FISHBOOST Results: one of the largest co-ordinated genotyping by sequencing efforts
FISHBOOST has successfully performed one of the largest co-ordinated genotyping by sequencing efforts in the world to date.
FISHBOOST has successfully performed one of the largest co-ordinated genotyping by sequencing efforts in the world to date.
Large-scale experiments
In work package 1 (WP1) of the project, large-scale disease challenge experiments were performed for several species and diseases (Table 1), and samples were collected for sequencing. For each disease challenge experiment, between 800 and 1600 individual fish samples were genotyped using various Restriction-site Associated DNA (RAD) sequencing approaches (RAD-Seq, ddRAD-Seq, 2bRAD-Seq).
The different RAD techniques were chosen according to the skills and experience of the laboratories involved and the characteristics of the experimental design and the genomes of the species.
Library preparation was performed by the University of Edinburgh (UK), University of Padova (Italy), and GeneAqua (Spain). The samples were sequenced by BMR Genomics (Italy), mainly using Illumina NextSeq 500 sequencing technology.
Table 1 Sequencing efforts
RAD Sequencing explained --- Source: http://www.floragenex.com/rad-seq/
What are RAD-Seq techniques?
RAD-Seq techniques combine the use of genome complexity reduction with restriction enzymes, and the high sequencing output of Next Generation Sequencing (NGS) technologies. Individual samples are given a nucleotide barcode, and many individuals are then sequenced in a single lane. This results in high coverage of sequence reads at specific sites in the genome, from which single nucleotide polymorphisms (SNPs) can be identified. These SNPs are concurrently genotyped, resulting in genome-wide genotypes for all animals in the sampled population (see Figure).
Why choose RAD-Seq?
There are several advantages of RAD-Seq versus alternative methods of genotyping, such as SNP arrays. RAD techniques are not dependent on prior genomic tools such as a reference genome assembly for the species of interest. The cost of RAD-Seq is lower per sample than for high density SNP arrays. RAD-Seq results in a SNP dataset that is specific to the population of interest, and is not dependent on a previously established SNP set used for an array.
There are also disadvantages to the RAD-Seq based approaches in comparison to SNP arrays. The quality of SNP data is typically lower than for a SNP array, and has a higher proportion of missing genotypes. In addition, there is a higher bioinformatics requirement for RAD-Seq versus SNP arrays. For the species studied in FISHBOOST, there were no SNP arrays available when the study began. However, the SNP marker resources generated in the project could be used for future SNP arrays in some of these aquaculture species.
Application of Results
These results have highlighted the utility of RAD techniques for cost-effective genotyping in aquaculture species. Analyses are now ongoing to map genes for disease resistance traits and calculate genomic breeding values. A comparison of the merits of each of the different RAD techniques will also be performed to guide future work in this area.
