GBS or ddRAD Library Preparation Guidelines

The following guidelines apply to those submitting DNA for GBS or ddRAD library preparation intended for sequencing on Illumina instruments.

All General Guidelines must be followed in addition to the guidelines specific to GBS/ddRAD library preparation.

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Sample Delivery Vessel - General Guidelines

  • Place in a 96-well skirted, translucent plate (e.g., Eppendorf twin.tec #00030129300).
    • Sample placement should be going down columns (A1,B1,C1…A2,B2,C2…) in contiguous wells (no skipping wells, rows, or columns unless “Blank” samples).
    • Plate should be sealed tightly with one of the following:
      • If shipping on dry ice, seal wells with tight-fitting 8-cap strips (e.g., Thermo Scientific #AB-0265)
      • If delivering from on campus, firmly seal wells with a clear adhesive seal (e.g., Bio-Rad Microseal ‘B’ Film #MSB1001).
    • Samples that are submitted in other containers will be transferred to a 96 well plate at the submitting lab’s expense. Please inquire for cost of transfer.

Sample Naming Conventions - General Guidelines

  • Each sample must be identified with a simple, unique identifier. If names have been used in previous submissions, don’t repeat in subsequent submissions.
  • The following conventions are strictly enforced:
    • 3-20 characters
    • A-Z, a-z, 0-9, dash (-) are the ONLY acceptable characters
    • Space, underscore, punctuation, and special characters are NOT ALLOWED
    • No replication

Sample Labeling Conventions - General Guidelines

  • All tubes and plates must include the following information.
    • PI name
    • Date
    • Sample name (tubes only)

Sample Quantification - General Guidelines

  • Upon sample receipt, the UWBC will quantify the DNA with a fluorometric assay. QC measures done by the UWBC are considered the gold standard, regardless of the measurements done by the submitting lab. Prior to proceeding with samples that fail to meet these requirements, the lab will be informed.

GBS and ddRAD - Specific Guidelines

  1. Follow ALL General Guidelines above.
  2. DNA Quantity: A minimum of 15 µL DNA at a concentration of 20-50 ng/µl is required. DNA should be measured by intercalating dye (e.g., PicoGreen, Qubit). Nanodrop and other spectrophotometers are not acceptable measures of DNA quantity. We will quantify the DNA once we receive it using PicoGreen. DNA concentrations do not need to be standardized across the plate; we will do this after they are received.
  3. Lyophilized DNA is acceptable, but we prefer to have DNA suspended in 10mM Tris or water. Please avoid EDTA.
  4. DNA Quality: To evaluate DNA quality, we require a gel image that includes a well-labeled ladder as well as an aliquot of each DNA prep. Load 100ng of each DNA sample on to a 1% agarose gel, and run long enough and slow enough that the size standards are clearly separated. DNA should all be high-molecular weight (above the highest size standard on the gel), with minimal smearing. If you can, please run the undigested sample next to the digested sample on the gel (see next step).
  5. In order to test how well the samples cut and to see if any inhibitors are present, all samples submitted should be digested using a common restriction enzyme that is NOT methylation sensitive (ex: HindIII or EcoRI). At the very minimum, 10% of the samples should be tested. 300-500ng of each sample should be digested for a minimum of 2 hrs at 37C, heat killed, then run on a 1% agarose gel as above. Gel images should be sent to nextgen-seq@biotech.wisc.edu for approval prior to submitting sample plates.
  6. Optimization: If this is your first GBS experiment, please check to see if your species has been previously validated for GBS by contacting nextgen-seq@biotech.wisc.edu. If it has not, we will need to create optimized assay conditions for your species. To do this, we will either need at least 10µg of DNA, from a single sample, or split between many samples. To optimize the species, we will test each set of samples with several restriction enzymes and adapter concentrations in order to find a “best case scenario” for your library. Optimized reactions create a library that has its highest concentration at 200-250 bp, with fragments tapering off to 500 bp, minimal residual adapter contamination, and no visible indication of repetitive DNA contamination (often visualized as a serrated effect on a Bioanalyzer chip). No sequencing is done on the GBS optimization.
  7. This service is provided by the plate, regardless of sample counts <96. Individual libraries within a single plate are pooled equal mass, when possible, prior to sequencing. Sequencing requests provided per sample will be summed per plate. Therefore any request will be met at the plate level but not necessarily at the sample level.