Tag Archives: RAD-Seq

Analysis of oaks using RAD-Seq

Excellent paper just out that dissects the phylogeny of North American oak trees using RAD-Seq.sequences-per-individual

A phylogenetic study of the American Oak clade based on RAD-seq data has recently be published by PLoS ONE. The study is one of the first to demonstrate the utility of RAD-Seq data for inferring phylogeny in a 23-33 million year-old clade.
Associated with the paper is a new R package for downstream analysis of phylogenetic RADseq data, RADami

A Framework Phylogeny of the American Oak Clade Based on Sequenced RAD Data >>

RAD-Seq for Genome Wide Association Studies

Dear NGS Expert Blog reader,

To kick off the 2014 discussion on RAD Sequencing for the NGS blog, I wanted to share some results from a recently published study describing the use of RAD for high throughput SNP genotyping in Miscanthus.

miscanthusThe tropical grass Miscanthus is an intriguing candidate for bioenergy crop development: It is well adapted to grow in environments worldwide, does not require intensive agricultural efforts to cultivate and is capable of producing large amounts of biomass. To illustrate this point, cultivars of Miscanthus giganteus are capable of growing over 3.5 meters in a single year! With such promise as a bioenergy solution, a number of research groups are working on modernizing breeding efforts in Miscanthus and integrating genomic technologies to help develop superior varieties.

Our group at Floragenex assisted in one recent published study, which illustrates how RAD sequencing was able to facilitate the rapid generation of sizeable molecular resources to aid in a genome wide association study (GWAS). The goal in a GWAS study is to identify a set of genetic variants that tend to be associated with specific traits that are observed in natural, unstructured populations. Some interesting highlights from this paper:

RAD-Seq was able to identify over 100,000 single nucleotide variants (SNVs) across 138 Miscanthus plants. The large number of markers is advantageous for association studies, where understanding the organization of the genome at high resolution is key.

Without an assembled Miscanthus genome, we accomplished variant calling with a two-pronged approach

  • a comparative genomics strategy using the Sorghum bicolor genome as a reference and
  • a de novo clustering approach using the Miscanthus RAD data.

Both were successful methods for high quality SNV discovery and genotyping.

After filtering, approximately 20,000 and 30,000 high quality markers, respectively, were genotyped across the Miscanthus population using the two approaches. After genotyping was complete, the comprehensive genome wide association analysis described in the paper showed statistically significant marker-trait associations for seven key Miscanthus treats, including lignin content, plant moisture and stem diameter. These traits are important for bioprocessing of plant material and the results suggest marker-assisted and genome selection studies could be effective tools in Miscanthus breeding.

The full article, entitled “Genome-wide association studies and prediction of 17 traits related to phenology, biomass and cell wall composition in the energy grass Miscanthus sinensis” can be found at New Phytologist:  http://www.ncbi.nlm.nih.gov/pubmed/24308815

As a co-author on this exciting publication, I would be happy to answer any of your questions on this paper, so do not hesitate to post them. For my next post, I will be comparing many of the new fractional sequencing technologies being utilized for NGS genotyping.

Rick Nipper,
President, Floragenex

RAD-Seq Publications in 2013

RADSeqDear NGS-Expert Blog reader,

As 2013 draws to a close, I wanted to take a few minutes and bring you a some interesting statistics on current trends in RAD sequencing publications and highlight the increasing use of this technology in genomics research.

2013 RAD-Seq Publications in Review

Through November 2013, there have been 45 peer-reviewed scientific papers featuring RAD-Seq as a major component of the published work. This value represents a 135% increase from the number of manuscripts published in 2012 and a 400% increase over the last two years.

RAD continues to be used extensively across a broad spectrum of genetic systems to answer fundamental questions in biology. This year, approximately 70% of the published work focused on applications of RAD-Seq in animal systems, while plant genomics efforts comprise roughly a quarter of the published scientific activity.

Finally, here are the three most-cited Rad-Seq Papers from 2013 (as reported by Google Scholar)

  • Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing.
    Ogden R, Gharbi K, Mugue N, Martinsohn J, Senn H, Davey JW, Pourkazemi M,
    McEwing R, Eland C, Vidotto M, Sergeev A, Congiu L.  Mol Ecol. 2013
  • Genomic patterns of introgression in rainbow and westslope cutthroat trout
    illuminated by overlapping paired-end RAD sequencing. Hohenlohe PA, Day MD, Amish SJ, Miller MR, Kamps-Hughes N, Boyer MC, Muhlfeld CC, Allendorf FW, Johnson EA, Luikart G.  Mol Ecol.
    2013 Jun;22(11):3002-13.
  • Mapping phenotypic, expression and transmission ratio distortion QTL using RAD markers in the Lake Whitefish (Coregonus clupeaformis). Gagnaire PA, Normandeau E, Pavey SA, Bernatchez L. . Mol Ecol.
    2013 Jun;22(11):3036-48.

