Hiseq max cluster density?

fffhiseq · 12-19-2011, 08:19 AM

Hey, any one came across the upper boundary of desired cluster density on Hiseq2000. Above what level of clusters there will not be any intensity detection?

csquared · 12-19-2011, 03:32 PM

A bit over 1M cluster/mm2

RPiddock · 12-20-2011, 01:59 AM

I would agree with that, above that and the passing filter bombs in the majority of cases.

vtosha · 12-20-2011, 03:03 AM

We got on GAIIx 1M/mm2, after filtration 600 K. Intensity was detected. Techsupport said about lane overload, but we got good results when assembly have done.

TonyBrooks · 12-20-2011, 03:43 AM

You seriously have to overload to get no data at all.
What happens as you increase concentration is that more and more reads (as a percentage of raw reads) fail the filters and you also start to get quality problems. The Q-score is based a number of statistics, some of which depend on being able to distinguish clusters from each other (which obviously is more difficult the more clusters there are).
Optimum cluster density is around 800-850k/mm^2 according to Illumina, but we've had over 1M/mm^2 and still got data loads of data out at the end with acceptable Q-scores. I guess some of that also depends on the nature of the library - a more biased or lower diversity library may cause problems at high density.
Routinely, I'd aim for 800k/mm^2 as you should get decent data regardless of the library.

fffhiseq · 12-20-2011, 10:52 PM

Thanks to all for the comments. basically we got cluster density in the range of 1100k+/-50/mm-2 on hiseq2000 with flw cell v3 and got 0% pass filter for all the sample except one with32% PF and PF reads 102M. samples dilution of10pM was used and Phix 8pM. Phix gave cluster density of 350k/mm2. it must be overloading but all Qc were performed on Bioanalyzer DNA1000 and qubit. it seems that it is important to run a titration flow cell before running the experiment.

vtosha · 12-20-2011, 11:21 PM

Yes, it is a problem with defining of concentration. I used 16 pM (Qubit) on all lanes and some lanes gave a 1M, phiX gave a 350k, and library on self-made adapters gave 400 K.
Real-time gave a some-fold concentration higher than Qubit. And we observe a different concentration of phiX libraries from some deliveries (eight-fold difference on Qubit and real-time).
TruSeq Sample Prep kits give very high cluster density from libraries, so be care of high concentration of libraries.

fffhiseq · 12-21-2011, 12:49 AM

@vtosha
yeah our qPCR showed four fold difference as compared to the qubit concentration. tech support shud redefine the parameters for the loading. else for first timers it is difficult without loading a titration flow cell.

vtosha · 12-21-2011, 03:08 AM

Our training teacher insisted on that Qubit data more valid.

fffhiseq · 12-21-2011, 03:34 AM

In our experiment we followed qubit readings but in real it underestimated or illumina recommendation is too large to be load on the flow cell. we too were told the same thing to follow fluoroscent based DNA quanti estimation.

GW_OK · 12-21-2011, 06:45 AM

You also can't really trust cluster scores that are over 1 million. The last time I overloaded a lane (after trusting the Qubit and not qPCR) I had clusters of ~1.2 million but ~60% passing filter. In talking with someone at illumina apparently the cluster finding algorithm starts not working properly at densities over ~ 1 million, and you may actually have a significantly higher cluster density that what it reports, it just can't handle it all.

LaoR · 01-19-2012, 03:11 PM

I remember Illumina mentioning in one of their talks that qPCR is the "gold" standard in terms of quantifying.

For the HiSeq 2000 that we are using, having a 2nM (from qPCR) and loading with 12pM on the cBot usually hits the 1 mil mark. It's risky hitting near max thought; you can get more data from higher cluster densities or nothing at all if you over cluster.

HESmith · 01-19-2012, 04:12 PM

You should be aware that high cluster densities (900-1000K) have a more deleterious effect on index reads than inserts. We've had several flow cells with good cluster calling (80-90% PF) and high quality scores (mean ~38), yet fewer than 50% of the indices were called accurately. In some cases, pseudotiles at the inflow side (which contain higher cluster densities) have completely dropped out (i.e., no basecalling) during the index read after producing high-quality insert reads. The problem can be mitigated by balancing the ratio of index bases at each position. It is not solved merely by having different bases at each position if those bases are excited by the same laser (A/C or G/T).

huguesparri · 01-26-2012, 02:02 AM

Same as HESmith here.
If you have multiplexed samples, you should not go higher than 800-850 k/mm² or the machine will have trouble reading the index and you'll loose information.

pmiguel · 01-26-2012, 09:53 AM

Quote:

Originally Posted by HESmith

You should be aware that high cluster densities (900-1000K) have a more deleterious effect on index reads than inserts. We've had several flow cells with good cluster calling (80-90% PF) and high quality scores (mean ~38), yet fewer than 50% of the indices were called accurately. In some cases, pseudotiles at the inflow side (which contain higher cluster densities) have completely dropped out (i.e., no basecalling) during the index read after producing high-quality insert reads. The problem can be mitigated by balancing the ratio of index bases at each position. It is not solved merely by having different bases at each position if those bases are excited by the same laser (A/C or G/T).

