Shelly’s Notebook: 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

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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.

uc?export=view&id=1y9QVMr6eXW140pNjZH2dxRqp4CZxXJsc

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. uc?export=view&id=1X0RSaGGf0pUokXWjMZfBCn96RgSmWIsH
  • 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!!!

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Shelly’s Notebook: Thur. Jul. 11, 2019 Salmon-Sea lice Zymo Pico Methyl prep

Zymo Pico Methyl kit prep

  • aliquoted sea lice DNA + water into the 48-well plate (wells 21 and 22) using the same quantities as posted yesterday

Following Zymo pico methyl kit protocol section 1:

  • Added 130uL of lightening conversion reagent to all wells and incubated at 98C 8 min, 54C 1 hour, hold @4C (program ZYM1 in STRIGG folder on PCR machine in 209 with heated lid)
  • followed the rest of section 1 as it’s written, except I used heated elution buffer (55C) and used a set of clean collection tubes to elute the samples in

For section 2:

  • On ice, I prepared the priming reaction mix (enough for 23 reactions, for 22 samples + 1 extra reaction for loss from pipetting) as follows:
    • 46uL PrepAmp Buffer (5x)
    • 23uL PrepAmp Primer (40uM)
    • I added 3uL to each well of the second half of the 48-well PCR plate
    • I added 7uL of purified Bisulfite-converted DNA from each sample to each well *** I accidentally added sample 21 DNA to well 20 which already had sample 20 DNA in it! So I discarded all contents from these wells
  • On ice, I prepared in 500uL tube the PrepAmp mix (enough for 22 reactions since I did not end up needing the extra reaction in the priming reaction mix):
    • 22uL PrepAmp Buffer (5x)
    • 82.5uL PrepAmp Pre-mix
    • 6.6uL PrepAmp Polymerase
  • I ran the thermocycler program specified in section 2 (program ZYM2 in STRIGG folder on PCR machine in 209 without heated lid)
    • after the first denaturation step, I added 5.05 uL of PrepAmp mix to all wells (except wells 20 and 21 which had no samples)
  • While ZYM2 was running, I prepared aliquoted samples 20 and 21 again:
    • For sample 20, I combined the size-selected gDNA digests from 7/9 (5uL of 4ng/uL = 20ng) and 7/10 (4.5uL of 3ng/ul = 13.5ng) + 10.5uL H2O for a total of 33.5ng to be on par with the other samples
    • For sample 21 (sea lice female #1): I combined 0.75uL + 19.25uL H2O (same quantities as previous
  • after the second denaturation step, I added 0.3uL of PrepAmp polymerase to all wells (except wells 20 and 21 which had no samples) *** this was quite the challenge. I tried using my P2, but I couldn’t get consistent quantities. I mainly eyeballed the volume using a P10 after first determining what 0.5uL looked like.
  • While ZYM2 continued running, I prepared bisulfite conversion reactions for samples 20 and 21 in 200uL PCR tubes:
    • 130uL of lightning conversion reagent
    • 20uL DNA
    • I ran ZYM1 on these tubes on block A of the PCR machine
  • After ZYM2 finished, I purified the PCR products following section 3, except I used heated elution buffer (55C), spun for 1 min during the wash and elution steps instead of 30 sec.
  • I transfered the purified PCR products to a new 48-well plate *** I only had ~9uL of each sample, not 11.5uL which the manual says I should have. So I added 2.5uL H2O to each well for a total sample volume of 11.5uL. I kept this plate on ice while waiting for samples 20 and 21 to catch up
  • After ZYM1 finished for samples 20 and 21, I followed the section 1 column clean up, I prepared the priming reactions in 200uL PCR tubes and rans ZYM2 on block A of the PCR machine. After the first denaturation step, I added the remaining PrepAmp mix (that I used for the other samples and had kept on ice), 5.05uL / PCR tube. During the 2nd denaturation step, I added 0.3uL PrepAmp polymerase to each tube.
  • After ZYM2 finished for samples 20 and 21, I followed the section 3 column clean up *** except I likely added sample 21 to the column labeled 20 (got distracted by Frida Taub). So the labels on these samples may be switched, but I’m keeping them as they are until I can confirm by the bioanalyzer (they should look different since one sample is undigested genomic DNA and the other sample is size selected digested genomic DNA. I transfered the purified PCR products to the plate (with 2.5uL H2O added to get a final sample volume of 11.5uL)

For section 4:

  • On ice, I prepared in a 500uL PCR tube amplification mix for 22 reactions as follows:
    • 275uL of LibraryAmp Master Mix(2x)
    • 22uL of LibraryAmp primers (10uM)
    • I added 13.5uL of amplification mix to each well containing 11.5uL of sample
  • I ran the thermocycler program specified in section 4 of the manual except I did a total 8 cycles since I had between 10-50ng starting DNA (specified in the appendix). This is the ZYM4 program on the PCR machine in room 209.
  • When ZYM4 finished, I followed the section 3 column clean up, except I used 1.7mL tubes as collection tubes for samples 11-22. *** this ended up being a mistake because in step 2 of section 3 because you don’t discard the wash buffer flow-through, there was residual wash buffer/ethanol in my elutions of these samples so I had to repeat the column clean up for these samples. I originally eluted in 14uL heated elution buffer (55C) to get about 12uL of product, however for samples 11-22 I ended up with ~20uL. Luckily, I had the columns and was able to use the same ones. In the end, I got 12uL of each sample from eluting with 14uL.

For section 5:

  • To the second half of the 48-well plate, I added:
    • 12.5uL LibraryAmp Master Mix (2x)
    • 1uL Index primers (10uM)
      • I used all 6 that were provided with the kit and used all 16 that we ordered through IDT. Samples 1-22 correspond to Index 1-22
    • 12uL of sample
  • I ran the thermocycler program specified in section 5 of the manual. This is the ZYM5 program on the PCR machine in room 209.
  • After ZYM5 finished, I followed the section 3 column clean up and had to break into the DNA clean-up and concentrator kit for more columns and buffer, but there are still plenty extra. I again did 1 minute spin time for wash and elution steps, and eluted in 14 uL of prewarmed elution buffer (55C).
  • I labeled samples ZMP Lib. 1-22 and stored in 209 -20C bottom drawer in box labeled “gDNA Salmon Skin sea lice infection (from Christian) S.Trigg”

All in all: I started around 9:30am and finished around 9:00pm. Without the mistakes/repeated steps, this would have likely taken 8 hours. Either way, awesome that it’s all done and hoping for a good QC tomorrow.

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