MiSeq v2 cluster density 1/2?

[pmiguel]

Did a run last week on a v1 hardware MiSeq using a v2 chemistry 500 cycle kit on qPCR titrated libraries. Clustered at 10 pM. Instead of the expected 800-1000 clusters/mm^2 we got 425.

Again these were with KAPA qPCR titrated libraries using the same protocol that usually allows us to nail cluster density. These TruSeq RNAseq libraries were a little odd in that the insert sizes were a mean of 700-800 bp to get the full value of the 250 base reads. But we have gotten dead-on accurate clustering results for libraries with much larger mean inserts sizes than these in the past. (Obviously this does require compensation for the larger amplicon sizes compared to the KAPA standards.)

This is the first time we have used v2 chemistry, however. But Illumina Tech support says there should be no major difference in cluster densities with v2. I have another run to do with another set of similar libraries, but I don't want to waste the reagents getting 1/2 the amount of data I should. On the other hand clustering at 20 pM seems too aggressive.

Anyone seeing much lower than expected cluster densities running v2 MiSeq chemistry? Especially with fairly long amplicons?

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Phillip

[Bucky]

We had the same problem here. We used to cluster at 8pM and that resulted in about 700k/mm2. Switching to v2, we ended up with 200k/mm2. Loaded the latest run at 15pm and that seemed to do the trick (1000k/mm2). Will probably set up the next run at 13 or 14pM.

[pmiguel]

Thanks Bucky,
I have been stripping my gears trying to figure out what was going on. That makes it seem like v2 performs more like a HiSeq with respect to raw cluster density[1].

Are you running v1 hardware, or has your instrument been upgraded to v2?

Anyone else see this or not see this?




[1]Of course as a matter of efficiency the cBot blows the MiSeq away -- 120 ul/lane for the HiSeq gets you the same density that 600 ul gets you for the MiSeq.

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Phillip

[Bucky]

Hey Philip,

We are using v1 hardware. I should also mention that I pooled my sample at 4pm and then diluted it out more than usual in order to get a lower NaOH final concentration. Maybe that helped as well..

[GenoMax]

Philip,

Disclaimer: I do not work in the lab. I am merely passing the following info along.

We are not seeing this problem with v.2. kits but the inserts in question are not long.

[pmiguel]

Originally Posted by Bucky

Hey Philip,

We are using v1 hardware. I should also mention that I pooled my sample at 4pm and then diluted it out more than usual in order to get a lower NaOH final concentration. Maybe that helped as well..

Not sure I follow you. Normal protocol is to mix 10 ul of 0.2 M NaOH with 10 ul of 2 nM (total concentration of all libraries), incubate, then neutralize with 980 ul (50xdilution) of HT1. That gives you 1 ml of 20 pM denatured libraries, that you can dilute further, as needed.

Could you elaborate, please?

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Phillip

[pmiguel]

Originally Posted by GenoMax

Philip,

Disclaimer: I do not work in the lab. I am merely passing the following info along.

We are not seeing this problem with v.2. kits but the inserts in question are not long.

Is this v2 kits on v2 hardware, or v1 hardware?

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Phillip

[GenoMax]

Originally Posted by pmiguel

Is this v2 kits on v2 hardware, or v1 hardware?

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Phillip

This is v.2. kits on v.2 hardware.

[pmiguel]

Originally Posted by GenoMax

This is v.2. kits on v.2 hardware.

Okay, this may be a v1 hardware/v2 reagent thing then. I heard from another core that they saw lower cluster densities ("like 1/2") using v2 chemistry on a v1 instrument. But that apparently went away when they were upgraded to v2 hardware.

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Phillip

[Bucky]

Originally Posted by pmiguel

Not sure I follow you. Normal protocol is to mix 10 ul of 0.2 M NaOH with 10 ul of 2 nM (total concentration of all libraries), incubate, then neutralize with 980 ul (50xdilution) of HT1. That gives you 1 ml of 20 pM denatured libraries, that you can dilute further, as needed.

Could you elaborate, please?

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Phillip

Right. Instead of starting with 10ul of 2nM, I start with 10ul of 4nM. So in order to get to my desired final concentration, I need to dilute out more, thus lower NaOH concentration. Does that make sense?

[pmiguel]

Originally Posted by Bucky

Right. Instead of starting with 10ul of 2nM, I start with 10ul of 4nM. So in order to get to my desired final concentration, I need to dilute out more, thus lower NaOH concentration. Does that make sense?

Ah, okay, got it.

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Phillip

[GW_OK]

Illumina put out a new methodology for denaturation with the v2 chemistry. It's in the new Rev E version of the Miseq users guide.

[pmiguel]

Originally Posted by GW_OK

Illumina put out a new methodology for denaturation with the v2 chemistry. It's in the new Rev E version of the Miseq users guide.

