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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0258906 |
| _version_ | 1819176019279478784 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-22T21:04:06Z |
| format | Article |
| id | doaj.art-72339b444d4f432eb7870439d2b30f63 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T21:04:06Z |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-72339b444d4f432eb7870439d2b30f632022-12-21T18:12:43ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-011610e025890610.1371/journal.pone.0258906Automated genotyping of microsatellite loci from feces with high throughput sequences.Isabel SaladoAlberto Fernández-GilCarles VilàJennifer A LeonardEcological 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.https://doi.org/10.1371/journal.pone.0258906 |
| spellingShingle | Isabel Salado Alberto Fernández-Gil Carles Vilà Jennifer A Leonard Automated genotyping of microsatellite loci from feces with high throughput sequences. PLoS ONE |
| title | 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_short | Automated genotyping of microsatellite loci from feces with high throughput sequences. |
| title_sort | automated genotyping of microsatellite loci from feces with high throughput sequences |
| url | https://doi.org/10.1371/journal.pone.0258906 |
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