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
Article first published online: 17 SEP 2013
© 2013 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
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Ecology and Evolution
Volume 3, Issue 11, pages 3947–3957, October 2013
How to Cite
Ecology and Evolution 2013; 3(11): 3947–3957
- Issue published online: 9 OCT 2013
- Article first published online: 17 SEP 2013
- Manuscript Accepted: 12 AUG 2013
- Manuscript Revised: 7 AUG 2013
- Manuscript Received: 2 JUL 2013
- Deutsche Bundesstiftung Umwelt
- OHM Haut-Vicdessos and the ERAnet. Grant Number: 1.5.040.10
- Fonds Spéciaux pour la Recherche. Grant Number: C11/23
- Calotriton arnoldi ;
- crested newt;
- cross-amplification success;
- genome size;
- Pyrenean mountain newt
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.