@article {26702,
	title = {High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model},
	journal = {Frontiers in Genetics},
	year = {2018},
	abstract = {
	Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple\&nbsp;loci\&nbsp;from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite\&nbsp;loci\&nbsp;for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite\&nbsp;loci\&nbsp;from the fish\&nbsp;Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per\&nbsp;locus\&nbsp;when genotyping eight\&nbsp;loci. Assuming a minimum requirement of 10 reads per\&nbsp;loci, two to four times more\&nbsp;loci\&nbsp;could be tested in each run, depending on the quality of the PCR reaction of each\&nbsp;locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.

},
	url = {https://doi.org/10.3389/fgene.2018.00073},
	author = {Juliana S. M. Pimentel and Anderson O. Carmo and Izinara C. Rosse and Ana P. V. Martins and Sandra Ludwig and et. al.}
}
