Genotyping by Sequencing

Based on the methods of: Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, et al. (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLOS ONE 6.

See also Rob Elshire’s protocol for the Buckler Lab.

  1. Assemble adapters with barcodes (this was done by Hardeep Rai in 2011).

  2. Assemble common adapter (done by Hardeep 2011).

  3. Mix each barcoded adapter with common adapter in exact 1:1 working mixes of 3ng/uL (done by Hardeep 2011).

  4. Quantify DNA samples on Qubit.

  5. Dilute DNA samples to 10ng/uL.

  6. Put one 100ng of a DNA sample (10ul of 10ng/uL) in each plate well.

  7. Spin down and dry down.

  8. Digestion:

      1. Make a digest master mix:
     Per sample56 X (54 samples +2 for pipette loss)
    NEB Buffer 32uL112uL
    1. Add 20uL master mix to each plate well. Capped tubes, vortexed briefly and spun back down to get any DNA that crept up the sides of the tubes in the drying process. If I do this again, I think I would NOT dry down the DNA, and just use 10uL less water in each reaction to adjust.
    2. Digest 2hrs @ 75 C then hold at 4C.
    3. Bioanalyze a subset of the results to verify digestion was complete. Contact Ninglin @ the CIB. Do 11 samples on one plate of Bio-Rad Experion with a 12K_DNA chip. Requires 3uL of each sample, but they ask for enough to rerun in case of technical issues. $80/chip and same day results.
    4. The virtual gel image from the bioanalyzer doesn’t have nearly as many dark bands in the 2000-5000bp range as Hardeep’s did, so I added 1uL of ApeKI to each tube, vortexed, spun down, and digested for another 2 hours at 75C. There ARE darker bands in this region indicating that there was digestion, just not complete, so I did not bioanalyze again.
  9. Ligation:

      1. Make ligation master mix:
     Per sample55X (54 +1 for pipette loss)
    10X T4 Ligase buffer5uL275uL
    T4 Ligase1.6uL88uL
    1. Add 28.8uL of master mix and 1.2uL of 3ng/uL adapter to each plate well, vortex, spin down.
    2. Ligate 1hr @ 22C then 30min @65C then hold at 4C.
  10. Pool samples (5uL from each well) into 1350uL (54 samples * 5uL * 5) PB binding buffer from the Qiagen QiaQuick PCR cleanup kit.

  11. Cleanup following the Qiagen PCR cleanup kit instructions with Qiagen PCR cleanup kit (one column needed) eluting into one 50uL EB solution.

  12. PCR enrichment:

    1. Setup one PCR reaction:
      • 25uL 2X NEB Taq master mix
      • 18uL ddH2O
      • 2.5uL PCR primer 1
      • 2.5uL PCR primer 2
      • 2uL cleaned pooled DNA
    2. Amplify 5min @ 72C, 30s @ 98C, 18 cycles of (10s @98C, 30s @65C, 30s @72C), 5min @72C, hold at 4C.
  13. Cleanup with Qiaquick PCR purification kit (one column needed) into 30uL of EB.

  14. Bioanalyze to compare the unenriched and PCR enriched library.