The Environmental Protection Agency, National Center for Computational Toxicology has a continuing requirement for support for targeted RNA-seq to evaluate treatment-related changes in gene expression in cells in culture. Examples of targeted RNA-seq approaches include, but are not limited to TempO-Seq and TruSeq RNA Exome. The EPA is interested in the contractor performing multiplexed assays that measure transcriptomic changes for up to 20,000 – 21,000 protein-coding genes using targeted RNA-seq directly on cell lysates or purified RNA samples.

EPA’s National Center for Computational Toxicology coordinates the High Throughput Toxicology (HTT) Research Program, which is part of EPA’s broader Chemical Safety for Sustainability (CSS) Strategic Research Action Plan. Chemicals are a lynchpin of innovation in the American economy. Moving toward sustainable development requires designing, producing, and using chemicals in safer ways. Information and methods are needed to make better-informed, more timely decisions about chemicals. The EPA’s HTT research program within CSS is designed to meet this challenge. Current chemical testing is expensive and time-consuming, only a small fraction of chemicals have been fully evaluated for potential adverse human health effects. NCCT is working to figure out how to change the current approach used to evaluate the safety of chemicals. NCCT research integrates advances in biology, biotechnology, chemistry, and computer science. Automated chemical screening technologies (called “high-throughput screening assays”) and informatics are being used to screen thousands of chemicals and identify important biological processes that may be disrupted by chemicals. Mathematical and advanced computer models are being developed to trace those disruptions to a related dose and human exposure and help link perturbations in biological processes to adverse health impacts. The combined information helps prioritize chemicals based on potential human health risks. Using HTT testing and computational methods, thousands of chemicals can be evaluated for potential risk at a small cost in a very short amount of time while also limiting the number of laboratory animal-based tests.

The contractor shall perform the following tasks:

• Targeted RNA-Seq Libraries and Sequencing
  • Targeted RNA-seq methods should provide multiplexed expression measurements for up to 20,000 – 21,000 protein-coding genes per sample.
  • The contractor shall provide options for flexible scaling of the number (ranging from 100 to 21,000) and identity (selected by EPA from vendor-supplied probe inventory) of protein-coding genes measured in the targeted RNA-Seq assay and incorporate such options into the pricing schedule.
  • The contractor shall be capable of generating supplemental read depth to previously prepared libraries and provide supplemental pricing for such services.
  • The contractor shall be capable of providing multiplexed expression measurement for a variety of species of interest to EPA including human (Homo sapiens), rat (Rattus norvegicus), mouse (Mus musculus), and zebrafish (Danio rerio). The contractor shall incorporate such options into the pricing schedule.
  • The contractor shall be able to generate targeted RNA-seq libraries directly from cell/tissue lysates shipped to the contractor without a separate column-based or Trizol-based RNA purification step. Automatable, mRNA capture approaches such bead- or plate-based methods or capture-free approaches that amplify and sequence the targeted region may be used.
  • The contractor shall be able to generate targeted RNA-seq libraries from purified total RNA samples shipped to the contractor using an appropriate method.
  • Targeted RNA-seq shall be collected to a minimum read depth specified based on the pricing table and based on an agreed-upon reference control sample or set of reference control samples.
  • A single probe or multiple probes per gene may be used.  However, if a single probe is used, the performance of that probe shall be validated.
  • The remaining sample volume and prepared libraries shall be frozen and shipped to EPA upon request.
  • The contractor shall provide an option to repress highly expressed genes to increase sensitivity of lower copy genes through probe attenuation or other methods
• Data Analysis
  • The contractor shall perform data quality control and analysis. The contractor shall remove adapters, contamination, and low-quality reads from the raw data.
  • The contractor shall perform the sequencing alignment to a reference genome.  The reference genome and alignment method (e.g., TopHat) will be specified by the EPA in consultation with the contractor. Alignment and distribution statistics shall be provided to the EPA.
  • A raw sample data file containing the total mapped counts per gene and the total mapped counts for each sample shall be provided to EPA. The contractor shall provide annotation regarding the association of each probe to the Entrez or EMBL-EBI Gene ID upon which the probe design was based.  In cases where more than one probe was included for each gene, the probes shall be uniquely annotated and associated with a corresponding Entrez or EMBL-EBI Gene ID upon which the probe design was based.
  • A normalized sample data file containing the normalized mapped counts per gene for each sample shall be provided to EPA. The normalization method will be specified by the EPA (e.g., FPKM, RPKM) in consultation with the contractor, but will depend on the technology platform selected. The contractor shall provide annotation regarding the association of each probe with an Entrez or EMBL-EBI Gene ID upon which the probe design was based.  In cases where more than one probe was included for each gene, the probes shall be uniquely annotated and associated with a corresponding Entrez or EMBL-EBI Gene ID upon which the probe design was based.
  • The raw data shall be provided as a FASTQ file on a per sample basis (i.e. 1 fastq file per sample)