenotypic sex was later identified. DNA and RNA will be extracted from oyster tissues. The RNA will be used to construct, QC and sequence RNA-seq libraries for next generation sequencing. The DNA will be used to construct, QC and sequence whole genome bisulfite sequencing (WGBS) libraries (or equivalent libraries produced enzymatically). USDA ARS will use the provided sequence data to identify differentially expressed transcripts or differentially methylated regions between male and female oysters.

Scope of work. The project requires DNA and RNA extraction (both from all samples), library preparation, and paired-end sequencing of 120 samples for both RNA-seq and WGBS. The RNA-seq libraries will be sequenced with 2 x 125 bp or 2 x 150 bp reads and target 20 million paired reads per sample (40 million reads total). The WGBS libraries will be sequenced with 2 x 150 bp reads and target 70 million paired reads per sample (140 million reads total). Sequenced data will be demultiplexed.

Detailed technical requirements (Contractor Requirements). USDA-ARS will submit 120 tissue samples preserved in RNA later and stored at -80C to the contractor. The contractor will perform and QC DNA and RNA extracts. The contractor will construct and QC mRNA libraries using polyA tail technology, index and pool samples appropriately, sequence pools to generate a minimum of 20 M paired-end reads (40M reads total) per sample, and demultiplex sequence data. The contractor will construct and QC WGBS libraries with inclusion of contractor supplied control DNA to evaluate conversion efficiency, index and pool samples appropriately, sequence pools to generate a minimum of 70 M paired-end reads (140M reads total) per sample, and demultiplex sequence data. The contractor will deliver the demultiplexed sequence data to USDA ARS.

Deliverables. The contract will be considered complete upon receipt of high quality, demultiplexed sequence data for each sample.

Reporting schedule. The sequencing data should be sent to Thomas A. Delomas, USDA ARS NCWMAC Shellfish Genetics, 483 CBLS, 120 Flagg Road, Kingston, RI 02881, or posted on a private server for download within 60 days of sample submission.

Period of performance. June 1st, 2022 – July 30th, 2022