Fri. Jul. 12, 2019 Salmon-Sea lice Zymo Pico Methyl prep QC and sequencing
Zymo Pico Methyl kit prep QC
I used the Chip Priming station, Agilent chip votex, and Bioanalyzer 2100 in the Seebs lab.
- 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.
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.
I measured concentrations with Qubit HS DNA assay and recorded concentrations here under the ‘sequencing’ tab.
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.
- 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.