Automated genotyping of microsatellite loci from feces with high throughput sequences

Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are en...

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Autores principales:	Isabel Salado, Alberto Fernández-Gil, Carles Vilà, Jennifer A. Leonard
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b2c3d68b82744710a8f1e5f85ba012e3
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spelling	oai:doaj.org-article:b2c3d68b82744710a8f1e5f85ba012e32021-11-04T06:09:18ZAutomated genotyping of microsatellite loci from feces with high throughput sequences1932-6203https://doaj.org/article/b2c3d68b82744710a8f1e5f85ba012e32021-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8544849/?tool=EBIhttps://doaj.org/toc/1932-6203Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.Isabel SaladoAlberto Fernández-GilCarles VilàJennifer A. LeonardPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Isabel Salado Alberto Fernández-Gil Carles Vilà Jennifer A. Leonard Automated genotyping of microsatellite loci from feces with high throughput sequences
description	Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.
format	article
author	Isabel Salado Alberto Fernández-Gil Carles Vilà Jennifer A. Leonard
author_facet	Isabel Salado Alberto Fernández-Gil Carles Vilà Jennifer A. Leonard
author_sort	Isabel Salado
title	Automated genotyping of microsatellite loci from feces with high throughput sequences
title_short	Automated genotyping of microsatellite loci from feces with high throughput sequences
title_full	Automated genotyping of microsatellite loci from feces with high throughput sequences
title_fullStr	Automated genotyping of microsatellite loci from feces with high throughput sequences
title_full_unstemmed	Automated genotyping of microsatellite loci from feces with high throughput sequences
title_sort	automated genotyping of microsatellite loci from feces with high throughput sequences
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/b2c3d68b82744710a8f1e5f85ba012e3
work_keys_str_mv	AT isabelsalado automatedgenotypingofmicrosatellitelocifromfeceswithhighthroughputsequences AT albertofernandezgil automatedgenotypingofmicrosatellitelocifromfeceswithhighthroughputsequences AT carlesvila automatedgenotypingofmicrosatellitelocifromfeceswithhighthroughputsequences AT jenniferaleonard automatedgenotypingofmicrosatellitelocifromfeceswithhighthroughputsequences
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Automated genotyping of microsatellite loci from feces with high throughput sequences

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