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Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato
Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction site...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833422/ https://www.ncbi.nlm.nih.gov/pubmed/26932983 http://dx.doi.org/10.1093/dnares/dsw004 |
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author | Shirasawa, Kenta Hirakawa, Hideki Isobe, Sachiko |
author_facet | Shirasawa, Kenta Hirakawa, Hideki Isobe, Sachiko |
author_sort | Shirasawa, Kenta |
collection | PubMed |
description | Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i) in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. |
format | Online Article Text |
id | pubmed-4833422 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-48334222016-04-18 Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato Shirasawa, Kenta Hirakawa, Hideki Isobe, Sachiko DNA Res Full Papers Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i) in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. Oxford University Press 2016-04 2016-02-29 /pmc/articles/PMC4833422/ /pubmed/26932983 http://dx.doi.org/10.1093/dnares/dsw004 Text en © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Full Papers Shirasawa, Kenta Hirakawa, Hideki Isobe, Sachiko Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title | Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title_full | Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title_fullStr | Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title_full_unstemmed | Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title_short | Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato |
title_sort | analytical workflow of double-digest restriction site-associated dna sequencing based on empirical and in silico optimization in tomato |
topic | Full Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833422/ https://www.ncbi.nlm.nih.gov/pubmed/26932983 http://dx.doi.org/10.1093/dnares/dsw004 |
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