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Old 05-04-2012, 01:11 PM   #1
pmiguel
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Default MiSeq vs HiSeq cluster densities

Those of you have just gotten MiSeq and intending to use them to help dial in cluster densities for their HiSeqs, check your MyIllumina: Bulletin. Especially

Quote:
Cluster efficiency Due to differences in the cluster generation chemistry, the MiSeq flowcells typically cluster at approximately 50% higher cluster density than the same library concentration loaded on a HiSeq flow cell (i.e. a library concentration that yields 750/mm2 clusters on a MiSeq will result in approximately 500/mm2 on a HiSeq).
Can anyone support or contradict this? We just got the MiSeqs, so this would be mission critical for us. I guess we tend to load our HiSeq (or, previously, HiScanSQ) at 12 pM [wrong, it was loaded at 10 pM] (assay by SYBR-green KAPA qPCR with Illumina phiX as our concentration standard.) That tended to land us in the 600-800 Kclusters/mm^2 on the HiScanSQ. On the HiSeq, the phiX control lanes at 12 pM came in way low (450 K clusters/mm^2) whereas the libraries we titrated with them came in higher than expected: 700-1100K clusters/mm^2. But that could just be normal variation.

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Phillip

Last edited by pmiguel; 05-05-2012 at 07:21 AM. Reason: Corrected loading concentration of phiX used.
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Old 05-04-2012, 08:34 PM   #2
Heisman
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Quote:
Originally Posted by pmiguel View Post
Those of you have just gotten MiSeq and intending to use them to help dial in cluster densities for their HiSeqs, check your MyIllumina: Bulletin. Especially



Can anyone support or contradict this? We just got the MiSeqs, so this would be mission critical for us. I guess we tend to load our HiSeq (or, previously, HiScanSQ) at 12 pM (assay by SYBR-green KAPA qPCR with Illumina phiX as our concentration standard.) That tended to land us in the 600-800 Kclusters/mm^2 on the HiScanSQ. On the HiSeq, the phiX control lanes at 12 pM came in way low (450 K clusters/mm^2) whereas the libraries we titrated with them came in higher than expected: 700-1100K clusters/mm^2. But that could just be normal variation.

--
Phillip
Our sequencing core has used a 1:1.4 ratio between MiSeq:HiSeq (similar to the 1:1.5 ratio you posted above), and they are considering going even higher in their recommendations. Not being part of the core I can't really give details beyond that.
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Old 05-15-2012, 06:34 PM   #3
megalodon
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Hi Philip,

we have followed Illumina's recommendation of using a 1:1.5 ratio between MiSeq:HiSeq and by doing that, we have been achieving roughly the same cluster densities on the HiSeq as for previous MiSeq runs for the same sample.

Daniela
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Old 05-17-2012, 02:18 AM   #4
RPiddock
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We've been loading our MiSeq at 6pM and HiSeq at 10pM and getting good results, so very similar all round.

Rachel
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