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


Possibility of Ideal Intestinal Remedy

I’m not able to keep intestinal condition without remedy which is prepared by Lactobacillus, Bifidobacterium, Lactococcus, and others. Eating yogurt is also okay for this purpose, ad personam I prefer to take these bacterial tablets and believe more effects. However, many people know these effects of current intestinal remedies are mild not fast-acting properties.

Several reports mentioned that intestinal bacterial flora and its regulation were not simple. It is starting discussion that natural immunity may regulate intestinal flora; e.g. antibacterial peptide α-defensins which is secreted from paneth cells on small-intestinal epithelium could regulate flora distribution (Salzman et al., 2010; Matsuda et al., 2011). By contrary, very simple strategy is reported as following: Clostridium difficile brings bad diarrhea that was resistant to antibiotic. Van Nood and co-workers (2013) injected healthy person’s feces into patient’s guts, and its curative effect was so good surprisingly, but we cannot call it a remedy!

Two NGS platforms, GS FLX/Junior and MiSeq, can perform distributional analysis via deep sequencing of 16S-rRNA amplicons. But it is still difficult for both platforms to do metagenome assembling for getting whole gene information in flora, because their read length is not long enough to make reliable contigs without chimeras between different bacteria one another. Therefore I strongly expect that super long read platforms including PacBio RS series and coming nano-pore technologies will break current limitations and will contribute to develop ideal intestinal remedy for my instable stomach.

Tumor Heterogeneity And The Underlying Genetics

Although this recorded seminar is promoted by Illumina, I still can recommend it as a very good talk about basic concepts on tumor heterogeneity and the underlying genetics. Kenneth Bloom, Chief Medical Officer at Clarient (GE Healthcare) and a pathologist, explains the needs for the application of NGS in clinical lab in a simple and very vivid manner. While watching this video I gained a better idea of the needs of a diagnostics lab and the challenges in developing a diagnostic NGS panel.

CSP for human exome sequencing with Ion Proton

Ion Proton System  - Front FacingEurofins MWG Operon joined the exclusive certified service provider program from Life Technologies. Using the Ion AmpliSeq Solution for human exome sequencing on Ion Proton offers several advantages:

  • Flexible number of samples: starting from 1 exome up to 96
  • Fast turnaround times: 5-10 workding days
  • High data quality: mean coverage ~ 100x;  mean read length ~ 160 bp
  • Low input material: starting from 250 ng / sample

Learn more about human exome sequencing at Eurofins here.Ion Proton

Transcriptome Sequencing In Translational Oncology Research

By using novel microfluidic tools, a team of researchers at Indiana University School of Medicine uncovered an unexpected ability of cancer cells to navigate and exit microscopic mazes along the shortest path. To explain this behavior, they propose a novel mechanism that guides cancer cell migration.

Find out how they have harnessed RNA-seq on tumor tissues to reveal efficacious drug targets and implement rational drug combinations in triple-negative breast cancer. Further, ongoing work on how RNA-seq is being used for biomarker discovery in retrospective cancer clinical trials will also be presented.

Genetic Differences Between “Identical” Twins Discovered

A multidisciplinary team in the Eurofins flagship Genomics laboratory in Ebersberg, Germany, has successfully completed a research project to genetically discriminate “identical” monozygotic twins.

So far there have been only theoretical considerations against the experimental finding and dogma that monozygotic twins are genetically fully identical. Statistically, around 6 of 1,000 males are identical twins. Up to now, forensic DNA fingerprinting testing could not be used in crime or paternity cases involving identical twins, as there was no possibility of genetically discriminating between them. Such cases are regularly discussed in the World’s press, including murder, child custody and heritage cases. Forensic laboratories around the world had accepted these analytical restrictions, but Eurofins scientists wanted to push these limits of DNA testing. They used the unique combination of leading forensics and genomics labs available at Eurofins to reach this milestone.

Technically, the scientists applied ultra-deep next generation sequencing and associated bioinformatics techniques. They sequenced DNA from sperm samples of two twins and from a blood sample of the child of one twin. Bioinformatics analysis revealed five mutations, so called Single Nucleotide Polymorphisms (SNPs) present in the twin father and the child, but not in the twin uncle. The SNPs were confirmed by classical Sanger sequencing. The results give experimental evidence for the hypothesis that rare mutations will occur early after or before the human blastocyst has split into two, the origin of twins, and that such mutations will be carried on into somatic tissue and the germ line.


The dataset in this project equaled a total of 241 human genomes, resulting from up to 94 fold genomic coverage of the involved three individuals.

The peer-reviewed study “Finding the needle in the haystack: Differentiating “identical” twins in paternity testing and forensics by ultra-deep next generation sequencing” is published in the renowned journal Forensic Science International: Genetics, Available online 8 November 2013, ISSN 1872-4973, http://dx.doi.org/10.1016/j.fsigen.2013.10.015.

1,000 Fish Transcriptome Project

The China National Genebank (CNGB) announces the official launch of the 1,000 Fish Transcriptome Project (Fish T1K). It marks the beginning of an amazing transcriptome study designed to unveil the mysteries of the origin, evolution, and diversification of the largest group of vertebrates.

The findings could enable scientists to pursue innovative approaches and strategies to address challenges in fish breeding, disease control and prevention, seafood safety and biodiversity conservation.

Read more at BGI news

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

Next Generation Sequencing in Lung Cancer

Dr. Benjamin Levy (Head of Thoracic Medical Oncology, Division of Hematology-Oncology, Beth Israel Medical Center, Continuum Cancer Centers of New York and Assistant Professor of Medicine, Albert Einstein College of Medicine) is talking with Selma Schimmel about next generation sequencing in lung cancer.

The interview was filmed at the American Society of Clinical Oncology Annual Meeting in Chicago 2013.

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.