Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
  • Problem Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

For each oyster sampled, animals were cleaned, measured and shucked to expose soft tissues. Gill and mantle were extracted from each oyster and preserved in 95% ethanol for genetic analysis. DNA was extracted using a QIAGEN QIAamp DNA Mini Kit. Genomic DNA concentration was measured with a Nanodrop spectrophotometer to ensure the concentration was ≥100 ng μL⁻¹. All positive samples in microcentrifuge tubes were randomly mixed before transport in case there was an error in future sequencing steps that could cause a loss in representation of an entire population. Once the microcentrifuge tubes containing the extracted DNA were thoroughly mixed, 20 μL of each of the 64 samples were used in subsequent library construction. The DNA of each individual oyster was digested separately with 2 restriction enzymes, EcoRI and MseI. The digested DNA fragments were then ligated to Illumina adaptors at the MseI end and with Illumina adaptors coupled with an 8–10 bp unique barcode to the EcoRI end to allow identification of the individual in silico. The restriction-ligation products were then PCR-amplified in 2 separate reactions using standard Illumina primers. The final PCR products were pooled and shipped for sequencing.

Fifty-nine P. marinus-positive samples were sequenced in 2020 at the Tufts University Genomic Services (200–400 bp fraction; single-end sequencing on an Illumina HiSeq 2500 using SE125), 42 samples were sequenced in 2022 at the University of Texas Genomic Sequencing and Analysis Facility (300–450 bp fraction; single-end sequencing on an Illumina NovaSeq SP using SR100) and 37 of each of those samples were sequenced in both runs.

Organism identifiers (Life Science Identifier (LSID)):
Crassostrea virginica (urn:lsid:marinespecies.org:taxname:140657)
Perkinsus marinus  (urn:lsid:marinespecies.org:taxname:562957)

Reads were aligned to the P. marinus genome (GCA_000006405.1) and the C. virginica genome (GCF_002022765.2). The median number of reads aligned to the P. marinus genome (0.13 M; a range of 0.10–1.01 M) was 0.37% of the median number aligned to the C. virginica genome (34 M; a range of 3.52–66.89 M). This yielded a ratio of 265 C. virginica to P. marinus reads per sample. Of the total number of reads per sample, a median of 0.80 and 91.30% of reads were P. marinus and C. virginica, respectively.

After alignment of 64 samples to the P. marinus genome, we kept 35 samples that had between 100 K and 1 M P. marinus reads. Another sample identified as 6837–16, from VA had 1.1 M reads, from which we randomly downsampled 500 K reads using samtools view. 

For P. marinus, we found all single-nucleotide polymorphisms, or SNPs [set minor allele frequency (MAF) threshold at 0] and then a custom script to find SNPs that had at least 1 read across 50% of individuals. This yielded 772 SNPs. An analysis of allele frequencies indicated that 393 SNPs were polymorphic between or within samples. Thus, all subsequent analyses are based on phred-scale genotype likelihoods of 393 polymorphic SNPs at 36 individuals. For C. virginica, we created genotypes from the same 36 individuals, and included SNPs with MAF > 1% and at least 1 read per sample, yielding genotypes at 52 100 SNPs.

Organism name, Life Science Identifier (LSID):
Crassostrea virginica, urn:lsid:marinespecies.org:taxname:140657
Perkinsus marinus, urn:lsid:marinespecies.org:taxname:562957

- Loaded CSV file "meta_forBCODMO.csv" into table "998962_v1_perkinsus-crassostrea-co-phylogeny"; empty strings and "NA" treated as missing values
- Set types for 18 columns: Accesion, BioProject, BioSample, geo_loc_name, host, isolate, isolation_source, organism, sample_name, siteName, State, Storage, tissue, Region, Region2 as string; Lat, Lon as number; date_collection as string
- Updated field metadata (descriptions, standard name IDs, supplied units) for all 18 columns, including NCBI accession identifiers (Accesion, BioProject, BioSample), geographic coordinates (Lat, Lon), collection date, organism (Crassostrea virginica and Perkinsus marinus), tissue type, storage method, and location fields
- Converted date_collection from format "%m/%d/%y" to ISO 8601 string format "%Y-%m-%d"
- Set date_collection type to date with format "%Y-%m-%d"
- Renamed column Accesion to Accession (correcting spelling)
- Output final table as "998962_v1_perkinsus-crassostrea-co-phylogeny.csv"

NSF abstract:

During the 20th century, the Pacific oyster Crassostrea gigas was deliberately introduced from its native range of coastal Asia to the estuaries of six continents. While the introduced Pacific oysters are widely aquacultured and thus can generate local economic wealth, they sometimes outcompete native oysters, and can carry microbial, animal and plant hitchhikers that negatively impact local economies and the ecological functioning of local estuaries. This study comprehensively assesses the pathways and sources of Pacific oyster introductions using a worldwide, population genetic survey. Simultaneously, the study also assesses the pathways and source of one hitchhiking protist (Haplosporidium nelsoni) that causes the disease MSX (multinucleated sphere X) in the Virginia oyster (Crassostrea virginica) along the eastern seaboard of the United States. One goal of this research is to generate management strategies that combat the negative impacts of the Pacific oyster and its associated invaders, and minimize future invasions. A second goal is to minimize some uncertainty about the population biology of the devastating Haplosporidium parasite, and thus, increase confidence of policy makers who are managing shellfish health, restoration and commerce. By quantifying the pathways and sources of C. gigas, this project may inform strategies to combat negative impacts of C. gigas and its associated invaders, as well as minimize future invasions. Moreover, quantifying dispersal within and among populations of H. nelsoni along the US East Coast will provide perspective on the effectiveness of regional biosecurity measures in preventing the ongoing dispersal of this destructive pathogen via aquaculture. In addition, the project lends itself well to programs that foster critical thinking and research experience among both undergraduate and K-12 students. The project provides opportunities for 6-9 undergraduates to perform research, includes a 2-day workshop on bioinformatics for the wider undergraduate community, and facilitates ongoing opportunities for K-12 students to participate in citizen-science research.

There is a wealth of information on the source, pathways and vectors of C. gigas based largely on historical documents but no study has comprehensively tested whether these historical accounts are correct using a worldwide, population genetic survey. Using >14K single-nucleotide polymorphisms (SNPs) from 41 populations across five continents a high level of spatial genetic differentiation was found within the native range and differences in source populations among non-native regions. Preliminary genetic data indicated that the parasitic protist, Haplosporidium nelsoni arrived with C. gigas imports to the US Atlantic coastline and then infected the native C. virginica, however the native source populations, the pathways and vector from which H. nelsoni arrived remain unknown. This project couples high-throughput sequencing technologies and Approximate Bayesian Computing (ABC)-based models to answer the following: What are the population genomic patterns among C. gigas from native and non-native regions? What are the population genomic patterns of Haplosporidium nelsoni among Asian and North American Crassostrea gigas and eastern North American C. virginica? What were the source populations and invasion pathways of C. gigas and H. nelsoni? Identifying source locations, pathways and vectors of introduction of C. gigas will provide researchers with a null-model of invasion history for dozens of other non-native species that were transported with C. gigas. Currently, there are no verified 'vector maps' for historical shipments of C. gigas that are similar to those generated from modern-day or historical shipping records.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.