Troubleshooting Zymo SwitchFree Library prep with Zymo team #97
Description
Conversation with Zymo about our issues with the Zymo SwitchFree Library prep kit.
We talked with Jeffrey Piña, Sr. Research Associate jpina@zymoresearch.com
I sent some of my RNA samples, Pacu-D8 (no library) and Mcap-E7 (low yield) so that he could try prepping the library. I've attached to this issue an Excel file with the details of the samples I sent.
Jeffrey replied on the 2nd of April 2025 with details about the tests he did (I've attached his report files to this issue). I'm summarizing his recommendations for things that worked:
- perform SwitchFree library preparation using 10 ng of RNA
- decreasing the volume of Poly A R1 Reagent from 5 ul to 3 ul (supplementing with 2 ul of DNase/RNase Free water), it can be helpful for samples that are challenging or more prone to dimer formation (it appears that the coral samples are more prone to dimer formation than the Hela control)
- libraries were amplified for 19 cycles, and the final elution was done in 20 ul
- the library concentrations ranged from 0.7 -1.5 ng/ul using the Qubit dsDNA High Sensitivity. He asked the services team, and libraries of this concentration should be fine to sequence
- to further reduce dimers he pooled together the coral libraries that used 3 ul of Poly A R1 based on the molarity of the library region (240 - 1000 bp). He then performed a cleanup on the remaining pool following the last paragraph in Appendix C in the kit protocol. The extra cleanup greatly reduces the dimer without impacting the library yield too much, as seen in the Tapestation report. Or if you prefer, you can perform the extra cleanup on individual libraries. With the extra cleanup, you can elute in a smaller volume to further concentrate the pool if needed.
- for the final elution, elute in 15 ul instead of 20 ul.
- timing for beads drying is critical during each of the cleanups. Typically, the bead cleanups are the most sensitive part of the protocol, and there may be room for improvement. The drying time for the first cleanup takes around 7-8 minutes, with subsequent cleanups taking slightly less time due to smaller bead volumes. Because of differences in temperature and humidity, the drying time between labs may vary. If you want to test out the bead drying times without using real sample, you can perform a mock cleanup following Appendix A using spare DNA as input. This is a quick way to assess how DNA recovery is affected by drying time.
Here is a post in my notebook with these recommendations.
QubitData_04-01-2025_10-37-20_SwitchFree 3' libraries_10 ng Federica Mcap and Pacu_10 ng Hela human controls.csv
2025-03-24_RNA integrity check of Federica Mcap-E7 and Pacu-D8_brought up to 20 ul_diluted 1to2.pdf
2025-04-01_Federica_Mcap and Pacu coral_3'libraries with human Hela control.pdf
2025-04-01_Federica_Mcap and Pacu library pool_before and after extra 0.85x cleanup.pdf