Best wishes for a safe holiday season.
Rick Nipper,
President, Floragenex

Do You Use RAD-Seq?

The last months we asked you if you are familiar with RAD-Seq, a new approach combining restriction site associated DNA marker genotyping (RAD) with next generation sequencing technology.

73 people did answer the questions. Nearly the half never heard of it.
For this reason we recommend some basic information about RAD-Seq.




Interview With Dr. Georg Gradl About Genome Sequencing

georg_gradl_lowBehind the scene…

You may know that Floragenex and Eurofins MWG Operon launched a new partnership to promote RAD sequencing.

As part of Floragenex’ series of interviews on genomic applications, they talked to our colleague and NGS expert Dr. Georg Gradl about his experiences with de novo genomic sequencing.

Check out the interview

De Novo Genome Sequencing of Sunflower Via RAD-Seq

sunflowerDear NGS Expert Blog reader,

As part of our ongoing series of posts on RAD sequencing, I wanted to share some results from a recently published study describing the use of RAD-Seq for high throughput SNP development in Helianthus annuus (Sunflower).

Sunflower is one of the leading oilseed and confectionery crops in North America, with an annual crop mass of approximately 1 billion kilograms and an economic value over 720 million USD. Despite the economic importance of sunflower, relatively modest genomic resources exist for molecular genetic and marker assisted breeding applications.

To accelerate genomics resource development in sunflower, Floragenex was tasked with rapidly identifying a large set of single nucleotide polymorphism (SNP) markers in North American sunflower through the use of RAD sequencing. The end goal was to translating those markers into a downstream genotyping assay, which could be used for high-throughput applications such as linkage and association mapping.
Some highlights on this study:

  • RAD-Seq was used to rapidly construct over 15.1 Mb of de novo sunflower genomic sequence, comparable in size to a small eukaryotic transcriptome.
  • There were over 94,000 putative SNP markers identified from analysis of six sunflower lines sequenced via RAD-Seq.
  • 16,467 of these variants were incorporated into an Illumina Infinium Genotyping Array.

The above study elegantly demonstrates how RAD is an incredibly efficient marker discovery tool. From just under half a lane of Illumina data (44M 2x80bp reads), a marker resource of over 16 thousand high quality variants could be rapidly generated and deployed for breeding applications.

The full article, entitled “De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach” can be found on BMC Genomics.

As a co-author on the publication, I would be happy to answer any of your questions on this paper, so don’t hesitate to post them. For my next NGS blog entry, I’ll be showing you some interesting publication trends seen with RAD sequencing.

Rick Nipper
President, Floragenex

RAD-Seq Expert Blogger: Welcome Rick Nipper

Dear reader,

As a new author here at the NGS Expert Blog, I wanted to take a few minutes to introduce myself, company and the cutting-edge genomics technology my team specializes in.

My name is Rick Nipper and I am the President of a company called Floragenex. You can learn a bit more about my scientific background on LinkedIn.

rad-seqOver the past 5 years, I’ve worked with the dedicated group at Floragenex to provide genomic services around a new and innovative technology called RAD Sequencing (RAD-Seq).

What is RAD-Seq? RAD stands for Restriction site Associated DNA Sequencing. At it’s core, RAD-Seq is a genomic sequencing strategy. The concept combines a unique library prep protocol with Illumina NGS, to sequence anywhere from 0.1 to 10% of a selected genome. This “complexity reduction” approach to sequencing a genome offers significant advantages compared to other common NGS approaches:

  1. Dynamic. RAD-Seq provides the ability to examine thousands to tens of thousands of genetic loci simultaneously across a genome, depending on your research need.
  2.  Flexible: RAD-Seq has been used as an effective tool across a variety of molecular studies across genetic marker identification, population genetics, linkage mapping, phylogenetics and genome selection
  3.  Speed: RAD-Seq can offer significant time-savings compared to alternative methods.
  4.  Bioinformatics: RAD-Seq projects usually require less next-gen sequencing than other approaches, leading to faster and more streamlined analysis.
  5.  Cost:  Because RAD-Seq is designed to examine a smaller fraction of a genome, it is an economical and cost-effective method for next-gen sequencing of hundreds of samples.

