[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?

--
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!!!

--
Phillip

[Vinz]

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!!!

--
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]

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.

--
Phillip

[Sylviane]

I got poor clustering with a 15pM library, contacted Illumina regarding the final NaOH concentration (I followed the new 0.2N NaOH protocol without thinking too much about it) and got this response:

"Regarding your library prep: I had to check myself and I must admit the user guide is misleading. For the sample you have prepared you obtained indeed a higher NaOH concentration as recommended (not more than 0.001 N (1 mM) in the final solution after diluting with HT1). So, you did not calculate the concentration wrong.
For your sample prep you should follow the steps for the preparation of PhiX"

(that is starting with 4nM)

Sylviane

[Triu10]

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.

We also experienced ~2x drop in cluster density after MiSeq hardware upgrade to V.2 compared to V1. After we adjusted the library conc. to 10 - 11 pM to achieve the recommended cluster density for MiSeq V2 + Chemistry V2 (880-965 K/mm2), the number of reads passing filter is always below 15M indicated in specs. We usually get 12 - 14M reads passing filter, i.e., 70 - 75% of total reads. We started with higher cluster densities, however, %PF drops down and we get the same number of reads as with lower cluster density.

Any ideas how can were get more reads passing filter?

Thanks

[pmiguel]

We also experienced ~2x drop in cluster density after MiSeq hardware upgrade to V.2 compared to V1. After we adjusted the library conc. to 10 - 11 pM to achieve the recommended cluster density for MiSeq V2 + Chemistry V2 (880-965 K/mm2), the number of reads passing filter is always below 15M indicated in specs. We usually get 12 - 14M reads passing filter, i.e., 70 - 75% of total reads. We started with higher cluster densities, however, %PF drops down and we get the same number of reads as with lower cluster density.

Any ideas how can were get more reads passing filter?

Thanks

Not sure how to bring the %PF up, but you can get higher cluster densities by starting with a higher conc library (eg 10 nM) for denaturation. This is because you end up having to dilute the NaOH to a lower concentration after denaturation to reach the desired cluster density. That is, the amount of NaOH in the denatured template effects ability of those templates to anneal to the flow cell.

By denaturing with a 10 nM library, I clustered at 12 pM on our most recent run and got 1100 Kclusters/mm^2. 80% PF and 15.5 million PF reads.

--
Phillip

[Triu10]

By denaturing with a 10 nM library, I clustered at 12 pM on our most recent run and got 1100 Kclusters/mm^2. 80% PF and 15.5 million PF reads.

--
Phillip[/QUOTE]

Thanks a lot Phillip. Our problem is not the cluster density as we also reach 1100 or so K/mm2 by starting denaturation with 2nM libraries. And we have quite low conc. of libraries obtained with limited PCR cycles in order to reduce the duplicate rate of the reads, it would not always be possible to start denaturation with 10nM.
But today I will pick up one library with high enough conc. and will try one run following your recommendations, though loading 10pM + 1% Phix. Will let you know about the results.

Andrius

[pmiguel]

Thanks a lot Phillip. Our problem is not the cluster density as we also reach 1100 or so K/mm2 by starting denaturation with 2nM libraries. And we have quite low conc. of libraries obtained with limited PCR cycles in order to reduce the duplicate rate of the reads, it would not always be possible to start denaturation with 10nM.
But today I will pick up one library with high enough conc. and will try one run following your recommendations, though loading 10pM + 1% Phix. Will let you know about the results.

Andrius

Okay, I will be interested to know.

One issue here is that we were just (2 weeks ago) hardware upgraded. When I originally was posting about this issue, it was in regards to using v2 kits on v1 hardware.

--
Phillip

[Triu10]

Okay, I will be interested to know.

One issue here is that we were just (2 weeks ago) hardware upgraded. When I originally was posting about this issue, it was in regards to using v2 kits on v1 hardware.

--
Phillip

Hi Philip,

We first got hardware upgrade and only later switched from v1 kit to v2. And from my experience, v1 kit required completely different settings compared to v2 kit when run on hardware v2. So, I assume, the hardware v1 behaves differently from v2, too.
Anyway, going back to my issue with kit v2 on hardware v2, we had three recent runs, and here are some numbers. I also give library dilutions
1. 10nM + 0.2N NaOH > 2nM > 20pM, loaded 10pM + 1% PhiX --> 836K/mm2, 86.04% PF, 13.46M PF reads
2. 5nM + 0.2N NaOH > 2nM > 20pM, loaded 12pM + 2% PhiX --> 888K/mm2, 83.66% PF, 13.86M PF reads
3. 5nM + 0.2N NaOH > 2nM > 20pM, loaded 13pM + 2% PhiX --> 955K/mm2, 77.94% PF, 13.52M PF reads

As I told before, we not always have high enough lib. conc. to start denaturation with 10nM. But looking at these three runs, it's clear that increase in cluster density does not compensate decrease in PF% and at the end we have more or less the same read numbers.
Looking forward for the scheduled tech. support visit from Illumina next week.

[yaximik]

We got our MiSeq already upgraded, so I cannot compare with the earlier version. Using the standard protocol for libraries of av. 300 nt length I clustered at 800 on average with 12 pM with 85-95% PF, for libraries of av. 460 nt and 600 nt used at 15 pM I clustered at ~1500 with 75% PF. The shorter libraries I could not get rid of adaptor dimers completely using agarose gel, perhaps this was the reason for a bit lower densities. Longer libraries were fractionated using LabChip XT. In either case I am using qPCR for quantitation, KAPA standard with different MM though.

[ECO]

Revisiting this...not sure why you guys are using 0.2N NaOH...all the MiSeq manuals (back to 2011, and the current one) I have recommend 0.1N NaOH, and a final denaturation concentration of 50mM NaOH.

[kcchan]

Actually, every revision of the manual since rev D has used 0.2N NaOH for the denaturation. The final concentration of NaOH after the final dilution (just before loading) should be no more than 1mM.

[ECO]

Actually, every revision of the manual since rev D has used 0.2N NaOH for the denaturation. The final concentration of NaOH after the final dilution (just before loading) should be no more than 1mM.

Wow, you're right! Based on this, they contradict their own advice if you load at anything higher than 10pM...(final NaOH is 2mM in their 20pM).