I have some hope that HCS 1.5.0 has improved index calling rates. (On the down side we were shut down for 3 weeks after installing 1.5.0. The upgrade failed to update a critical line in a config file. The result of this was that flow cells frequently, but not always, failed to find the edge of lane 8 and refused to start cycle 1 scanning.)

We always try to "MK balance" (M=A,C ; K=G,T) our indexes in a lane. By which I mean (as you imply above) you want a good number of clusters in the A or C channel AND plenty in the G or T channels as well. But a response to my post about this made me think this was not really an issue for the HiSeq, only for HiScanSQs.

Anyway, we have a lane with a mean cluster density of 950 K/mm2. 94% of the PF reads demultiplexed. (3 indexes.) That does seem better than what we were getting with previous versions of HCS.

--
Phillip

Robby · 03-31-2012, 01:44 AM

Hello everyone,

I have a look for the cluster densities for the first time. Do you use for the cluster density the number out of the first base report or the cBot number or any other source? I am just asking because our number of clusters vary between the cBot and the first base report.

Thanks
Robby

pmiguel · 03-31-2012, 02:28 PM

Quote:

Originally Posted by Robby

Hello everyone,

I have a look for the cluster densities for the first time. Do you use for the cluster density the number out of the first base report or the cBot number or any other source? I am just asking because our number of clusters vary between the cBot and the first base report.

Thanks
Robby

Unless I am missing something, the cBot has no capability to assay cluster density.

On the instrument there is a very inaccurate attempt at cluster density determination made after the first cycle. This is included in the first base report, or "FBR". Then after the 4th cycle another determination is made and for all intents and purposes that is the cluster density.

However, the cluster density, as measured by the instrument software is not always accurate. At very low, or very high (>1000 Kclusters/mm2) densities the cluster density will be substantially below what the "real" density is.

None of the above considers the next layer of processing that determines the "PF" or Pass Filter clusters.

--
Phillip

Robby · 04-03-2012, 02:53 AM

Thanks Philip. You wrote, that after the 4th cycle another cluster density determination is made. Is that cluster density the same as written in the final run statistics? Or where can we find the cluster densities after the 4th cycle?

Robby · 04-03-2012, 05:17 AM

OK, I found the answer: The cluster density will not change after cycle 4, so the final cluster density is the same like the 4th cycle cluster density.

pmiguel · 04-03-2012, 05:41 AM

Quote:

Originally Posted by Robby

OK, I found the answer: The cluster density will not change after cycle 4, so the final cluster density is the same like the 4th cycle cluster density.

Yes, that is correct.

--
Phillip

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12-19-2011, 08:19 AM	#1
fffhiseq Junior Member Location: india Join Date: Dec 2011 Posts: 4	Hiseq max cluster density? Hey, any one came across the upper boundary of desired cluster density on Hiseq2000. Above what level of clusters there will not be any intensity detection?

12-19-2011, 03:32 PM	#2
csquared Member Location: Huntsville, AL Join Date: May 2008 Posts: 67	A bit over 1M cluster/mm2 __________________ HudsonAlpha Institute for Biotechnology http://www.hudsonalpha.org/gsl

12-20-2011, 01:59 AM	#3
RPiddock Lab Monkey Location: UK Join Date: Jun 2011 Posts: 16	I would agree with that, above that and the passing filter bombs in the majority of cases.

12-20-2011, 03:03 AM	#4
vtosha Member Location: Moscow Join Date: May 2010 Posts: 36	We got on GAIIx 1M/mm2, after filtration 600 K. Intensity was detected. Techsupport said about lane overload, but we got good results when assembly have done.

12-20-2011, 03:43 AM	#5
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	You seriously have to overload to get no data at all. What happens as you increase concentration is that more and more reads (as a percentage of raw reads) fail the filters and you also start to get quality problems. The Q-score is based a number of statistics, some of which depend on being able to distinguish clusters from each other (which obviously is more difficult the more clusters there are). Optimum cluster density is around 800-850k/mm^2 according to Illumina, but we've had over 1M/mm^2 and still got data loads of data out at the end with acceptable Q-scores. I guess some of that also depends on the nature of the library - a more biased or lower diversity library may cause problems at high density. Routinely, I'd aim for 800k/mm^2 as you should get decent data regardless of the library.

