Abstract:

Marine mammal health researchers use many standard veterinary panels, methods, and immune biomarkers, but there are likely additional biomarkers of health and toxicity that would further inform health assessments. Transcriptomic analysis is a powerful tool examining the molecular alterations that underlie toxic responses and disease and can yield extremely sensitive biomarkers. Archived blood (n = 76) and skin (n = 28) samples collected during common bottlenose dolphin health assessments in Barataria Bay, Louisiana and Sarasota, Florida from 2013-06-17 to 2018-07-20, were analyzed by RNA-seq to support and enhance the assessment of animal health. The raw sequence data and FPKM (fragments per kilobase of transcript per million mapped reads) values generated by sequence processing can be found by searching https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi for GSE138765 (blood) and GSE138935 (skin) or the individual accession numbers provided in the read me information.

Suggested Citation:

Morey, Jeanine, Randall Wells, Teresa Rowles, Cynthia Smith, Eric Zolman, Ryan Takeshita, Brian Balmer and Lori Schwacke. 2021. RNA-seq analysis of archived blood and skin from common bottlenose dolphin (Tursiops truncatus) health assessments in the northern Gulf of Mexico, 2013-06-17 to 2018-07-20. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/JYNCX7RQ

Purpose:

In the wake of the Deepwater Horizon oil spill, numerous adverse health impacts have been observed in bottlenose dolphins, including compromised reproductive, pulmonary, cardiac, immune, and adrenal functions. Transcriptomic-based approaches allow for a robust analysis of the molecular mechanisms involved in disease and stress response and may allow for earlier detection of changes in health than standard veterinary assessment. The transcriptomic data were analyzed in conjunction with the substantial pool of health and environmental data collected during health assessments to investigate the utility of transcriptomic data in an overall assessment of dolphin health and/or as markers of specific health concerns.

Data Parameters and Units:

Accession.number Gene Expression Omnibus (GEO) Accession number Sample.type Sample tissue type (Blood or Skin) Sex Sex of dolphin (Male or Female) Location General location of sample collection Sample.collection.date Date of sample collection (m/d/y) Field.ID Unique identifier for each dolphin Lat Latitude of sample collection (decimal degrees) Lon Longitude of sample collection (decimal degrees)

Methods:

Illumina-based RNA-seq analyses were carried out on RNA extracts of whole blood from 76 archived samples and skin from 28 archived samples using the Tursiops truncatus NIST Tur_tru v1 genome as a guide. Blood samples were collected in Barataria Bay, LA (n=60) or Sarasota Bay, FL (n=16) from 2013-2018. Skin samples were collected in Barataria Bay, Louisiana during 2016 and 2017. Samples from Sarasota Bay, Florida served as a reference population that had not been exposed to substantial oil, whereas the samples from Barataria Bay come from a population directly impacted by the Deepwater Horizon oil spill. Total RNA was extracted from blood using the PAXgene Blood RNA kit or skin using Qiagen Rneasy Lipid Tissue Mini kits with on-column Dnase digestion. Libraries were constructed using a NEBNext ultra Directional RNA Library Prep kit for Illumina and indexed with the NEBNextMultiplex Oligos for Illumina. Sequencing was performed on an Illumina NovaSeq 6000, generating 150 base paired-end reads to a targeted depth of at least 50M (blood) or 25M (skin) reads per sample. The Illumina BCL output files were converted to fastq-sanger file format and sequence quality trimming was performed on CyVerse Discovery Environment with the HTProcess-prepare_directories-and-run_fastqc-0.1 and HTProcess_trimmomatic_0.33 apps. The following Trimmomatic parameters were used: ILLUMINACLIP:TruSeq3-SE.fa 2:30:10; LEADING:10; TRAILING:10; SLIDINGWINDOW:4:20; HEADCROP:6; MINLEN:36. Reads were mapped to the Tursiops truncatus genome, NIST Tru_tru v1 (GCF_001922835.1), on CyVerse Discovery Environment with the HTProcess_Tophat-2.1.1 app using Bowtie as the alignment engine. Read counts as FPKM (fragments per kilobase of transcript per million mapped reads) were generated on CyVerse Discovery Environment with the HTProcess_Cufflinks-2.2.1 app with the NIST Tur_tru v1 genome as a reference. Highly expressed hemoglobin sequences (HBB, HBM, HBZ) were masked from analysis of blood samples.

Individual Files: