<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Juliana S. M. Pimentel</style></author><author><style face="normal" font="default" size="100%">Anderson O. Carmo</style></author><author><style face="normal" font="default" size="100%">Izinara C. Rosse</style></author><author><style face="normal" font="default" size="100%">Ana P. V. Martins</style></author><author><style face="normal" font="default" size="100%">Sandra Ludwig</style></author><author><style face="normal" font="default" size="100%">et. al.</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model</style></title><secondary-title><style face="normal" font="default" size="100%">Frontiers in Genetics</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2018</style></year></dates><urls><web-urls><url><style face="normal" font="default" size="100%">https://doi.org/10.3389/fgene.2018.00073</style></url></web-urls></urls><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p style=&quot;text-align: justify;&quot;&gt;
	Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;&amp;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&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;&amp;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&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;&amp;nbsp;from the fish&amp;nbsp;&lt;i&gt;Prochilodus costatus&lt;/i&gt;, 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&amp;nbsp;&lt;i&gt;locus&lt;/i&gt;&amp;nbsp;when genotyping eight&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;. Assuming a minimum requirement of 10 reads per&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;, two to four times more&amp;nbsp;&lt;i&gt;loci&lt;/i&gt;&amp;nbsp;could be tested in each run, depending on the quality of the PCR reaction of each&amp;nbsp;&lt;i&gt;locus&lt;/i&gt;. 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.
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