Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species.
Sequencing of pooled samples (Pool-Seq) using next-generation sequencing technologies has become increasingly popular, because it represents a rapid and cost-effective method to determine allele frequencies for single nucleotide polymorphisms (SNPs) in population pools. Validation of allele frequenc...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3820589?pdf=render |
| _version_ | 1829509505253113856 |
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| author | Christian Rellstab Stefan Zoller Andrew Tedder Felix Gugerli Martin C Fischer |
| author_facet | Christian Rellstab Stefan Zoller Andrew Tedder Felix Gugerli Martin C Fischer |
| author_sort | Christian Rellstab |
| collection | DOAJ |
| description | Sequencing of pooled samples (Pool-Seq) using next-generation sequencing technologies has become increasingly popular, because it represents a rapid and cost-effective method to determine allele frequencies for single nucleotide polymorphisms (SNPs) in population pools. Validation of allele frequencies determined by Pool-Seq has been attempted using an individual genotyping approach, but these studies tend to use samples from existing model organism databases or DNA stores, and do not validate a realistic setup for sampling natural populations. Here we used pyrosequencing to validate allele frequencies determined by Pool-Seq in three natural populations of Arabidopsis halleri (Brassicaceae). The allele frequency estimates of the pooled population samples (consisting of 20 individual plant DNA samples) were determined after mapping Illumina reads to (i) the publicly available, high-quality reference genome of a closely related species (Arabidopsis thaliana) and (ii) our own de novo draft genome assembly of A. halleri. We then pyrosequenced nine selected SNPs using the same individuals from each population, resulting in a total of 540 samples. Our results show a highly significant and accurate relationship between pooled and individually determined allele frequencies, irrespective of the reference genome used. Allele frequencies differed on average by less than 4%. There was no tendency that either the Pool-Seq or the individual-based approach resulted in higher or lower estimates of allele frequencies. Moreover, the rather high coverage in the mapping to the two reference genomes, ranging from 55 to 284x, had no significant effect on the accuracy of the Pool-Seq. A resampling analysis showed that only very low coverage values (below 10-20x) would substantially reduce the precision of the method. We therefore conclude that a pooled re-sequencing approach is well suited for analyses of genetic variation in natural populations. |
| first_indexed | 2024-12-16T11:46:53Z |
| format | Article |
| id | doaj.art-5993173d71724cbf8fc2befe9153a70e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T11:46:53Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-5993173d71724cbf8fc2befe9153a70e2022-12-21T22:32:48ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01811e8042210.1371/journal.pone.0080422Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species.Christian RellstabStefan ZollerAndrew TedderFelix GugerliMartin C FischerSequencing of pooled samples (Pool-Seq) using next-generation sequencing technologies has become increasingly popular, because it represents a rapid and cost-effective method to determine allele frequencies for single nucleotide polymorphisms (SNPs) in population pools. Validation of allele frequencies determined by Pool-Seq has been attempted using an individual genotyping approach, but these studies tend to use samples from existing model organism databases or DNA stores, and do not validate a realistic setup for sampling natural populations. Here we used pyrosequencing to validate allele frequencies determined by Pool-Seq in three natural populations of Arabidopsis halleri (Brassicaceae). The allele frequency estimates of the pooled population samples (consisting of 20 individual plant DNA samples) were determined after mapping Illumina reads to (i) the publicly available, high-quality reference genome of a closely related species (Arabidopsis thaliana) and (ii) our own de novo draft genome assembly of A. halleri. We then pyrosequenced nine selected SNPs using the same individuals from each population, resulting in a total of 540 samples. Our results show a highly significant and accurate relationship between pooled and individually determined allele frequencies, irrespective of the reference genome used. Allele frequencies differed on average by less than 4%. There was no tendency that either the Pool-Seq or the individual-based approach resulted in higher or lower estimates of allele frequencies. Moreover, the rather high coverage in the mapping to the two reference genomes, ranging from 55 to 284x, had no significant effect on the accuracy of the Pool-Seq. A resampling analysis showed that only very low coverage values (below 10-20x) would substantially reduce the precision of the method. We therefore conclude that a pooled re-sequencing approach is well suited for analyses of genetic variation in natural populations.http://europepmc.org/articles/PMC3820589?pdf=render |
| spellingShingle | Christian Rellstab Stefan Zoller Andrew Tedder Felix Gugerli Martin C Fischer Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. PLoS ONE |
| title | Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. |
| title_full | Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. |
| title_fullStr | Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. |
| title_full_unstemmed | Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. |
| title_short | Validation of SNP allele frequencies determined by pooled next-generation sequencing in natural populations of a non-model plant species. |
| title_sort | validation of snp allele frequencies determined by pooled next generation sequencing in natural populations of a non model plant species |
| url | http://europepmc.org/articles/PMC3820589?pdf=render |
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