iMSAT: a novel approach to the development of microsatellite loci using barcoded Illumina libraries

Illumina sequencing with its high number of reads and low per base pair cost is an attractive technology for development of molecular resources for non-model organisms. While many software packages have been developed to identify short tandem repeats (STRs) from next-generation sequencing data, these methods do not inform the investigator as to whether or not candidate loci are polymorphic in their target populations.

We provide a python program iMSAT that uses the polymorphism data obtained from mapping individual Illumina sequence reads onto a reference genome to identify polymorphic STRs. Using this approach, we identified 9,119 candidate polymorphic STRs for use with the parasitoid wasp Trioxys pallidus and 2,378 candidate polymorphic STRs for use with the aphid Chromaphis juglandicola. For both organisms we selected 20 candidate tri-nucleotide STRs for validation. Using fluorescent-labeled oligonucleotide primers, we genotyped 91 female T. pallidus collected in nine localities and 46 female C. juglandicola collected in 4 localities and found 15 of the examined markers to be polymorphic for T. pallidus and 12 of the examined markers to be polymorphic for C. juglandicola.

We present a novel approach that uses standard Illumina barcoding primers and a single Illumina HiSeq run to target polymorphic STR fragments to develop and test STR markers. We validate this approach using the parasitoid wasp T. pallidus and its aphid host C. juglandicola. This approach, which would also be compatible with 454 Sequencing, allowed us to quickly identify markers with known variability. Accordingly, our method constitutes a significant improvement over existing STR identification software packages.