SNP Discovery in European Anchovy (Engraulis encrasicolus, L) by High-Throughput Transcriptome and Genome Sequencing

Abstract

Increased throughput in sequencing technologies has facilitated the acquisition of detailed genomic information in non-model species. The focus of this research was to discover and validate SNPs derived from the European anchovy (Engraulis encrasicolus) transcriptome, a species with no available reference genome, using next generation sequencing technologies. A cDNA library was constructed from four tissues of ten fish individuals corresponding to three populations of E. encrasicolus, and Roche 454 GS FLX Titanium sequencing yielded 19,367 contigs. Additionally, the European anchovy genome was sequenced for the same ten individuals using an Illumina HiSeq2000. Using a computational pipeline for combining transcriptome and genome information, a total of 18,994 SNPs met the necessary minor allele frequency and depth filters. A series of further stringent filters were applied to identify those SNPs likely to succeed in genotyping assays, and for filtering of those in potential duplicated genome regions. A novel method for detecting potential intron-exon boundaries in areas of putative SNPs has also been applied in silico to improve genotyping success. In all, 2,317 filtered putative transcriptome SNPs suitable for genotyping primer design were identified. From those, a subset of 530 were selected, with the genotyping results showing the highest reported conversion and validation rates (91.3% and 83.2%, respectively) reported to date for a non-model species. This study represents a promising strategy to discover genotypable SNPs in the exome of non-model organisms. The genomic resource generated for E. encrasicolus, both in terms of sequences and novel markers, will be informative for research into this species with applications including traceability studies, population genetic analyses and aquaculture.

Population Genomics of Marine Fishes: Next-Generation Prospects and Challenges

“…Alternative approaches combine transcriptome and genome sequence data (e.g., Lamichaney et al,. 2012;Montes et al, 2013) or split variant discovery and genotyping in two steps, applying various sequencing methods to identify genetic variation that is subsequently genotyped on high-throughput platforms (e.g., Limborg et al, 2012;Milano et al, 2014; see also Table 1 for examples of next-generation sequencing studies in marine fishes).…”

Development of gene-associated single nucleotide polymorphisms for Japanese anchovy Engraulis japonicus through cross-species amplification

“…A total of 482 SNPs previously developed for E. encrasicolus [31,32], comprising 467 nSNPs and 15 mtSNPs, were selected for designing primers and probes (Table S1). SNPs were genotyped with TaqMan OpenArray Genotyping System (Life Technologies, CA) (see below) at the Sequencing and Genotyping Service (SGIker) of the University of the Basque Country (UPV/EHU).…”

“…Based on genotyping calls, SNPs were classified into five categories previously defined by Montes et al [32]: no signal (no amplification), disperse (< 80% of individuals assigned to a cluster), paralogous sequence variants or multi-site variants (PSV/MSV) (≥ 99% of heterozygous individuals), monomorphic [minor allele frequency (MAF) < 0.01], and polymorphic (MAF ≥ 0.01) (Fig. 1).…”

“…This approach is a good choice for developing genetic markers in species with no reference genome but abundant genomic resources available in closely related species. This is the case for E. japonicus, since hundreds of genetic markers are available in the closely related E. encrasicolus, including mitochondrial DNA (mtDNA) sequences [29], microsatellites [30], and nuclear (nSNP) and mitochondrial single nucleotide polymorphisms (mtSNPs) discovered from cloning, PCR and subsequent comparative sequencing [31], and from transcriptome sequencing [32]. Any of these classes of markers could be cross-amplified in E. japonicus to develop new genetic markers for this species.…”

Development of genomic resources for Nothofagus species using next‐generation sequencing data

“…; Montes et al . ). For Fagaceae species, a draft reference genome has been released in 2014 for Castanea mollisima (http://www.hardwoodgenomics.org/), although gene databases and other genome sequencing projects have been under development in the last few years (Neale & Kremer ; Neale et al .…”