Since a couple of months our HiSeq 2000 shows inconstant Cluster Density.
We started having troubles when some library pools (we multiplex our libraries in pools and then run each pool on multiple lanes) showed very low cluster density (generally around 200 K/mm2), and then when we re-run the exact same pools (same concentration, same mix, same tube) on another flowcell we obtain optimal density (up to 800 to 1000 K/mm2). We have troubleshoot this problem extensively with Illumina Tech Support but we were not able to point out what the problem is.
We use the HiSeq 2000 machine since 2 years with good result and we have a robust protocol for library quantification and QC. We do Paired End Sequencing (2 x 200 cycles). The problem is always "pool dependant" and when the libraries are re-run it usually gives good results, so we tend to think the problem might be somewhere between the libraries denaturation and the cBot. We've tried to do a re-hyb on a Flowcell showing poor density at the First Base Report, but the density remains the same afterwards. Only repeating the cBot completely with a new flowcell seems to work. We have consider a flowcell batch problem, but it was ruled out since two flowcells of the same batch did not react the same way (one was perfect and the other showed density problems). The HiSeq dont seem to be part of the problem either, we've run many tests.
For an example, see in attachment the density of 2 runs, where the first was problematic for Pool E (first 4 lanes, 200 K/mm2) and the re-run was good (around 800 K/mm2). The re-run was done using the same tube of pooled libraries.
Any ideas? Anybody experience something similar? Any input would be greatly appreciated, we are running out of explanations...
Many thanks!
We started having troubles when some library pools (we multiplex our libraries in pools and then run each pool on multiple lanes) showed very low cluster density (generally around 200 K/mm2), and then when we re-run the exact same pools (same concentration, same mix, same tube) on another flowcell we obtain optimal density (up to 800 to 1000 K/mm2). We have troubleshoot this problem extensively with Illumina Tech Support but we were not able to point out what the problem is.
We use the HiSeq 2000 machine since 2 years with good result and we have a robust protocol for library quantification and QC. We do Paired End Sequencing (2 x 200 cycles). The problem is always "pool dependant" and when the libraries are re-run it usually gives good results, so we tend to think the problem might be somewhere between the libraries denaturation and the cBot. We've tried to do a re-hyb on a Flowcell showing poor density at the First Base Report, but the density remains the same afterwards. Only repeating the cBot completely with a new flowcell seems to work. We have consider a flowcell batch problem, but it was ruled out since two flowcells of the same batch did not react the same way (one was perfect and the other showed density problems). The HiSeq dont seem to be part of the problem either, we've run many tests.
For an example, see in attachment the density of 2 runs, where the first was problematic for Pool E (first 4 lanes, 200 K/mm2) and the re-run was good (around 800 K/mm2). The re-run was done using the same tube of pooled libraries.
Any ideas? Anybody experience something similar? Any input would be greatly appreciated, we are running out of explanations...
Many thanks!
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