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Old 04-29-2019, 09:16 PM   #1
mamor
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Default Consistent low MiSeq cluster density and PhiX alignment with high quality reads (PF%)

Hi everyone,

I am struggling to understand an issue I am having with sequencing on an Illumina MiSeq. I have had success in the past with this exact protocol at a different institution - with another machine and reagents etc.

I am sequencing custom 20 pM ddRADseq libraries using a V3 600 cycle kit.
My workflow is;
library prep > pippin prep > AMPure > Tape station > Bioline library QC qPCR kit > dilute to 10 nM > side by side denature and dilute 10 nM library and PhiX to 20 pM > spike in 10% PhiX.

On my last run I got low cluster densities (365K/mm2) and low PhiX alignment (0.91% - aiming for 10%) with good sequence quality (91.51% >=Q30). This seems to be the way each run goes.

Any help would be appreciated

Michael.
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Old 04-30-2019, 05:34 AM   #2
microgirl123
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One possibility that would explain this is that your phiX and library are not denaturing properly. How old is your NaOH? I always denature with NaOH, and then heat-denature my final combined library/phiX dilution.
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Old 04-30-2019, 03:13 PM   #3
mamor
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Thanks for the reply. My 1N NaOH is about 8 months old now. I make up my working solution as I am denaturing. This issue occurred 1 day after making my stock and 8 months later on my second run.
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Old 05-01-2019, 12:40 PM   #4
Jessica_L
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Following up on microgirl123's post, do you periodically check the pH of the NaOH? The stock should be high of course, but even the 0.2N should be 13 or so.

The only other immediate thing I can think of for low cluster density is that you're carrying over too much NaOH into cluster generation, but that usually only happens if you're using too much OH or if there's an issue with the HT1 buffer. It could happen because of the pipettes if they're not calibrated or an o-ring has gone bad on one of them.
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Old 05-01-2019, 03:25 PM   #5
mamor
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I haven't been checking the pH. It seems like the best place to start.
I do use calibrated pipettes, but I also have an addition dilution step that reduces the final concentration of NaOH to 0.4 mM. Does anyone have any thoughts on my process?

For denaturing and dilution:
- start with a 10 nM library and 0.2N NaOH (library: 10,000 pM, NaOH: 200 mM)
- 1:1 dilution with 0.2N NaOH (library: 5000 pM, NaOH: 100 mM)
- Add 990 uL HT1 (library: 50 pM, NaOH: 1 mM)
- Dilute library to 20 pM = 240 uL library:360 uL HT1 (library: 20 pM, NaOH: 0.4 mM)
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Old 05-07-2019, 10:38 AM   #6
Jessica_L
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The NaOH concentrations are about what they should be for each individual step of the process. The only thing I haven't seen before is starting with a 10nM library-- the standard protocols I've seen all recommend starting with 4nM. I'm not sure that makes a difference, but it might be worth trying. Starting with 4 can still get you to 20pM if you load at that concentration.


If you need to get the library to a higher concentration, you can follow the procedure that ArcherDx uses in their protocols, which generate a 40pM denatured library:
10uL of 4nM library + 10uL of 0.2N NaOH
Incubate 5 minutes at RT, then add 10uL of 200mM Tris pH 7
+970uL of HT1
The final concentration is a 40pM library and ~2mM NaOH. Since the library is more concentrated at this step, you use less of it, and dilute to a final loading volume of 1000uL.

I've used both the standard Illumina protocol and this one, and had no problems with the cluster generation part of things, but again, we start with 4nM.

Good luck!
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