High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model

Abstract

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci 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 loci 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 loci from the fish 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 locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each 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.

The Guppy Sex Chromosome System and the Sexually Antagonistic Polymorphism Hypothesis for Y Chromosome Recombination Suppression

“…Now that molecular markers can be developed for non-model organisms such as the guppy, and, using a genome assembly, for any desired genome region [ 56 , 57 ], it should be possible to test whether the genetic maps differ between up- and down-river populations of guppies. A major advantage of molecular markers in the guppy system is that they can be genotyped in both sexes without the need for hormone treatments.…”

Genetic evaluation of migratory fish: Implications for conservation and stocking programs

“…All individuals were taxonomically identified based on Britski, Sato, and Rosa (1984) and then returned to the river. In the laboratory, fragments (12.5 mm 2 ) of all caudal fin samples were placed into 96‐well plates to start the genomic DNA extraction protocol (detailed in Pimentel et al, 2018). Sampling was conducted as permitted by permanent collecting licenses issued by the Brazilian Instituto Chico Mendes de Conservação da Biodiversidade (protocol no.…”

“…The NGS approach was chosen in this study due to its high performance with large numbers of samples, low cost–benefit ratio, and rapid data acquisition. NGS genotyping and treatment of data were performed following Pimentel et al (2018). Briefly, samples were genotyped using a pseudomultiplex strategy for eight microsatellite loci with tri‐ or tetra‐nucleotide repeats: ProC10, ProC18, ProC22, ProC36, ProC37, ProC44, ProC48, and ProC49 (Dryad Repository: https://doi.org/10.5061/dryad.tqjq2bvtq).…”

Fast sequence-based microsatellite genotyping development workflow

“…Studies published so far have explored specific technical or analytical aspects of SSRseq. Several bioinformatics approaches have been developed (Hoogenboom et al, 2016;Suez et al, 2016;Zhan et al, 2017;Barbian et al, 2018), different laboratory protocols tested (Vartia et al, 2016;Pimentel et al, 2018) and means to account for molecular variation on population genetic inference compared (Neophytou et al, 2018;Šarhanová et al, 2018;Viruel et al, 2018;Curto et al, 2019). Together, these studies explored numerous issues surrounding the technical and analytical advantages of SSRseq over traditional methods.…”