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Old 06-04-2014, 05:51 AM   #12
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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Quote:
But I need some mechanism that allows qPCR to accurately quantitate cluster density for a pool of long amplicons -- that is what I see.
To continue the discussion I would like to question the quantification of a large insert library (in this case 1-5Kb) by QPCR. I doubt that during QPCR such large fragments will amplify efficiently to enable accurate quantification unless extension time is increased to 4-5 mins in which case I am not sure if KAPA polymerase used for QPCR would be capable of such relatively long range PCR. The default KAPA cycling program is sufficient for amplifying up to 1 kb and in such condition it will only amplify a portion of large fragments (<1kb). The result will be underestimation of library concentration because only a portion of it is quantified. Other issue would be differences in amplification efficiency of standards (~400 bp) with library. I have verified this by running QPCR product on DNA Chip and comparing its size to input amplicon size in large insert libraries. I have found that the amplicon peak of QPCR is substantially lower than actual input DNA.

Quote:
But I see no reason at all to favor the RTA-mediated explanation for which, other than unsubstantiated claims from Illumina, there is no evidence for.
I have seen this many times and have heard from others about it as well. But I never knew that Illumina explains the observation similar to what I independently came up with.
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