Fri. Jul. 12, 2019 Salmon-Sea lice Zymo Pico Methyl prep QC and sequencing

Zymo Pico Methyl kit prep QC

Bioanalyzer

I used the Chip Priming station, Agilent chip votex, and Bioanalyzer 2100 in the Seebs lab.

PROCEDURE:

  • I ran the Bioanalyzer 2100 high sensistivity DNA assay on all the samples. To do this, I followed the quick start guide.
    • I first rinsed the electrodes with an electrode cleaning chip following protocol on page 118 of the maintenance manual
    • I did not adjust the base plate of the chip priming station since it was already set at C and it did not appear that anyone had done that before.
    • I wasted my first chip because I did not prime it correctly:
      • I got the error message listed on page 73 of the troubleshooting manual so I followed the directions on page 139 to perfom the seal test in the priming station and I realized the lock hadn’t latched with an audible click when I first did it!
      • This was for the better anyways because when I vortexed this chip at 2400rpm as the quick guide specifies, I thought I saw a little liquid spraying so I turned it down to 2200rpm NOTE FOR FUTURE: 2200rpm is enough
    • It took about 6 minutes for the software to start showing the traces from the ladder, then each subsequent sample took about 3 minutes. There were a total of 11 samples (samples 1-11) on one chip.
    • Right before running the next chip with samples 12-20
      • rinsed the electrodes again with the same cleaning chip following page 118 of the maintenance manual
      • the software froze and would only reopen in demo mode so I restarted the laptop, reopened the program, and it finally behaved. This took about 10 minutes, and the manual says to use the chip within 5 minutes. But it still ran fine judging by the ladder and how the samples looked similar to the first run.

RESULTS:

img

CONCLUSIONS:

  • Similarity between lanes 20 and 22 confirms samples 21 and 20 labels did get swapped as previously suspected. So sample #20 is Sea lice Female 1.

  • Gel images generally look in the size range of the example in the zymo pico methylseq manual so that’s encouraging.

Qubit

I measured concentrations with Qubit HS DNA assay and recorded concentrations here under the ‘sequencing’ tab.

CONCLUSIONS:

  • The yields ranged from 25-134ng, which suggest the library prep worked, considering I only started with between 20-50ng of DNA and went through all those steps.

  • Bioanalyzer and Qubit concentrations generally agree, but in the past I’ve relied on Qubit and gel to determine the amount of DNA in the library, so I used those to calculate the nanomolar concentrations.

Pooling samples

  • I calculated nanomolar concentrations using the calculator under the tools menu in the application EnzymeX
    • you can enter in the average size(bp) of your library and 10nM (it says pmol, but it’s the same for nM if you consider the weight to be ng/uL instead of ug). This is the protocol followed by the Ecker lab.
    • Example: for a 240bp library, a 1.584ng/uL concentration is a 10nM concentration.
  • I entered these concentrations into this spreadsheet under the column ‘Conc_for_10nM(based on Qubit and gel avg)’.
  • I calculated dilutions volumes in the same spreadsheet under columns ‘vol_dna_for_10nM’ and ‘vol_water_for_10nM’ and prepared these dilutions to get every sample at 10nM.
  • Because I wanted each salmon sample to get 3.5% of reads and each sea lice sample to get 15% of reads, I made a 30uL pool by adding 1.05uL of each salmon sample and 4.5uL of each sea lice sample.

Submitting samples to NW Genomics Core

  • I submitted a signed quote for a NovaSeq SP 300 cycle flowcell (to get 1.6B 150bp PE reads) and a filled out library submission form
  • I dropped off my 30uL sample pool with Dolores at NWGC (ground floor of Genome Sciences).
    • She said the kit is coming next Tuesday and she will try to start it next week if the machine is available
    • She is going to load my library at 270pM
    • It only takes 1.5 days to run so she’s hoping to send the data by the end of the week after next.

FINGERS CROSSED!!!

Written on July 12, 2019