Frequently Asked Questions:
Answers to the most commonly asked questions about Floragenex, RAD-Seq, ddRAD, SBG and AmpliSeq can be located below, but if you can't find what you're looking for, please send us your questions using the Contact-Us page.
our team and organization
Floragenex is a privately held biotechnology company that provides solutions for genomic analysis in human, plant and animal systems. We focus on applications combining next-generation DNA sequencing in combination with fractional sequencing technologies such as RAD-Seq , ddRAD-Seq / SBG and Ion AmpliSeq. With multiple offices located on the US Pacific coast, our technologies allow investigation of genomes at unprecedented levels for academic, governmental and commercial researchers worldwide.
Who we work with
Floragenex works with academic, governmental and non-profit researchers worldwide. Since 2007, our genomics service team has proudly provided support for RAD sequencing projects in over 350 projects, spanning 25 countries and 6 continents. Our dedicated team can help manage each step of every project from DNA sequencing to data analysis, providing an integrated solution to help scientists advance their research and publish impact results.
RAD-Seq / ddRAD-Seq / SBG
restriction enzyme based sequencing
What ARE RAD-Seq / dd-rAD-Seq / SBG and why should I use them?
RAD-Seq, ddRAD and SBG stands for Restriction-site Associated DNA Sequencing, Double Digest RAD and Sequence Based Genotyping, respectively. At their core, each of these technoligies are "complexity reduction" protocols are designed to examine a small percentage of the target genome instead of the entire genomic sequence. RAD-Seq involves the digestion of a template genome with a specific enzyme called a restriction endonuclease, followed by several shearing and molecular biological processing steps. ddRAD and SBG use two restriction enzymes instead of the a cut and shear process. The resulting mixture from a RAD / ddRAD / SBG library is then sequenced using a next-generation DNA sequencing platform. The short fragments (or tags) of genomic DNA that flank each digestion site are screened for the presence of genetic variation such as single nucleotide polymorphisms (SNPs). The principal advantage of these approaches is the ability to simultaneously examine tens of thousands of genetic loci with vastly reduced sequencing costs versus whole genome approaches. Additionally, RAD / ddRAD / SBG offer other advantages in high-multiplex sequencing operations and enables more streamlined bioinformatic analysis.
Are there papers on RAD-Seq?
There are over 100 peer-reviewed scientific studies employing RAD-Seq. Check out our science page for more information, or read about RAD-Seq in the October 2011 Illumina iCommunity newsletter.
What restriction enzymes are available for RAD-Seq and ddRAD-Seq / SBG?
Floragenex uses a defined panel of restriction enzymes for RAD sequencing that have been validated in dozens of plant, animal and microbial genomes. To date we have used the following restriction enzymes for RAD: SbfI, SgrAI, NotI, EcoRI, BamHI and PstI. (Note some enzymes have limited barcode sets). For ddRAD / SBG, (a two enzyme protocol) PstI and MseI are the only nucleases available.
What is the maximum multiplexing limit with RAD-Seq / ddRAD-Seq / SBG?
Multiplexing of up to 380 samples is possible with RAD-Seq. ddRAD-Seq / SBG multiplexing is capped at 95 samples.
What next-gen sequencing platforms and read lengths are available for RAD-Seq / ddRAD-Seq / SBG?
RAD-Seq is available across the entire Illumina family of sequencing platforms, including the MiSeq, HiSeq and NextSeq systems. Read lengths of between 50 to 150 base pairs are possible.
DO YOU HAVE EXPERIENCE USING RAD-SEQ IN MY SPECIES OF INTEREST?
Floragenex has sequencing experience in over 100 plant, animal, and microbial species. Our team brings over 30 years of combined experience in RAD library construction, Illumina next-generation DNA sequencing and high-throughput bioinformatics analysis. Our group is also familiar with other sequencing strategies (whole genome shotgun, capture sequencing, etc.) so we can help in planning how to integrate RAD-Seq into an overall research program or larger study.
