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

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.

Microsatellite discovery in an insular amphibian (Grandisonia alternans) with comments on cross-species utility and the accuracy of locus identification from unassembled Illumina data

“…RADseq; Davey and Blaxter 2011), identifying SSRs from longer read sequence data (e.g. Drechsler et al 2013;Wei et al 2014), or restricting shotgun Seq-to-SSR searches to so called ''Best PALs'' ([6 repeats for 4-6 mers; Castoe et al 2012a) when researchers need to perform population genetics assessments using NGS with limited resources.…”

“…The use of unassembled reads is a timesaving characteristic (Castoe et al 2012a;Lance et al 2013), however, several studies have cautioned against this approach when using short read data, such as from the Illumina platform. These studies have found that Illumina paired-end identified microsatellite loci can have an order of magnitude smaller success rate (in terms of amplification across multiple individuals and sequencing error) than those loci identified from longer 454 or PacBio reads (Drechsler et al 2013;Wei et al 2014). Despite this, the use of PALFINDER to identify SSR loci from short, unassembled Illumina reads is widespread (e.g.…”

“…Despite this, the use of PALFINDER to identify SSR loci from short, unassembled Illumina reads is widespread (e.g. amphibians, Drechsler et al 2013;Peterman et al 2013;bivalves, O'Bryhim et al 2012a; crustaceans, Stoutamore et al 2012;fish, O'Bryhim et al 2012b;Nunzuata et al 2013;mammals, Barthelmess et al 2013;reptiles, Castoe et al 2012b). Interestingly, few studies have performed validation experiments using SSR loci identified from unassembled reads.…”

“…Those that have (e.g. Mikheyev et al 2010;Delmas et al 2011;Castoe et al 2012b) have typically used unassembled 454 reads, thus leaving the question of whether SSR loci can be reliably identified from shorter reads poorly addressed outside of a few studies (Drechsler et al 2013;Wei et al 2014).…”

Range contraction and loss of genetic variation of the Pyrenean endemic newt Calotriton asper due to climate change

“…The use of higher resolution markers such as microsatellites (e.g. Drechsler et al 2014;Valbuena-Ureña et al 2014) and a range-wide sampling of all populations combined with promising new methods (e.g. Dubey et al 2013;Pfenninger et al 2012) should provide improved insights into the effects of climate change on the loss of genetic diversity and evolutionary history in C. asper from the Pyrenees.…”