Pico Methyl-Seq library prep checks

Pico Methy-Seq Library Prep checks for E5 samples

I have been doing the pico prep for 15 e5 deep dive samples and I want to sum up all the information here, as I have had to redo 6 samples.

Prep dates

  • 6/13/24
    • Input amount: 25 ng for all species
    • Number of PCR cycles (Section 4): 9
    • Successful libraries: 403 (POC), 413 (ACR), 417(POC), 423 (ACR), 427 (ACR), 439 (ACR), 467 (ACR), 471 (POR), 491 (POR)
    • Failed libraries: 385 (POC), 393 (POC), 401 (POC), 421 (POR), 487 (POR), 489 (POR)
    • Tapestation results
  • 8/7/24
    • Input amount: 25 ng for all species
    • Number of PCR cycles (Section 4): 9
    • Modifications: new PrepAmp polymerase
    • Successful libraries: none
    • Failed libraries: 385 (POC), 393 (POC), 401 (POC), 421 (POR), 487 (POR), 489 (POR)
    • Tapestation results
  • 8/28/24
    • Input amount: 25 ng for POC, 10 ng for POR
    • Number of PCR cycles (Section 4): 12 for POC, 9 for POR
    • Modifications: Different input amount for POR, changed number of cycles for POC
    • Successful libraries: none
    • Failed libraries: 385 (POC), 393 (POC), 401 (POC), 421 (POR), 487 (POR), 489 (POR)
    • Tapestation results
  • 10/15/24
    • Input amount: 50-60 ng for POC, 2-20 ng for POR
    • Number of PCR cycles (Section 4): 15 for POC, 9 for POR
    • Modifications: Different input amount for both species, changed number of cycles for POC. For POR, used extractions done by myself (on 9/25/24) and Kristina (in 2021). Also cleaned the POR DNA before using a Zymo PCR adapter clean-up kit (see post)
    • Successful libraries: none
    • Failed libraries: 385 (POC), 393 (POC), 401 (POC), 421 (POR), 487 (POR), 489 (POR)
    • Tapestation results

Failed libraries

Let’s take a closer look at the Tapestation results for the libraries that failed

385 (POC)

6/13/24

8/7/24

8/28/24

10/15/24

393 (POC)

6/13/24

8/7/24

8/28/24

10/15/24

401 (POC)

6/13/24

8/7/24

8/28/24

10/15/24

421 (POR)

6/13/24

8/7/24

8/28/24

10/15/24 (top is Kristina extraction, bottom is my extraction)

487 (POR)

6/13/24

8/7/24

8/28/24

10/15/24 (top is Kristina extraction, bottom is my extraction)

489 (POR)

6/13/24

8/7/24

8/28/24

10/15/24 (top is Kristina extraction, bottom is my extraction)

Written on October 16, 2024