Project deliverable Open Access
Carlsson, Jens; Carlsson, Jeanette E L; Boavida, Joana; Arnaud-Haond, Sophie
The deliverable includes:
1. Species ID Sheets for Genotyping By Sequencing, GBS, (RAD-seq and microsatellite analyses)
The objective of this deliverable was to pave the road for further analyses on species for which we hope to gather samples allowing population genomics/ connectivity assessment through genome scan analysis (several geographic locations, with 15-50 specimens per location). The present document thus reports tests performed during the first two years of Atlas on selected species and on the performance of restriction site-associated DNA sequencing (RAD-seq) on cold-water coral species and Genotyping by Sequencing (GBS) of microsatellites of three commercial fisheries species. RAD protocols are standard, once the suitability of the genome is tested and the enzyme selected, the ID sheets attached therefore contain the required information using the same RAD protocol as other labs. As for the genome scan using microsatellites, the protocol is described in the first publication (Farrell et al. 2017) and the same protocol was used for the two other fisheries species. While the microsatellite loci deployed on boarfish (Capros aper) are available in Farrell et al. 2017 (open access), loci used for horse mackerel (Trachurus trachurus) and Dublin bay prawn (Nephrops norvegicus) will be released on publication.
This test stage comprised successful RAD-seq efforts for five coral species, including the reef-forming and solitary scleractinian corals and one octocoral. Two enzymes were assessed for performance. The appendix only includes information obtained from the enzyme delivering the best coverage of the genome (depth of coverage generally above 15x; thousands of loci recovered). Future steps include increased sample numbers to allow stricter data filtering, and complete population genomics on a subset of those species (e.g. Lophelia pertusa, Madrepora oculata and Dendrophyllia cornigera). Major constraints include availability of samples (tissue), poor preservation conditions of pre-Atlas collections (room temperature, ethanol evaporation, mold and freeze thawed), causing degraded DNA and the need to further optimise DNA extraction protocols for individual species.
GBS of microsatellites has been targeted for three commercial fisheries species, boarfish, horse mackerel and Dublin Bay prawn. The initial detection of microsatellites has been performed for all three species through shotgun sequencing (MiSeq 300PE or 250PE) and primers for microsatellites have been designed for these species. These primers are subsequently used on a large number of individuals (c.1,000 individuals per species) in a single MiSeq run per species and subsequently genotyped. The study focused on boarfish has been completed and published (Farrell et al. 2017), while the GBS data for horse mackerel and Dublin bay prawn are currently being analysed.