What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches
[en] The development of microsatellite loci has become more efficient using next-generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra-nucleotide motif-enriched species-specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra-nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species-specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454-sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.
Research center :
AFFISH-RC - Applied and Fundamental FISH Research Center - ULiège
Drechsler, Axel; Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld
Geller, Daniel; Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld
Freund, Katharina; Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld
Schmeller, Dirk S.; Helmholtz Center for Environmental Research & Universit e de Toulouse
Künzel, Sven; Max Planck Institute for Evolutionary Biology
Rupp, Oliver; Center for Biotechnology, University of Bielefeld
Loyau, Adeline
Denoël, Mathieu ; Université de Liège - ULiège > Département de Biologie, Ecologie et Evolution > Biologie du comportement - Ethologie et psychologie animale
Valbuena-Ureña, Emilio; Universitat Autonoma de Barcelona & Centre de Fauna Salvatge de Torreferrussa
Steinfartz, Sebastian; Department of Behavioral Biology, Unit of Molecular Ecology and Behavior, University of Bielefeld
Language :
English
Title :
What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper, Lissotriton helveticus, and Triturus cristatus) and comparison with Illumina-based approaches
Publication date :
2013
Journal title :
Ecology and Evolution
eISSN :
2045-7758
Publisher :
John Wiley & Sons
Volume :
3
Issue :
11
Pages :
3947-3957
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE] ULg FSR - Université de Liège. Fonds spéciaux pour la recherche [BE]
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