Disparity between cycle 1 and 2 thumbnails

[matth431]

Hi,

I thought I gotten a feeling from looking at live thumbnails during scan as to the average cluster density we would obtain, but our latest run has thrown me - density looked similar at cycle 1 to successful runs previous (HiScanSQ) but at cycle 2 the thumbnails show FAR fewer clusters than cycle 1 - if I were judging on C1 I would expect ~900k/mm2, on C2 I'd say less than 300k/mm2!!.

The clusters are nowhere near as bright as they were at C1 either. Anyone seen such a dramatic dropoff in density across cycles before? These are low diversity libraries (with 5% PhiX spike) but that shouldn't affect the live thumbnail, should it? It looks green no matter which base is added...

Matt

[pmiguel]

Hi,

I thought I gotten a feeling from looking at live thumbnails during scan as to the average cluster density we would obtain, but our latest run has thrown me - density looked similar at cycle 1 to successful runs previous (HiScanSQ) but at cycle 2 the thumbnails show FAR fewer clusters than cycle 1 - if I were judging on C1 I would expect ~900k/mm2, on C2 I'd say less than 300k/mm2!!.

The clusters are nowhere near as bright as they were at C1 either. Anyone seen such a dramatic dropoff in density across cycles before? These are low diversity libraries (with 5% PhiX spike) but that shouldn't affect the live thumbnail, should it? It looks green no matter which base is added...

Matt

Hey Matt,
IIRC the HiScanSQ shows you the combined clusters for 2 of the bases at a time. Low diversity could definitely affect the apparent cluster density of a given scan.

You really just need to wait for after the 4th base to find out how things are going.

--
Phillip

[matth431]

Hey Matt,
IIRC the HiScanSQ shows you the combined clusters for 2 of the bases at a time. Low diversity could definitely affect the apparent cluster density of a given scan.

You really just need to wait for after the 4th base to find out how things are going.

--
Phillip

Thanks - on checking this morning, cycle 2 had an extremely high percentage of T accordingly to the intensity plot. The other nucleotides shot back up on cycles 3-4. I am a bit shocked as we are using 24 separate barcodes across the first 5 nucleotides, so these should have a fair amount of each nucleotide at position 2 - will have to see how that base gets called, I suppose. Cluster density is about smack in the middle of the two values I estimated - 600k/mm2. On the low side but then the reduced diversity tends to mean more severe dropoff in cluster PF (we usually lose ~20-30% at densities of 800k/mm2) so we may be trading lower raw clusters for more PF with this run.