Floragenex Frequently Asked Questions (FAQs):
- Tell me about Floragenex
- What genomic applications and services does Floragenex provide?
- What is RAD-Seq and why should I use it?
- Are there papers on RAD-Seq?
- What types of scientific studies are possible with RAD-Seq?
- Do you have experience in my organism of interest?
- What type of restriction enzymes do you use?
- How many fragments or tags does each enzyme generate?
- What types of next-gen sequencing do you use?
- How is single-end RAD-Seq different from paired-end RAD-Seq?
- What depth of sequencing coverage do you achieve?
- There is no reference genome for my species. Does RAD still work?
- My plant of interest is a polyploid. Is that a problem?
- How many genetic markers will RAD identify?
- Can I use a pooled DNAs to facilitate SNP discovery?
- Can I use a pooled sequencing approach for paired-end RAD-Seq?
- I want to take SNPs identified from RAD-Seq and construct a genotyping chip or array. Is this possible?
- I want to use RAD-Seq to genotype a population. Is this practical?
- How much does a Floragenex project cost?
- Are there minimum sample numbers needed for RAD-Seq?
- How much DNA do you need for RAD-Seq?
- Do you provide bioinformatics support?
- What are the deliverables for a Floragenex project?
- How long will my project take?
- What do I need to send to begin my project?
About Floragenex
Floragenex is a contract research organization founded in 2007 that specializes in the application of next-generation DNA sequencing to further genomic studies in plant and animal biology. We support the global academic research community and commercial agricultural biotechnology sector by providing end-to end outsourced genome research services, high-throughput sequencing capabilities and bioinformatics analysis.
What genomic applications and services does Floragenex provide?
Our firm provides end-to-end expertise in a single platform technology: Restriction site Associated DNA (RAD-Seq). RAD-Seq is a versatile multiplex sequencing protocol designed to provide maximum data at minimum cost. The Floragenex team provides over 20 years of combined experience in 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 deciding how to integrate RAD-Seq into an overall research program or larger study.
What is RAD-Seq and why should I use it?
RAD-Seq is what is known as a genome “complexity reduction” protocol, which is designed to reliably interrogate a small percentage of the target genome instead of the entire genome sequence. To achieve this, the genome is digested with a specific restriction endonuclease, sheared and then sequenced. The short fragments (or tags) of genomic DNA that flank each digestion site are then screened for the presence of genetic variation such as single nucleotide polymorphisms (SNPs). The principal advantages RAD-Seq provides are reduced sequencing cost versus whole genome approaches, high-multiplex sequencing and more streamlined bioinformatic analysis. Want to know a bit more? Read about RAD-Seq in the October 2011 Illumina iCommunity newsletter.
Are there papers on RAD-Seq?
Yes. There are dozens of peer-reviewed scientific studies employing RAD-Seq which have been published. Check out our publications for more information.
What types of scientific studies are possible with RAD-Seq?
RAD-Seq is a genetic variant discovery and genotyping system. Depending on your interest, several applications can be achieved in a single sequencing project:
Genetic Marker Discovery
Identify and catalog large numbers of genetic variants
such as microsatellites, SNPs and InDels.
Local Genome Assembly
De novo genome assembly for survey sequencing,
comparative genomics and variant detection.
Genotyping-by Sequencing (GBS)
Develop genotype information for linkage maps
association mapping or population genetic analysis.
Do you have experience using RAD-Seq in my crop or species of interest?
Ask us! Floragenex has sequencing experience in over 30 unique plant species. We have four years of knowledge working with poaceae, asterids, solanacea, trees and a variety of other major row and vegetable crops.
What type of restriction enzymes do you use for RAD-Seq?
Floragenex uses a predefined panel of methylation sensitive restriction enzymes for RAD sequencing in plants. Use of these methyl-sensitive enzymes helps restrict sequencing to endonuclease sites in the hypomethylated, low-copy fraction of most angiosperm genomes. This technique allows us to efficiently sequence both small, gene-rich genomes and plants with large genomes harboring massive retrotransposon content.
How many fragments or tags does each enzyme generate?
It varies between species, but low density enzymes will typically generate 5,000 to 10,000 unique fragments from whole genome digestion, while high-density enzymes usually generate over 50,000 tags.
What types of next-generation sequencing do you use with RAD?
Floragenex uses only Illumina GAIIx/HiSeq 200 sequencing platforms for RAD-Seq. Current read lengths range between 50 and 150bp with both single and paired-end sequencing possible.
How is single-end RAD-Seq different from paired-end RAD-Seq?
