Abstract
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.
Highlights
Microsatellite loci are still considered valuable tools for addressing basic questions in ecology, evolution, and behavior in nonmodel organisms, despite the fact that other molecular markers have become increasingly popular due to next-generation sequencing (NGS) approaches (e.g., genotyping or sequencing of single-nucleotide polymorphisms (SNPs))
The species classification by Multiplex Identifier (MID) tag identification of all sequences performed by PHOBOS (Methods, step A) led to 19,562 sequences for T. cristatus (15.37% of all sequences), 52,075 sequences for C. asper (40.92%), and 55,626 sequences for L. helveticus (43.71%)
While the 15 L. helveticus loci were analyzed without any additional labeling (Methods, step G), the Type-it multiplex PCR of the T. cristatus and C. asper loci resulted in the detection of 13 (T. cristatus) and 21 (C. asper) polymorphic loci (Methods, step F)
Summary
Microsatellite loci are still considered valuable tools for addressing basic questions in ecology, evolution, and behavior in nonmodel organisms, despite the fact that other molecular markers have become increasingly popular due to next-generation sequencing (NGS) approaches (e.g., genotyping or sequencing of single-nucleotide polymorphisms (SNPs)). By using NGS approaches on genomic libraries enriched for microsatellite motifs, the isolation process has become much simpler and more cost-effective (e.g., Abdelkrim et al 2009) These approaches result in tens of thousands of sequence reads, which are expected to lead to a large amount of suitable microsatellite loci (e.g., Yang et al 2012). The correlation between the initial number of sequence reads obtained and the number of usable polymorphic microsatellite loci may be low as the number of potentially amplifiable loci (PALs) is negatively influenced by many factors These factors include sequence read quality (cut-off score values), motif length (type and number of repeat), and the presence, quality, and necessary length of the primer region, in addition to the amplification success and confirmed polymorphism of loci across the studied populations. NGS-based microsatellite loci development approaches should be efficient in obtaining a sufficient number of loci in such species (e.g., amphibians)
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