Dataset Information

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

ABSTRACT: Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

SUBMITTER: Ali OA

PROVIDER: S-EPMC4788223 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Ali Omar A OA O'Rourke Sean M SM Amish Stephen J SJ Meek Mariah H MH Luikart Gordon G Jeffres Carson C Miller Michael R MR

Genetics 20151229 2

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a m ...[more]

PMID: 26715661

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Project description:BackgroundThe development of genome-wide genotyping resources has provided terrestrial livestock and crop industries with the unique ability to accurately assess genomic relationships between individuals, uncover the genetic architecture of commercial traits, as well as identify superior individuals for selection based on their specific genetic profile. Utilising recent advancements in de-novo genome-wide genotyping technologies, it is now possible to provide aquaculture industries with these same important genotyping resources, even in the absence of existing genome assemblies. Here, we present the development of a genome-wide SNP assay for the Black Tiger shrimp (Penaeus monodon) through utilisation of a reduced-representation whole-genome genotyping approach (DArTseq).ResultsBased on a single reduced-representation library, 31,262 polymorphic SNPs were identified across 650 individuals obtained from Australian wild stocks and commercial aquaculture populations. After filtering to remove SNPs with low read depth, low MAF, low call rate, deviation from HWE, and non-Mendelian inheritance, 7542 high-quality SNPs were retained. From these, 4236 high-quality genome-wide loci were selected for baits-probe development and 4194 SNPs were included within a finalized target-capture genotype-by-sequence assay (DArTcap). This assay was designed for routine and cost effective commercial application in large scale breeding programs, and demonstrates higher confidence in genotype calls through increased call rate (from 80.2?±?14.7 to 93.0%?±?3.5%), increased read depth (from 20.4?±?15.6 to 80.0?±?88.7), as well as a 3-fold reduction in cost over traditional genotype-by-sequencing approaches.ConclusionImportantly, this assay equips the P. monodon industry with the ability to simultaneously assign parentage of communally reared animals, undertake genomic relationship analysis, manage mate pairings between cryptic family lines, as well as undertake advance studies of genome and trait architecture. Critically this assay can be cost effectively applied as P. monodon breeding programs transition to undertaking genomic selection.

Project description:Background2b-RAD (type IIB endonucleases restriction-site associated DNA) approach was invented by Wang in 2012 and proven as a simple and flexible method for genome-wide genotyping. However, there is still plenty of room for improvement for the existent 2b-RAD approach. Firstly, it doesn't include the samples pooling in library preparation as other reduced representation libraries. Secondly, the information of 2b-RAD tags, such as tags numbers and distributions, in most of species are unknown. The purposes of the research are to improve a new 2b-RAD approach which possesses samples pooling, moreover to figure out the characteristic and application potentiality of 2b-RAD tags by bioinformatics analysis.ResultsTwelve adapter1 and an adapter2 were designed. A library approach comprising digestion, ligation, pooling, PCR and size selection were established. For saving costs, we used non-phosphorylated adapters and indexed PCR primers. A F2 population of rice (Oryza sativa .L) was genotyped to validate the new approach. On average, 2000332 high quality reads of each sample were obtained with high evenness. Totally 3598 markers containing 3804 SNPs were discovered and the missing rate was 18.9%. A genetic linkage map of 1385 markers was constructed and 92% of the markers' orders in the genetic map were in accordance with the orders in chromosomes. Meanwhile, the bioinformatics simulation in 20 species showed that the BsaXI had the most widespread recognition sites, indicating that 2b-RAD tags had a powerful application potentiality for high density genetic map. Using modified adapters with a fix base in 3'end, 2b-RAD was also fit for QTL studies with low costs.ConclusionsAn improved 2b-RAD genotyping approach was established in this research and named as I2b-RAD. The method was a simple, fast, cost-effective and multiplex sequencing library approach. It could be adjusted by selecting different enzymes and adapters to fit for alternative uses including chromosomes assembly, QTL fine mapping and even natural population analysis.

Project description:Reduced-representation genome sequencing represents a new source of data for systematics, and its potential utility in interspecific phylogeny reconstruction has not yet been explored. One approach that seems especially promising is the use of inexpensive short-read technologies (e.g., Illumina, SOLiD) to sequence restriction-site associated DNA (RAD)--the regions of the genome that flank the recognition sites of restriction enzymes. In this study, we simulated the collection of RAD sequences from sequenced genomes of different taxa (Drosophila, mammals, and yeasts) and developed a proof-of-concept workflow to test whether informative data could be extracted and used to accurately reconstruct "known" phylogenies of species within each group. The workflow consists of three basic steps: first, sequences are clustered by similarity to estimate orthology; second, clusters are filtered by taxonomic coverage; and third, they are aligned and concatenated for "total evidence" phylogenetic analysis. We evaluated the performance of clustering and filtering parameters by comparing the resulting topologies with well-supported reference trees and we were able to identify conditions under which the reference tree was inferred with high support. For Drosophila, whole genome alignments allowed us to directly evaluate which parameters most consistently recovered orthologous sequences. For the parameter ranges explored, we recovered the best results at the low ends of sequence similarity and taxonomic representation of loci; these generated the largest supermatrices with the highest proportion of missing data. Applications of the method to mammals and yeasts were less successful, which we suggest may be due partly to their much deeper evolutionary divergence times compared to Drosophila (crown ages of approximately 100 and 300 versus 60 Mya, respectively). RAD sequences thus appear to hold promise for reconstructing phylogenetic relationships in younger clades in which sufficient numbers of orthologous restriction sites are retained across species.

Project description:Accurate methods for tracking individuals are crucial to the success of fisheries and aquaculture management. Management of migratory salmonid populations, which are important for the health of many economies, ecosystems, and indigenous cultures, is particularly dependent on data gathered from tagged fish. However, the physical tagging methods currently used have many challenges including cost, variable marker retention, and information limited to tagged individuals. Genetic tracking methods combat many of the problems associated with physical tags, but have their own challenges including high cost, potentially difficult marker design, and incompatibility of markers across species. Here we show the feasibility of a new genotyping method for parent-based tagging (PBT), where individuals are tracked through the inherent genetic relationships with their parents. We found that Rapture sequencing, a combination of restriction-site associated DNA and capture sequencing, provides sufficient data for parentage assignment. Additionally, the same capture bait set, which targets specific restriction-site associated DNA loci, can be used for both Rainbow Trout Oncorhynchus mykiss and Chinook Salmon Oncorhynchus tshawytscha. We input 248 single nucleotide polymorphisms from 1,121 samples to parentage assignment software and compared parent-offspring relationships of the spawning pairs recorded in a hatchery. Interestingly, our results suggest sperm contamination during hatchery spawning occurred in the production of 14% of offspring, further confirming the need for genetic tagging in accurately tracking individuals. PBT with Rapture successfully assigned progeny to parents with a 98.86% accuracy with sufficient genetic data. Cost for this pilot study was approximately $3 USD per sample. As costs vary based on the number of markers used and individuals sequenced, we expect that when implemented at a large-scale, per sample costs could be further decreased. We conclude that Rapture PBT provides a cost-effective and accurate alternative to the physical coded wire tags, and other genetic-based methods.

Dataset Information

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Publications

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

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