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Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato
- Source :
- DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
- Publication Year :
- 2015
-
Abstract
- 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.
- Subjects :
- 0301 basic medicine
in silico prediction
In silico
Sequence assembly
Genomics
Biology
Breeding
Polymorphism, Single Nucleotide
DNA sequencing
03 medical and health sciences
Solanum lycopersicum
single nucleotide polymorphism
Genetics
Computer Simulation
genetic linkage map
Molecular Biology
Whole genome sequencing
restriction-associated DNA sequencing
High-Throughput Nucleotide Sequencing
General Medicine
DNA Restriction Enzymes
Sequence Analysis, DNA
Full Papers
Restriction enzyme
Restriction site
030104 developmental biology
tomato (Solanum lycopersicum)
Genome, Plant
Reference genome
Subjects
Details
- ISSN :
- 17561663
- Volume :
- 23
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- DNA research : an international journal for rapid publication of reports on genes and genomes
- Accession number :
- edsair.doi.dedup.....9dd2a0c0af4b61f135aa2a17b93fffcb