title: Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake.
abstract: The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).
authors: Department of Experimental and Evolutionary Biology, University of Bologna, Bologna, Italy. ilaria.milano2@unibo.it
authors: Ilaria
authors: I
authors: Milano
authors: Massimiliano
authors: M
authors: Babbucci
authors: Frank
authors: F
authors: Panitz
authors: Rob
authors: R
authors: Ogden
authors: Rasmus O
authors: RO
authors: Nielsen
authors: Martin I
authors: MI
authors: Taylor
authors: Sarah J
authors: SJ
authors: Helyar
authors: Gary R
authors: GR
authors: Carvalho
authors: Montserrat
authors: M
authors: Espiñeira
authors: Miroslava
authors: M
authors: Atanassova
authors: Fausto
authors: F
authors: Tinti
authors: Gregory E
authors: GE
authors: Maes
authors: Tomaso
authors: T
authors: Patarnello
authors: Luca
authors: L
authors: Bargelloni
mesh_terms: D000818:Animals; D003247:Conservation of Natural Resources / Q000379:methods*; D030541:Databases, Genetic; D005060:Europe; D048388:Gadiformes / Q000235:genetics*; D005787:Gene Frequency / Q000235:genetics; D023281:Genomics / Q000379:methods*; D005843:Geography; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D058977:Molecular Sequence Annotation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012372:ROC Curve; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D059467:Transcriptome / Q000235:genetics*