Attachment 2 Specs_NextGenSequencing.docx

Specifications for next generation sequencing project 4/29/2022 The USDA ARS National Cold Water Marine Aquaculture Center’s Shellfish Genetics Lab (Kingston, RI) requires the acquisition of next generation sequencing services to better characterize transcriptomic and epigenetic processes during gonad development of eastern oysters.

Minimum service requirements include:

· DNA and RNA Extraction and QC of 120 oyster tissue samples (approximately 10mg in size, range of approximately 5-10mg) preserved in RNAlater and stored at -80C. Note that both DNA and RNA will need to be extracted from each sample.

· Preparation and QC of 120 high-quality, indexed mRNA next generation sequencing libraries using polyA tail technology

· Preparation and QC of 120 high-quality, indexed whole genome bisulfite sequencing (or equivalent libraries produced using methylation sensitive enzymes, commonly referred to as “Enzymatic Methyl-seq”) next generation sequencing libraries with inclusion of control DNA during the library prep to measure efficiency of bisulfite (or enzymatic) conversion

· Appropriate, equimolar pooling of high-quality libraries for sequencing

· 2 x 125 paired-end or 2 X 150 paired-end sequencing of mRNA library pools with a minimum of 20 M paired-end reads per library (40M reads total)

· 2 X 150 paired-end sequencing of whole genome bisulfite sequencing library pools with a minimum of 70M paired end reads (140M reads total) per sample

· Demultiplexed data for each pool

· 60 day turnaround time