Low Cluster Densities in MiSEQ?? Possible explanations???

[abyss]

Dear All,
I have recently observed very low cluster densities when sequencing libraries on the MiSeq.
Briefly, I had loaded the Flow cell with 17.2 pM libraries along with a 5% PhiX Spike in (Also 17.2pM). This I thought was very high concentration but went ahead since my last run gave me a low cluster density figures as well.
The Average size of my libraries are 350bp including the adaptor length. (so approx. 210-215bp insert size).
The quality of the data is great (98%>Q30; 94% PF). But, the output gives me 176K/mm2 cluster density with 5% PhiX as % aligned. This leads me to believe that my concentration calculations were accurate but the number of fragments sticking to the Flow Cell were way lesser than my previous experiences. The 2N NaOH stock was made fresh and accurately.
The only difference that I can think of from my previous runs and the current one (and penultimate run; where also I got low cluster densities) is that I used QIAEXII Gel Extraction instead of QiaQuick Gel Extraction.
Has anyone been in a similar situation? What could be the possible reasons for this? Does the gel extraction kit matter?
Thanks for your help.

[barrmur]

17.2 seems really high. Our miseq performs really well at 10pm giving us 10Gb plus for a 2 x 250 run. I guess most of the answers you will get will contain the question, " how did you quantify your libraries?" with Qpcr being the best but we find the bio analyser just fine. Cannot help with the gel extractions as we use Invitrogen e gels followed by pcr and all is good. Have you tried a FC with just PhiX to get some metrics? One thing we have noticed is that it helps if you cluster straight away after denaturing. A long delay can reduce cluster numbers.

[abyss]

I guess most of the answers you will get will contain the question, " how did you quantify your libraries?" with Qpcr being the best but we find the bio analyser just fine.

Hi Barrmur,
Thanks for your reply.
I used Qubit 2.0 Fluorometer to estimate DNA concentrations, and have a good correlation of Qubit values and KAPA qPCR kit values. Since the former is easier and faster, I just use that.
I have also tried the E-Gels, but for my application (ChIP-Seq) it gave me really poor results (data-wise, not run-wise); not a clue as to why. So, I stuck to Gel Purification because all my previous experiments that have been gel purified worked really well.
As to loading the samples, they were denatured for 5 min. Diluted in the prechilled HT1 buffer and loaded straight away, without much delay.
So still at sea.

[barrmur]

Hmmm, puzzling alright. FYI some of our customers have run chip sample without size selection and have gotten good results. I assume you are taking the tailed adapters into account when size selecting. If the selection is carried out before pcr the adapters can make the products run slower than you think.

Cluster numbers are a puzzle. I would suggest a PhiX flow cell. It might have just been a bad batch of clustering reagents in the kit.

[Calluna]

Hi abyss,

I've been having some issues with cluster density lately, and was wondering if you've had any luck figuring out the cause of your low-clustering samples.

Briefly, I'm running some small RNA libraries that were run on High Sensitivity BA and quantitated with KAPA qPCR. They've were run on 50 cycle kits received about 2 weeks ago, targeting 12.5pM for the final concentration. I loaded 90% sample to 10% 12.5pM PhiX. I've had: 1 run cluster at about 750, 2 runs cluster in the 300's, 1 run cluster in the 100's, and another completely flatlined.

I've run some Nextera XT development/testing samples recently that were also quantitated with KAPA. I targeted 12.5pM and loaded 99% sample to 1% PhiX on old expired 50 cycle Miseq kit lots, average clusters have been decent at ~800 k/mm2.

Let me know if you do a PhiX run and how it turned out. I'm completely puzzled as to why my small RNA samples are clustering so low.

[abyss]

Hey Calluna,
Just a few questions. I guess if we can figure out some of the common methods between us we may be able to find the culprit.
As I mentioned in my previous posts, I suspect Qiagen's QIAEX II gel extraction kit to be the culprit. Did you use that?
As for the runs that had a low cluster density, did it give back an equivalent % of PhiX reads in accordance to how much you had put in? e.g. for the 10% PhiX dope in gave back what % aligned of the PhiX. This might be important to differentiate a problem between the library prep or cluster generation problem.
Thirdly what is the Lot# of your kits. If we are using similar lot# kits, then we had better call Illumina and hopefully get some product replacement.
The Lot# of the reagent kits I am using is 9291077

[salkh]

2N NaoH was used for denaturation?

[abyss]

2N NaoH was used for denaturation?

The 2N NaOH stock was made fresh, but the working denaturation concentration was 0.1N NaOH, i.e. adding equal volume of libraries and 0.2N NaOH.

