Factors affecting cluster density

SeqTruth · 11-05-2010, 08:32 AM

Apart from the obvious factor -- library concentration -- I was wondering what other variables affect cluster density. In other words, if one were to use qPCR for library quantification and load all samples at the same pM concentration, what factors would affect cluster densities? Which factors have the biggest influence i.e. are the biggest source of cluster density variability?

Here are some of my ideas, but I was hoping to draw on the experience here. Please comment and/or add to my list:

1. Cluster station or cbot?
2. Average fragment length of library
3. GC-content of library
4. Type of sample
- gDNA
- Methyl-seq
- mRNA (cDNA)
- small RNA
- GEX
- ChIP-seq
- multiplexed/barcoded
- others?

5. Type of flow-cell (SE or PE)
6. Lot number of flow-cell
7. Type of cluster generation kit
8. Version of cluster generation kit
9. Lot number of cluster generation kit

Anything else?

GW_OK · 11-05-2010, 01:12 PM

Conc. of NaOH in your denatured library going on to the flowcell. I think this is one of the reasons the HCl neutralization method works best.

SeqTruth · 11-07-2010, 11:40 PM

Thanks, GW_OK. I was not even aware that people use different NaOH concentrations. Would you care to elaborate at all?

Does anyone have any insight into how any of those things on my list impact on cluster density?

GW_OK · 11-09-2010, 06:59 AM

I recall somewhere in the manuals a mention that cluster generation is impaired if the NaOH concentration is above 800uM, which is one of the reasons I think they have you doing such a massive dilution after the denaturing step. I don't really use a different NaOH concentration per se, but I do neutralize with an equivalent of HCl. This lets me use very very dilute libraries.

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11-05-2010, 08:32 AM	#1
SeqTruth Member Location: South Africa Join Date: Apr 2009 Posts: 12	Factors affecting cluster density Apart from the obvious factor -- library concentration -- I was wondering what other variables affect cluster density. In other words, if one were to use qPCR for library quantification and load all samples at the same pM concentration, what factors would affect cluster densities? Which factors have the biggest influence i.e. are the biggest source of cluster density variability? Here are some of my ideas, but I was hoping to draw on the experience here. Please comment and/or add to my list: 1. Cluster station or cbot? 2. Average fragment length of library 3. GC-content of library 4. Type of sample - gDNA - Methyl-seq - mRNA (cDNA) - small RNA - GEX - ChIP-seq - multiplexed/barcoded - others? 5. Type of flow-cell (SE or PE) 6. Lot number of flow-cell 7. Type of cluster generation kit 8. Version of cluster generation kit 9. Lot number of cluster generation kit Anything else?

11-05-2010, 01:12 PM	#2
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Conc. of NaOH in your denatured library going on to the flowcell. I think this is one of the reasons the HCl neutralization method works best.

11-07-2010, 11:40 PM	#3
SeqTruth Member Location: South Africa Join Date: Apr 2009 Posts: 12	Thanks, GW_OK. I was not even aware that people use different NaOH concentrations. Would you care to elaborate at all? Does anyone have any insight into how any of those things on my list impact on cluster density?

11-09-2010, 06:59 AM	#4
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	I recall somewhere in the manuals a mention that cluster generation is impaired if the NaOH concentration is above 800uM, which is one of the reasons I think they have you doing such a massive dilution after the denaturing step. I don't really use a different NaOH concentration per se, but I do neutralize with an equivalent of HCl. This lets me use very very dilute libraries.