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Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers

Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (S...

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Autores principales: Alves, Filipe, Martins, Filipa M. S., Areias, Miguel, Muñoz-Mérida, Antonio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741888/
https://www.ncbi.nlm.nih.gov/pubmed/34997147
http://dx.doi.org/10.1038/s41598-021-04275-8
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author Alves, Filipe
Martins, Filipa M. S.
Areias, Miguel
Muñoz-Mérida, Antonio
author_facet Alves, Filipe
Martins, Filipa M. S.
Areias, Miguel
Muñoz-Mérida, Antonio
author_sort Alves, Filipe
collection PubMed
description Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.
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spelling pubmed-87418882022-01-10 Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers Alves, Filipe Martins, Filipa M. S. Areias, Miguel Muñoz-Mérida, Antonio Sci Rep Article Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies. Nature Publishing Group UK 2022-01-07 /pmc/articles/PMC8741888/ /pubmed/34997147 http://dx.doi.org/10.1038/s41598-021-04275-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Alves, Filipe
Martins, Filipa M. S.
Areias, Miguel
Muñoz-Mérida, Antonio
Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title_full Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title_fullStr Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title_full_unstemmed Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title_short Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers
title_sort automating microsatellite screening and primer design from multi-individual libraries using micro-primers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741888/
https://www.ncbi.nlm.nih.gov/pubmed/34997147
http://dx.doi.org/10.1038/s41598-021-04275-8
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