Genotyping-by-sequencing without prior genome information.


RAD (Restriction-site Associated DNA) sequencing is a method by which certain restriction enzymes are used to fragment genomic DNA samples, followed by size selection and sequencing of molecules within a certain size range. This enables reproducible enrichment of a particular set of genomic loci, where the loci retained for sequencing are more or less randomly distributed over the genome. The sequence data resulting from this application may be used for downstream applications including, but not limited to: Linkage analysis, inference of population genetics metrics and polymorphism discovery.

Sample requirements

InputPurified EcoRI-cut DNA. Gel results must be provided.
Amount>200 ng
Concentration16–100 ng/µL
Volume16–170 µL
Sample Buffer10 mM Tris, pH 8–8.5 or similar (e.g. Qiagen’s EB).

How to evaluate the sample quality

We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples. For this library prep method, we require the following to be included when submitting sample information:

  • The EcoRI-cut DNA should be run on a gel with digested/undigested samples side-by-side, to confirm successful digestion.

If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.

What we do with your samples

EcoRI-digested samples are prepared using the Agilent Bravo system (protocol available at Github: https://github.com/ngi-automation/rad-seq).

Library preparation

During preparation a custom adapter with complementary bases to the EcoRI overhang is ligated to the ends of the digested DNA using T4 DNA ligase and 10X T4 DNA ligase (New England Biolabs). The ligated adapter contains the partial Illumina adapter. Samples are purified using AMPure XP bead purification (Beckman Coulter), strand displacement is performed using Bst 2.0 polymerase, 10X ThermoPol buffer (New England Biolabs) and dNTPs (Thermo Fisher Scientific), another bead purification follows before amplification and indexing by PCR. Finally, the libraries are cleaned up in another bead purification.

Library QC and sequencing

To assess the quality of the library, the concentration is measured with the Qubit dsDNA HS assay (Thermo Fisher Scientific) and the fragment size distribution is checked using the Fragment Analyzer (Agilent). A calculated concentration in the range of 420–720 bp is used to even out the different samples contribution to the pool before subjecting the pool to size selection with the BluePippin (Sage Science). Concentration and size of the pool is analyzed with Qubit and Fragment Analyzer (or Bioanalyzer) before sequencing  according to agreed upon setup.

Expected results

It is expected that the resulting sequence data from each sample is enriched for loci in the genome that were generated by EcoRI cuts and that the libraries from each sample will feature mostly the same sequenced loci.


Analysis with STACKS: http://catchenlab.life.illinois.edu/stacks/

Relevant Technologies
Bioinformatics Pipelines
Method Status


We are routinely running this method. Please visit the Order Portal to place an order.