TY - JOUR
T1 - Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake
AU - Milano, Ilaria
AU - Babbucci, Massimiliano
AU - Panitz, Frank
AU - Ogden, Rob
AU - Nielsen, Rasmus O
AU - Taylor, Martin I
AU - Helyar, Sarah J
AU - Carvalho, Gary R
AU - Espiñeira, Montserrat
AU - Atanassova, Miroslava
AU - Tinti, Fausto
AU - Maes, Gregory E
AU - Patarnello, Tomaso
AU - Bargelloni, Luca
AU - FishPopTrace Consortium
PY - 2011/11/22
Y1 - 2011/11/22
N2 - 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).
AB - 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).
KW - Animals
KW - Conservation of Natural Resources
KW - Databases, Genetic
KW - Europe
KW - Gadiformes
KW - Gene Frequency
KW - Genomics
KW - Geography
KW - Heterozygote
KW - High-Throughput Nucleotide Sequencing
KW - Molecular Sequence Annotation
KW - Polymorphism, Single Nucleotide
KW - ROC Curve
KW - Reproducibility of Results
KW - Sequence Analysis, DNA
KW - Transcriptome
U2 - 10.1371/journal.pone.0028008
DO - 10.1371/journal.pone.0028008
M3 - Article
C2 - 22132191
SN - 1932-6203
VL - 6
SP - e28008
JO - PloS one
JF - PloS one
IS - 11
ER -