Abstract
Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct “Seq-to-SSR” approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample – a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.
Highlights
Constant advances in DNA sequencing technology and lower costs are driving innovation in the life sciences, and are having an especially large impact on the study of ecology, evolution, and population genetics
For detailed comparisons of performance of 454 versus Illumina-based sequencing, we focused analyses on the python, for which we had both types of data from the same individual
In a previous study [7], we had collected 28.5 million base-pairs (Mbp) from 118,973 reads from shotgun genomic libraries using the 454 platform, and we use these data to evaluate the performance of the 454 reads in the Seq-to-simple sequence repeats (SSRs) approach
Summary
Constant advances in DNA sequencing technology and lower costs are driving innovation in the life sciences, and are having an especially large impact on the study of ecology, evolution, and population genetics. Simple sequence repeats (SSRs; known as microsatellite loci) have long been important in population genetic studies, but the identification of SSRs from non-model species previously required substantial and costly technical effort, and often returned far fewer loci than were required to address most population genetics questions adequately. This effort included creating libraries enriched for SSR loci, cloning, hybridization to detect positive clones, plasmid isolation, and Sanger sequencing. SSRs are rare in the genomes of some species, and the prohibitive cost of sufficient 454 sequencing in such cases often necessitates a pre-sequencing SSR enrichment step
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