FREQUENTLY ASKED QUESTIONS

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GENERAL INFO

  • 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 technologies are "complexity reduction" protocols and 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 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 enable more streamlined bioinformatic analysis.

  • We accept DNA extracted from anything but humans, virus', or bacteria.

    That means plants, animals, coral, fungi, ect can be sent to our labs! For processing we require samples be extracted and normalized to a consistent concentration. Samples should show good quality high molecular weight dsDNA with no to minimal contamination. Watch our video with recommendations on how to ship samples to our lab.

  • We include our own control in processing which goes in well H12. This is why we request that H12 always be left empty.

  • Yes, though each species has a unique number of restriction enzyme cutsites and as a result, some species may be underrepresented or overrepresented in the final data set. Whenever possible, sample plates should be grouped by species.

  • No, not at this point in time.

  • 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, and PstI. (Note some enzymes have limited barcode sets). For ddRAD / SBG, (a two enzyme protocol) we utilize PstI/MseI, EcoRI/MseI, and Sbfl/Msel.

  • Multiplexing of up to 380 samples is possible with RAD-Seq. ddRAD-Seq / SBG multiplexing is capped at 95 samples.

  • 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, target sequencing, etc.) so we can help in planning how to integrate RAD-Seq into an overall research program or larger study.

  • 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.

  • There are over 100 peer-reviewed scientific studies employing RAD-Seq. Check out our project page for more information, or read about RAD-Seq in the October 2011 Illumina iCommunity newsletter.

PROJECT INFO

  • Submitting clean, high-quality genomic DNA is one of the most important factors for sequencing success, and also one of the trickier parts of DNA extractions. While there are kits and/or specialized protocols for yielding pure, high-molecular weight DNA, contamination of samples can still occur. Whenever contamination is suspected, the first step is to identify a likely cause and then take steps to remove it. The most common culprits are RNA, degraded DNA, and protein, and as a first step, RNase treatment can often help and is unlikely to cause any issues even if RNA contamination is not the issue. That failing, we recommend looking back at the extraction protocol and/or performing a secondary cleanup if there appear to be multiple contaminants.

    We are always happy to help ensure the success of your project, so if would like a second opinion feel free to reach out!

  • While set(s) of 95 are the standard project size we do have options for projects with fewer samples. Our pilot project option, for instance, can have as few as 8 samples. We can also see about setting up a specific quote for your project.

  • Project turnaround various depending on each project needs. Projects can take up to 12 weeks before data is ready to be delivered.

  • Raw sequence data is provided in FASTQ format. FASTQ files are filtered for adapter sequences prior to delivery. If a project includes bioinformatics analysis you will receive an archive with all files produced in the analysis, including FASTQ files demultiplexed by sample, any reference sequences created or used, alignment files, and genotype files in VCF format, and reports for each step. Optionally, the genotype files can be converted to STRUCTURE, Genepop, IUPAC, strataG, or Join Map formats. FASTQ files and analysis archives are delivered through a secure time sensitive link.

  • We do not perform extractions or normalization in our lab however we have a partner that does. If you are interested in this service let us know and we can get you more detailed information.

  • 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.

  • 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.

  • No you cannot. Our processing requires samples to be in 96-well PCR plate format.

SEQUENCING INFO

  • Next-Gen sequencing is available on the following platforms: Illumnia NovaSeq 6000, Illumnia MiSeq, and the PacBio Sequel II system.

  • Read lengths of between 50 to 150 base pairs are possible. Only single-end reads (SR) are available for ddRAD-seq / SBG projects (1x100bp), while RAD-seq projects are read in a paired-end (PE) format (typically 2x150bp).

  • 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.

  • Depth of sequencing varies based on the amount of sequencing purchased.

  • 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.