[pmiguel]

A few possible issues:

(1) Final concentration of NaOH in the diluted, denatured sample you load into the MiSeq cassette can be no higher than 0.01 M. [Note added later: this is wrong, it should be 0.001M] So, if you want to load the MiSeq at >10pM template concentration, you must denature your templates at >2nM. If you have a final NaOH concentration of >10 mM [Should be >1mM] your cluster density will be lower.

(2)Ethidium bromide fluoresces. So if you stained your gels with EtBr, you would need to get rid of that prior to assaying the concentration of your gel extracted libraries with a fluorimeter. Probably the same story for other dyes. We also saw issues with qPCR/EtBr.

(3)One big problem with fluorimetry over qPCR is that intact amplicons look no different than non-intact amplicons. So if your library is only 10% full amplicons through a failure in ligation, etc. then qPCR will tell you that. But fluorimetry will not.

--
Phillip

[Calluna]

abyss,

For our samples I think it might be something with the libraries themselves. We ran the same samples (new pools) on the Hi-Seq wtih much more PhiX (i.e. ~30-50%); they again clustered low.

If you haven't already, give Illumina Tech support a call. They can go over your data with you to see if there may be any obvious explanation for failure. They may even send you a replacement kit

[kmcarr]

(1) Final concentration of NaOH in the diluted, denatured sample you load into the MiSeq cassette can be no higher than 0.01 M. So, if you want to load the MiSeq at >10pM template concentration, you must denature your templates at >2nM. If you have a final NaOH concentration of >10 mM your cluster density will be lower.

Phillip,

Isn't the recommended maximum final [NaOH] 1mM, not 10?

[abyss]

A few possible issues:

(1) Final concentration of NaOH in the diluted, denatured sample you load into the MiSeq cassette can be no higher than 0.01 M. So, if you want to load the MiSeq at >10pM template concentration, you must denature your templates at >2nM. If you have a final NaOH concentration of >10 mM your cluster density will be lower.

Dear Philip,
Thanks for your suggestions.
I denature my libraries at 0.1M conc. NaOH for 5 min. then dilute the solution to 0.001M NaOH, which is also 20pM (pooled libraries). I further dilute this to 17.2pM

(2)Ethidium bromide fluoresces. So if you stained your gels with EtBr, you would need to get rid of that prior to assaying the concentration of your gel extracted libraries with a fluorimeter. Probably the same story for other dyes. We also saw issues with qPCR/EtBr.

(3)One big problem with fluorimetry over qPCR is that intact amplicons look no different than non-intact amplicons. So if your library is only 10% full amplicons through a failure in ligation, etc. then qPCR will tell you that. But fluorimetry will not.

On your suggestion, I simultaneously did a Qubit quantification and a qPCR quantification. It seems like the Qubit was underestimating the library concentrations by quite a bit. So, I was certainly loading more DNA on the flow cell if the qPCR numbers are to be trusted.
The libraries after ligation are cleaned up with AMPure beads then PCR amplified, again cleaned up with AMPure beads, then loaded on a gel and purified. I guess I shouldn't be seeing unligated product, and even if there is some it should be abysmally low in comparison to the PCR amplified products.

[pmiguel]

Dear Philip,
Thanks for your suggestions.
I denature my libraries at 0.1M conc. NaOH for 5 min. then dilute the solution to 0.001M NaOH, which is also 20pM (pooled libraries). I further dilute this to 17.2pM

Not sure I understand. If you do the typical 10 ul of 2nM sample + 10 ul of 0.2N NaOH, then you are denaturing at 0.1N NaOH, as you say. To dilute this to 1 mM, you would need to dilute 20 ul to 2 ml. If you do that, then your pool would be at 10 pM, not 20pM.

I presume you are diluting to 1000 ul, which means your NaOH concentration is 2mM. Illumina wants the final concentration of NaOH to be no higher than 1mM NaOH. But you can't get that low without going below 10pM template concentration.

Anyway, although this would result in somewhat lower cluster density, I don't think it would fall as low as you are seeing.

--
Phillip

[pmiguel]

Phillip,

Isn't the recommended maximum final [NaOH] 1mM, not 10?

Yes, you are correct.

--
Phillip

[abyss]

Yes, that is true. The final NaOH conc. will be 1.72mM @ 17.2pM library loading volume.

Not sure I understand. If you do the typical 10 ul of 2nM sample + 10 ul of 0.2N NaOH, then you are denaturing at 0.1N NaOH, as you say. To dilute this to 1 mM, you would need to dilute 20 ul to 2 ml. If you do that, then your pool would be at 10 pM, not 20pM.

I presume you are diluting to 1000 ul, which means your NaOH concentration is 2mM. Illumina wants the final concentration of NaOH to be no higher than 1mM NaOH. But you can't get that low without going below 10pM template concentration.

Anyway, although this would result in somewhat lower cluster density, I don't think it would fall as low as you are seeing.

--
Phillip