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Old 05-15-2015, 06:08 PM   #1
soleulloa
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Red face Calculating phiX concentration...

Hi all!
I'm a new user of MiSeq and I'm confused about phiX concentrarion to spike in.
In Nextera XT DNA protocol they show a list of dilutions to prepare a final solution of phiX 12,5 pM. Then you have to mix 30 uL of phiX 12,5 pM with 570 uL of pooled libraries, so you are diluting phiX 20 times: the new concentration will be less than 1 pM.

Is that right??!!! Please anyone can explain to me?
I'm not sure how much phiX I have to see in %aligned to know that my run was ok.

Thank youuuuuuu
Soledad
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Old 05-18-2015, 06:24 AM   #2
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The phiX is just there as a control. You don't want it present at high concentrations.
The MiSeq usually needs at least 1% phiX or it won't show error rate results. But if you have .125pM phiX in a 8pM pool, that will be plenty high enough.

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Old 05-18-2015, 06:32 AM   #3
soleulloa
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Thanks Phillip!!!

I need to understand how much phiX (in percentage) I have in my final pool (including phiX spike in). For example, in my first run I did exactly I described above for spike-in and I have 1,2% aligned of phiX. So, I want to know if using that dilutions I put really 1,2% of phiX in the 600 uL that I used to charge the cartridge.

I hope you can help me!!
Thanks

Soledad
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Old 05-18-2015, 10:12 AM   #4
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Originally Posted by soleulloa View Post
Thanks Phillip!!!

I need to understand how much phiX (in percentage) I have in my final pool (including phiX spike in). For example, in my first run I did exactly I described above for spike-in and I have 1,2% aligned of phiX. So, I want to know if using that dilutions I put really 1,2% of phiX in the 600 uL that I used to charge the cartridge.

I hope you can help me!!
Thanks

Soledad
I guess you did, or you would not have gotten 1.2% aligned. Or am I missing something?

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Old 05-20-2015, 12:49 AM   #5
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It's not exactly on topic but close enough:

I recently had some trouble getting my desired % of PhiX clusters. I'm doing amplicon sequencing (16 S, 2x300 v3 on MiSeq) needing higher PhiX concentrations. In one run, I spiked in 5 % but I had only around 1.5 % of PhiX clusters. Another run had 15 % spiked in, and resulted in a mere 4 % of PhiX clusters. This wasn't enough to get solid diversity, which is why the quality of the first few bases was pretty poor and the overall intensities weren't that high. The run was still fine overall but I can't say I'm too happy with it.
The lot numbers of PhiX used were different for both runs and both weren't the lot numbers of faulty PhiX that circled around some time ago.
What might be the cause for this? I'll perform some more amplicon sequencing shortly, and it would be great to know what I can do to improve this situation.
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Old 05-20-2015, 01:03 AM   #6
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@dfhdfh: I seem recall a recent email from Illumina about quality problems with some phiX lots (at least in the US). May want to check with your local FAS, if you did not email an email.
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Old 05-20-2015, 01:11 AM   #7
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@dfhdfh: I seem recall a recent email from Illumina about quality problems with some phiX lots (at least in the US). May want to check with your local FAS, if you did not email an email.
Thanks for your reply. However, like I said, the used lot numbers were not listed by Illumina.
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Old 05-20-2015, 01:17 AM   #8
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The email I recall was from last couple of months (sorry I don't work in the lab). Is that the one you are thinking of?

If you suspect more lots may be affected, it would not be bad to report this lot to illumina (probably that is how the other lots were identified as bad). You can at least get a free replacement.
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Old 05-20-2015, 01:28 AM   #9
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Yes, this is the email I'm thinking of.

I called Illumina, they said they haven't received any notifications with issues concerning these lot numbers.

I bought another new PhiX and hope it'll work this time around.
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Old 05-20-2015, 06:14 AM   #10
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Quote:
Originally Posted by dfhdfh View Post
It's not exactly on topic but close enough:

I recently had some trouble getting my desired % of PhiX clusters. I'm doing amplicon sequencing (16 S, 2x300 v3 on MiSeq) needing higher PhiX concentrations. In one run, I spiked in 5 % but I had only around 1.5 % of PhiX clusters. Another run had 15 % spiked in, and resulted in a mere 4 % of PhiX clusters. This wasn't enough to get solid diversity, which is why the quality of the first few bases was pretty poor and the overall intensities weren't that high. The run was still fine overall but I can't say I'm too happy with it.
The lot numbers of PhiX used were different for both runs and both weren't the lot numbers of faulty PhiX that circled around some time ago.
What might be the cause for this? I'll perform some more amplicon sequencing shortly, and it would be great to know what I can do to improve this situation.
Yes, we've had this problem as well -- phiX percents way below what we expected.

We actually check every batch of phiX we get on the bioanalyzer and generally they are much lower than the expected concentration. But rarely are they less than 50% of their specified concentration of 10nM. Even taking this into account we were sometimes seeing much, much lower % than expected phiX in MiSeq runs. Several fold lower as dfhdfh mentions above.