Want to know a bit more about RAD-Seq and how it is being used? There are over 70 publications showcasing the use of the technology. Here are a few I’ve personally been involved in over the years:

Drop me a line if you have any questions about these publications. In the next few months, I’ll continue to post here, highlighting interesting uses of RAD-Seq in the literature, how the technology can help drive research and talking more about some publications in which we’ve been involved.

Rick Nipper
President, Floragenex

Whose Genome Has Been Sequenced? Cicer Arietinum

de-novo-sequencingWith this new bi-weekly series we would like to highlight some if not all genomes that have been sequenced in the last 6 to 12 months. And at this point of time I am still uncertain if the diversity of organisms and species will be the “eye-opener” or the different technologies and strategies that have been used…

We started this series off in January where we reported about the de novo sequencing of the domestic goat Capra hircus.

Today I would like to report about a plant genome, the Cicer arietinum:

According to the GenomeWeb article this de novo genome sequencing approach is only the 3rd one for crop legume plants. For me that is kind of astonishing since breeding and optimisation of crop is already done since years. Maybe this is due to the huge genomes of plants that outperform animal genomes by far. For our chickpea plant with 740 million base pairs we talk about a medium size plant genome. But let’s focus on the sequencing approach for now (Varshney et. al):

What was sequenced?

De novo sequencing of one reference chickpea plant and re-sequencing of 90 cultivated & wild chickpea lines from 10 different countries

Sequencing strategy:

  1. De novo genome sequencing on HiSeq 2000 (paired-end module) of 1 genome with 11 shotgun and mate-pair libraries (insert sizes: ~ 170; 500; 800; 2,000; 5,000; 10,000; 20,000 bp) and BAC end sequencing
    Data output: 153.01 Gb; after filtering & correction steps only 87.65 Gb data were used for de novo assembly
  2. Re-sequencing of genomes
    • Whole genome re-sequencing on 29 varieties using Illumina 100 bp paired-end sequencing on HiSeq 2000
    • RAD-sequencing of 61 genotypes on HiSeq 2000 (48x ApeKI; 24x HindIII)

According to D. Cook “the sequencing of the chickpea provides genetic information that will help plant breeders develop highly productive chickpea varieties that can better tolerate drought and resist disease — traits that are particularly important in light of the threat of global climate change”. (Davis Enterprise).

Read the complete publication here.

RAD-Seq – A brief technical overview

Some time ago I was introducing a new approach combining restriction site associated DNA marker genotyping (RAD) with next generation sequencing technology. Originally this method was developed for microarray platforms. However, the combination of RAD and NGS (Illumina) – resulting in RAD sequencing (RAD-Seq) – enabled the massivly parallel and multiplexed sample sequencing. RAD-Seq is becoming more and more powerful and has the potential to revolutionize agrigenomics, because one can discover and screen thousands of SNP’s and genotype large populations in a high throughput manner at the same time. The scope of the following section is to give a short technical overview how this can be accomplished:

Genomic DNA of each sample is digested in parallel with a certain restriction enzyme and a specific P1 adapter is ligated to the restriction fragments. Thereby each sample will be equipped with an individual P1-adapter containing a sample-specific molecular identifier (Barcode) and Illumina adapter sequences (forward amplification primer site and Illumina sequencing primer site, respectively). If multiplexing is desired, the adapter-ligated fragments of a number of samples can now be pooled. The level of multiplexing depends on the number of differed P1-adapters which have been used before. In a further step the RAD pool will be sheared, size-selected and ligated with a second adapter (P2). The P2 adapter comprises a divergent “Y” adapter containing the reverse amplification primer sites. However, the P2 adapter is special such that fragments lacking the P1 adapter cannot be amplified. This guarantees, that only fragments containing a P1 and a P2 adapter will be selectively and robustly enriched during amplification step following next. The overall length of RAD-tags which can be further analysed mainly depend on the size selection step and sequencing run mode (single vs. paired end), respectively.


RAD-Seq – A Powerful Tool for Non-Reference Organisms

What is RAD-Seq?

Restriction site associated DNA sequencing (RAD-Seq) using the Illumina technology was initially published in PLoS One and in PLoS Genetics. Along with other studies, these studies demonstrated that this smart technique can be very useful for the identification and further analysis of a high number of genetic markers distributed over the genome. This is especially advantageous if working with non-model organisms where no reference sequence is available. Briefly, RAD-Seq results in a reduced representation of the respective genome, because only fragments near a specific type of restriction site are sequenced to deep coverage. Such fragments are called ‘RAD tags’ and serve in subsequent analysis steps as a reference for the design of genetic markers. The overall number of possible RAD tags within an organism strongly depends on the restriction enzyme of choice and the genome of interest.