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Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata

Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequenc...

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Autores principales: Fracassetti, Marco, Griffin, Philippa C., Willi, Yvonne
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604096/
https://www.ncbi.nlm.nih.gov/pubmed/26461136
http://dx.doi.org/10.1371/journal.pone.0140462
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author Fracassetti, Marco
Griffin, Philippa C.
Willi, Yvonne
author_facet Fracassetti, Marco
Griffin, Philippa C.
Willi, Yvonne
author_sort Fracassetti, Marco
collection PubMed
description Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq) of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual). The validation was based on comparing single nucleotide polymorphism (SNP) frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS). Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14) and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual), which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05).
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spelling pubmed-46040962015-10-20 Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata Fracassetti, Marco Griffin, Philippa C. Willi, Yvonne PLoS One Research Article Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq) of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual). The validation was based on comparing single nucleotide polymorphism (SNP) frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS). Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14) and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual), which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05). Public Library of Science 2015-10-13 /pmc/articles/PMC4604096/ /pubmed/26461136 http://dx.doi.org/10.1371/journal.pone.0140462 Text en © 2015 Fracassetti et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Fracassetti, Marco
Griffin, Philippa C.
Willi, Yvonne
Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title_full Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title_fullStr Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title_full_unstemmed Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title_short Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata
title_sort validation of pooled whole-genome re-sequencing in arabidopsis lyrata
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604096/
https://www.ncbi.nlm.nih.gov/pubmed/26461136
http://dx.doi.org/10.1371/journal.pone.0140462
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