Single-end RAD-Seq employs standard single-read sequencing chemistry and is designed to only interrogate the genomic sequence immediately adjacent to a restriction enzyme digestion site. The advantage with this approach is that any genetic variation within the read can be readily identified or scored. Single end RAD-Seq tends to be most useful for marker discovery and resequencing studies in species with a reference genome, or for large-scale genotyping studies.
Paired-end RAD-Seq is a technical analog to the 454 sequencing platform. PE RAD-Seq allows us to generate much longer sequence information, up to 500-600 basepairs than with typical single-read chemistries.This has great utility for de novo assembly and marker discovery in species without a reference genome, developing marker content for SNP genotyping assays or for comparative genomics studies.
What is the sequencing coverage you obtain with RAD-Seq?
Depth of sequencing varies by application and the project design. For paired-end RAD-Seq, because of the nature of de novo assembly, >80x is desired. For single-read sequencing, anywhere from 15x-40x may be appropriate, again depending on the specifics of the project.
Wait! There is no reference genome for my species. What can RAD do there?
RAD-Seq is incredibly flexible. If you need genomic or marker data from a plant, we can generate it, regardless of genome status. The exact strategy and project design selected will be based on your research goals.
My plant of interest is a polyploid. Is that a problem?
RAD-Seq is applicable to both diploids and polyploids. Our team has worked in a variety of complex, challenging systems, including species with large, duplicated genomes.
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 for RAD-Seq. Typically low-density scans can uncover several hundred markers, while high-density scans can catalog thousands to tens of thousands of markers. As you might expect, more genetically diverse species will generate more markers than crops that have been bottlenecked or have a narrow genetic base.
Can you use a pooled sequencing approach with RAD-Seq to facilitate SNP discovery?
Yes, sequencing of pooled samples is possible with RAD-Seq. Typically the objective is identification of major alleles (MAF > 0.05) in the pooled DNA. Depending on the number of samples present in the DNA pool, sequencing coverage may be adjusted to achieve the goals of the project.
If pooled sequencing works for variant detection, can you use a pooled sequencing approach with the paired-end RAD-Seq technique?
Not for sequence assembly. By definition, pooling DNA introduces significant sequence heterozygosity into a sample. This can dramatically affect the efficiency of sequence assembly and shorten N50 contig lengths. However, once an assembled reference is obtained, pooled paired-end sequencing is possible. Again, sequencing coverage will need to be adjusted to facilitate accurate variant detection.
I want to take SNPs identified from RAD-Seq and construct a genotyping chip or array. Is this possible?
Yes. For SNP marker development, which is compatible with other genotyping platforms (ie. Illumina GoldenGate, Sequenom MassARRAY, Roche TaqMan), an appropriate sequencing strategy is selected based on the available genomic resources. With an existing genome, only the single-end RAD-Seq approach is necessary for marker development. Absent this reference, the more complex, paired end, de novo assembly approach is required. To date our group has successfully applied these approaches for SNP development in over 20 species.
I want to use RAD-Seq to genotype a population. Is this practical?
Absolutely. This approach is called genotyping-by-sequencing (or RAD-Seq GBS). Most GBS studies use a single-end RAD-Seq approach and are designed to catalog and score genetic variants from either a biparental mapping or unstructured population.
How much does a Floragenex RAD-Seq project cost?
Each project is unique. Depending on the project size, complexity and application the budget for a RAD-Seq effort can fall anywhere between $10,000 and $200,000 (USD).
Are there minimum sample numbers needed for RAD-Seq?
For SNP discovery, we usually need only 2 or more samples to begin work. For RAD-Seq GBS work, we require 48 or more samples to initiate a project (we work in increments of 48 and 96).
What quality and quantity of genomic DNA do you need for RAD-Seq?
Floragenex input requirements for genomic DNA are strict. RAD-Seq library production requires several micrograms of purified, high molecular weight genomic DNA. Please see our sample input requirements for more specific information.
Do you provide bioinformatics support?
Yes. General bioinformatics support is included in the cost for each sample. We also provide advanced analysis services for any project requiring more detailed analysis.
What are the deliverables for a Floragenex project?
Our standard deliverables for sequencing efforts depend on the research application. As an example, for projects focused on SNP discovery we provide:
1) The raw Illumina sequence data and associated quality reports
2) Assembled sequence contigs or scaffolds (fasta format)
3) BAM alignment files
4) Genotype and SNP calls annotated in a Variant Call Format (VCF) 4.1 file.
How long will my project take?
Turn-around times for most FGX projects typically range between 12-24 weeks, depending on the application, and scale of effort. GBS projects take a minimum of 20-24 weeks to complete.
What do I need to begin my project?
Just provide us some information about your project and we’ll guide you through the project design phase and provide information on what our laboratory needs to get started.
