After getting satisfactory Qubit results post-PCR, my final step in the ddRAD/EpiRAD library prep was to run the samples on the Agilent TapeStation to make sure fragments of correct and consistent size were selected earlier by the Pippin Prep.

1. Spin down samples and vortex

2. Obtain High Sensitivity D1000 Screentape and reagents. Spin down and vortex reagents. Note that ladder is in an extremely small volume.

3. Obtain 13 strip tubes (12 plus one for ladder) and place on a tube rack

4. Label 1st tube “L” for ladder, then 1-12 for each pool (only need to indicate order, not every tube)

5. Pipet 2 uL buffer into each tube

6. Pipet 2 uL ladder into “L” tube

7. Pipet 2 uL sample into each respective tube

8. Place caps on tubes, spin down, and vortex

9. Remove caps from tubes **Important – do not run TapeStation with lids on tubes

10. Place tubes in instrument, making sure ladder is in well position #1 (top left)

11. Place High Sensitivity D1000 Screentape in instrument with label facing towards the front of the instrument and the barcode facing right.

12. Ensure there are new pipet tips in machine and no used ones remaining

13. Open Agilent Tapestation Controller software

14. Select the tubes that will be used on the diagram

15. Rename the samples. These need to be the final names that will be sent to the sequencing facility. For example, my run was named JD001, and each pool was named A-L, such as JD001_A, JD001_B, and so on.

16. Press “START”

17. Save to JayDimond folder on desktop

18. When run is done, save output file as a MS Word document by creating a report under the file menu; click option to create thumbnail of each sample. This needs to be saved using the final plate name sending to the sequencing facility. For example, my file was named 2016-08-08_JD001_A-L.docx. If the run contains only part of a plate or parts of different plates name accordingly.

My results looked good, showing consistent size selection and quantities that matched well with the prior Qubit results. The fragment sizes were a bit larger than they should have been, averaging about 495 bp instead of 465 bp. If the resulting fragments are to be compared to fragments generated in another set of libraries, it is important that the fragment sizes are as consistent as possible, because the loci chosen will vary depending on their size. Thus it is particularly essential that the Pippin Prep settings are the same.

These libraries will be sent to UC Berkeley within the next two weeks or so along with Greg’s frog libraries. Once there, the facility will perform qPCR to ensure the libraries are combined in equimolar ratios in a single lane.

Library metadata:

Note that two samples were compromised due to pipetting errors: sample 102, digested by PstI/MspI, pool 3, well C; and sample 120, digested by PstI/MspI, pool 8, well E. These samples were completely excluded early in the process and no sequences should be obtained from them.