How many genomic sequences does each enzyme generate?
Enzyme performance will vary between species, but enzymes that digest a genome infrequently (low density) will typically generate 5,000 to 10,000 unique fragments from whole genome digestion, while high-density enzymes usually generate over 50,000 tags. Note that digestion performance can vary widely between species due to genome organization.
What is the sequencing coverage you obtain with RAD-Seq / ddRAD-Seq / SBG?
Depth of sequencing varies based on the amount of sequencing purchased.
There is no reference genome for my species. Will RAD sequencing work in my system?
RAD-Seq, ddRAD-Seq or SBG will work with or without an available reference genome. The exact strategy and project design selected will be based on your research goals.
My species of interest is a polyploid. Is that a problem?
RAD-Seq, ddRAD and SBG are applicable to both diploids and polyploids. Our team has worked in a variety of complex, challenging systems, including species with large, duplicated genomes. Contact our service team if you have any questions.
How many genetic markers will I obtain using RAD-Seq?
The number of markers will depend on the diversity in the target species and enzyme selected. Typically low-density scans can uncover several hundred markers, while high-density scans can catalog thousands to tens of thousands of markers. More genetically diverse species will generate more markers than species that have been bottlenecked or have a narrow genetic base.
WHAT TYPES OF SCIENTIFIC STUDIES ARE POSSIBLE WITH RAD-SEQ / ddRAd-Seq / SBG?
RAD is a versatile system for genetic marker discovery and genotyping. Floragenex clients have used the technology for SNP and SSR marker discovery, linkage mapping, population genetics, ecological and conservation genomics research, association mapping and genome selection.
analysis of next-gen sequencing data
DO YOU PROVIDE BIOINFORMATICS SUPPORT?
Bioinformatics analysis using Floragenex's cloud based pipeline is available to clients who need additional support. This robust analytical package has been optimized over hundreds of projects to produce a variety of reports, file types and outputs.
What bioinformatics analysis do you perform?
Floragenex provides a range of bioinformatic solutions focused on next-gen sequencing, including, sequence quality assessment, de novo assembly, NGS alignment, variant calling (SNPs) and conversion of variant calls into a multitude of software formats (such as STRUCTURE and JoinMap™). Visit our Analytics page for more detailed information or to view an example report.
choose from a variety of service options
What genomic products and services does Floragenex provide?
We provide services with RAD, ddRAD / SBG and Ion Torrent AmpliSeq technology solutions to support client needs in advanced scientific research. Bioinformatics and analytical support are available for all of these technologies. These options provide a tiered system of genomic solutions in genetic marker discovery, genotyping and analysis. Floragenex also provides Asanté products for sample collection and DNA extraction.
What are the deliverables and turn around times for a Floragenex project?
Deliverables are contingent on the work specified, but can range from library preparation to NGS data and / or analysis files. Turn around times also vary based on the complexity of work, however, many projects are completed in under 12 weeks
What quality and quantity of genomic DNA do you need for RAD-Seq?
To achieve optimal project outcomes, Floragenex has stringent input requirements for genomic DNA. RAD-Seq library production requires several micrograms of purified, high molecular weight genomic DNA. Please see our sample input requirements for more specific information.
how to get your project running
What do I need to plan or begin my project?
If you are unsure about how to use RAD, ddRAD, SBG or AmpliSeq in your project, please send us a message via our Contact Us page below and provide the following information.
- What species are you studying? Are all samples in this project from the same species?
- Briefly describe your project's scientific goals. (Example: population genetics, phlyogenetics / geographics, linkage / QTL map, association mapping, breeding or parentage analysis)
- Approximately how many genetic variants / markers do you require to meet the project goals above? Please provide estimated numbers you are looking for in the final ready to analyze dataset.
- How many individual samples will you be submitting?
- Do you require bioinformatics support?
- Any other special requests?
One of our staff will be in touch to guide you through the process and provide information on what our laboratory needs to get started.