12-20-2011, 10:52 PM	#6
fffhiseq Junior Member Location: india Join Date: Dec 2011 Posts: 4	Thanks to all for the comments. basically we got cluster density in the range of 1100k+/-50/mm-2 on hiseq2000 with flw cell v3 and got 0% pass filter for all the sample except one with32% PF and PF reads 102M. samples dilution of10pM was used and Phix 8pM. Phix gave cluster density of 350k/mm2. it must be overloading but all Qc were performed on Bioanalyzer DNA1000 and qubit. it seems that it is important to run a titration flow cell before running the experiment.

12-20-2011, 11:21 PM	#7
vtosha Member Location: Moscow Join Date: May 2010 Posts: 36	Yes, it is a problem with defining of concentration. I used 16 pM (Qubit) on all lanes and some lanes gave a 1M, phiX gave a 350k, and library on self-made adapters gave 400 K. Real-time gave a some-fold concentration higher than Qubit. And we observe a different concentration of phiX libraries from some deliveries (eight-fold difference on Qubit and real-time). TruSeq Sample Prep kits give very high cluster density from libraries, so be care of high concentration of libraries.

12-21-2011, 12:49 AM	#8
fffhiseq Junior Member Location: india Join Date: Dec 2011 Posts: 4	@vtosha yeah our qPCR showed four fold difference as compared to the qubit concentration. tech support shud redefine the parameters for the loading. else for first timers it is difficult without loading a titration flow cell.

12-21-2011, 03:08 AM	#9
vtosha Member Location: Moscow Join Date: May 2010 Posts: 36	Our training teacher insisted on that Qubit data more valid.

12-21-2011, 03:34 AM	#10
fffhiseq Junior Member Location: india Join Date: Dec 2011 Posts: 4	In our experiment we followed qubit readings but in real it underestimated or illumina recommendation is too large to be load on the flow cell. we too were told the same thing to follow fluoroscent based DNA quanti estimation.

12-21-2011, 06:45 AM	#11
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	You also can't really trust cluster scores that are over 1 million. The last time I overloaded a lane (after trusting the Qubit and not qPCR) I had clusters of ~1.2 million but ~60% passing filter. In talking with someone at illumina apparently the cluster finding algorithm starts not working properly at densities over ~ 1 million, and you may actually have a significantly higher cluster density that what it reports, it just can't handle it all.

01-19-2012, 03:11 PM	#12
LaoR Junior Member Location: California Join Date: Jan 2012 Posts: 3	I remember Illumina mentioning in one of their talks that qPCR is the "gold" standard in terms of quantifying. For the HiSeq 2000 that we are using, having a 2nM (from qPCR) and loading with 12pM on the cBot usually hits the 1 mil mark. It's risky hitting near max thought; you can get more data from higher cluster densities or nothing at all if you over cluster.

01-19-2012, 04:12 PM	#13
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	You should be aware that high cluster densities (900-1000K) have a more deleterious effect on index reads than inserts. We've had several flow cells with good cluster calling (80-90% PF) and high quality scores (mean ~38), yet fewer than 50% of the indices were called accurately. In some cases, pseudotiles at the inflow side (which contain higher cluster densities) have completely dropped out (i.e., no basecalling) during the index read after producing high-quality insert reads. The problem can be mitigated by balancing the ratio of index bases at each position. It is not solved merely by having different bases at each position if those bases are excited by the same laser (A/C or G/T).

01-26-2012, 02:02 AM	#14
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Same as HESmith here. If you have multiplexed samples, you should not go higher than 800-850 k/mm² or the machine will have trouble reading the index and you'll loose information.

03-31-2012, 01:44 AM	#16
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	Hello everyone, I have a look for the cluster densities for the first time. Do you use for the cluster density the number out of the first base report or the cBot number or any other source? I am just asking because our number of clusters vary between the cBot and the first base report. Thanks Robby

04-03-2012, 02:53 AM	#18
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	Thanks Philip. You wrote, that after the 4th cycle another cluster density determination is made. Is that cluster density the same as written in the final run statistics? Or where can we find the cluster densities after the 4th cycle?

04-03-2012, 05:17 AM	#19
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	OK, I found the answer: The cluster density will not change after cycle 4, so the final cluster density is the same like the 4th cycle cluster density.