Note that these same batches of phiX gave us no such issues on our HiSeq runs. What seemed to work, although I'm not sure why, was to add the phiX to the library to be run, in the correct proportion, prior to denaturation.

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Old 05-20-2015, 06:35 AM   #11
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Quote:
Originally Posted by pmiguel View Post
What seemed to work, although I'm not sure why, was to add the phiX to the library to be run, in the correct proportion, prior to denaturation.

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+1 Phillip.

Consider creating a new post with an appropriate title to make this information readily visible. Buried deep in this post it may not show up easily in a search.
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Old 05-20-2015, 10:29 AM   #12
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I almost always see (within 1 or 2%) the "correct" proportion of phiX relative to my amplicon library. However, I am adding 10% of 12.5 pM phiX to a 9.5 pM library to get this. And relative to what pmiguel said - I add it after the NaOH denaturation and dilution down to 9.5 pM, but I heat denature the final diluted pool to be sure of complete denaturation.
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Old 05-20-2015, 11:03 PM   #13
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Well, within 1 or 2 % is just not good enough. When I'm doing amplicon sequencing and want to cut down on "wasting" reads on PhiX, I don't want to get 3 % instead of the 5 % I'm putting in. You say you're adding 10 % of 12.5 pM PhiX to a 9.5 pM library. This means you're adding way more PhiX than specified for 10 % in order to get to the cluster numbers you want? What percentage of PhiX clusters do you observe?

@pmiguel: That sounds interesting. Unfortunately, we're a rather small lab and can't afford to just do things like this for testing. But it shouldn't really make a difference whether you denature PhiX and your library seperately and then put them together or whether you put them together and then denature, should it?

At least I don't just seem to be too dumb to pipet together the correct percentages of two solutions

Last edited by dfhdfh; 05-20-2015 at 11:06 PM.
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Old 05-21-2015, 05:33 AM   #14
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Originally Posted by dfhdfh View Post
@pmiguel: That sounds interesting. Unfortunately, we're a rather small lab and can't afford to just do things like this for testing. But it shouldn't really make a difference whether you denature PhiX and your library seperately and then put them together or whether you put them together and then denature, should it?
"Should" it? No. But we are not in "should" territory here. Things aren't working which suggests that something we think is true, isn't.

Since we were getting the expected cluster density from the library in the main denaturation and not getting what we expected from the phiX dentaturation, I decided we should just add the phiX to the main denaturation. That seemed to work. But it may have been a way to avoid some sort of error that was being made with the phiX denaturation. Actually, that is what I thought the issue was.

But now that I hear of what sounds like the same issue in another lab, I am not so sure.
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Old 05-21-2015, 06:26 AM   #15
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Two weeks from now, I'll run some bacterial total RNA libraries which shouldn't have too much of an issue with diversity. Maybe I'll try one the "Illumina way" and one the "pmiguel way" and see what the results will bring. Have to think about this.
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Old 05-21-2015, 07:15 AM   #16
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Originally Posted by dfhdfh View Post
Two weeks from now, I'll run some bacterial total RNA libraries which shouldn't have too much of an issue with diversity. Maybe I'll try one the "Illumina way" and one the "pmiguel way" and see what the results will bring. Have to think about this.
Perhaps the "Purdue Way" rather than "pmiguel way" would make me sound more modest?

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Old 05-21-2015, 11:15 PM   #17
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Haha, ok

I'll report as soon as I know more.
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Old 06-08-2015, 05:33 AM   #18
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Ok, these are the results:

First run (1x150 v3, high diversity sample), I spiked in 5 % PhiX (Illumina way), which resulted in 3.85 %. As I was quite happy with this result, I performed the second run (also 1x150 v3, high diversity sample) with 1 % spike-in (also Illumina way), which resulted in 0.8 %.

Today, I started an amplicon run (2x300 v3) with a spike-in of 10 % and used the same PhiX lot as for the samples from last week (Illumina way). However, this time I only achieved 5.02 % of PhiX (it's still running and only 50 cycles in but I don't think it'll change).
Well, that's rather disappointing and I have no clue what the difference is. Somehow the library seems to have an influence? I'm not sure.
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Old 06-08-2015, 05:53 AM   #19
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The percentage of phiX you end up with is also dependent on how accurate the quantification of your actual library is. If your library is underquantified, you will end up with a lower percentage of phiX. If it's overquantified, you'll end up with a higher percentage. That's why I usually aim for a higher percentage than 5%. I'd hate to lose a low diversity run over the phiX concentration.

You also need to keep in mind that you are pipetting really small volumes, both in loading your library and in quantifying it. Even with perfect pipetting skills, it's impossible to be totally accurate. One thing that I find helpful is, if I end up with a very high concentration library (based on the KAPA quant), to dilute it down to less than 10 nM and quantify again before loading the MiSeq.
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Old 06-08-2015, 06:36 AM   #20
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That's a really good point. However, the libraries were all quantified in the same way and I can predict pretty well what cluster densities I'll get. So I'm actually quite confident with my measurements.
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