I downloaded it to take a look. Not seeing the difference in the denaturation method though. Looks pretty much the same to me.

It is different from the HiSeq -- there the concentration of NaOH used is 1/2 that than for the MiSeq denaturation. Is that new with Rev E of the MiSeq UG?

Anyway, we were using the 0.2 N NaOH, just as called for in Rev E.

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Phillip

[pmiguel]

After complaining the Illumina about this issue they told me they did not equipment in-house to test it out. (Meaning, I presume, all their in-house MiSeq have v2 hardware.) So they offered to send a couple of MiSeq kits out to test this hypothesis. Just started the v1 kit this morning.

Clustered at 10 pM and got 758 Kclusters/mm^2. That seems a little lower than normal, I think. Actually, I ran the same library on v2 chemistry 2 days ago at 15 pM and got a cluster density less than 500 Kclusters/mm^2. But Illumina wants the experiment to be done by the same person, same day, etc.

I'll let it run until cycle 26 and then abort the run, so I can get the second run started today.

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Phillip

[Bucky]

Cool, let us know what you find out!

[bbeitzel]

We've been getting low cluster densities since our MiSeq was upgraded. I used to load at 8 pM (by Kapa qPCR) and get ~500-600K / mm2. Our last 2 runs were loaded at 15 pM and only gave ~150-175K / mm2. I tried taking the number of reads obtained from those low density runs to generate a "corrected" concentration. Started that today and am getting 1400K / mm2 with 5% PF. So much for that idea....

[pmiguel]

My v2 run did cluster at a lower density, but nowhere near the difference I saw previously (~50%). V1 and v2 runs were clustered at 758 vs. 652 Kclusters/mm^2, respectively for 10 pM.

I changed a few things. Not sure if they were salient:

(1) My library had been over-diluted down to 1 nM. So, for my previous load (a couple of days ago) at 15 pM I had added 1 ul of 2M NaOH to 19 ul of my library to reach the desired denaturation conditions. (0.1 M NaOH.) This clustered at 430 on a v2 500 cycle run.
So, I used a speed vap (vacuum only, no heat) to bring 80 ul of the sample split among 4 tubes down to 40 ul. Hence 2nM. This I could use the standard MiSeq denaturation technique on.
(2) Our lab is a little chilly -- 19 oC. So I actually set a heat block at 25 oC for the denturation incubation.
(3) I pre-chilled the HT1 (on ice) to be used to neutralize the denaturation reaction.

So, what is the difference?

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Phillip

[pmiguel]

Just to underline the implications of what Bucky wrote upthread:

Note from pp 74-75 of the rev. E MiSeq manual:

If your application requires higher than a 20 pM final concentration of your library,
make sure that your concentration of NaOH is equalto 0.2N in the denaturation solution and not more than 0.001N (1mM) in the final solution after diluting with
HT1. Higher concentrations of NaOH in the library will inhibit library hybridization to
the flowcell and decrease cluster density.

20 ul at 200 mM diluted to 1 ml (50x). So 4 mM. To get down to 1 mM you would need to do a further 4x dilution! Starting at the recommended 2 nM, that means your maximum load concentration would be 5 pM!

So to follow both the recommended v2 load concentration (~12.5 pM) and load at a final NaOH concentration of no more than 1 mM would require an initial library concentration of > 5nM!!!

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Phillip

[Vinz]

Originally Posted by pmiguel

Just to underline the implications of what Bucky wrote upthread:

Note from pp 74-75 of the rev. E MiSeq manual:



20 ul at 200 mM diluted to 1 ml (50x). So 4 mM. To get down to 1 mM you would need to do a further 4x dilution! Starting at the recommended 2 nM, that means your maximum load concentration would be 5 pM!

So to follow both the recommended v2 load concentration (~12.5 pM) and load at a final NaOH concentration of no more than 1 mM would require an initial library concentration of > 5nM!!!

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Phillip

I am reading the instructions as: Use 0.2N NaOH as denaturation solution. Concentration during denaturation should be 0.1N NaOH. Then dilute that at least 100 fold to get NaOH below 1mM.
Starting with 2.5nM
Denaturation: 1.25 nM
Dilute 100 fold to 12.5 pM

[pmiguel]

Originally Posted by Vinz

I am reading the instructions as: Use 0.2N NaOH as denaturation solution. Concentration during denaturation should be 0.1N NaOH. Then dilute that at least 100 fold to get NaOH below 1mM.
Starting with 2.5nM
Denaturation: 1.25 nM
Dilute 100 fold to 12.5 pM

Thanks Vinz. Looks like my calculations were off 2 fold.
That is not as bad. But does mean using the standard protocol, that calls for 2nM starting concentration, the maximum you can cluster at is 10 pM. To cluster at above that concentration, you should start with more concentrated library.

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Phillip