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Old 03-15-2018, 08:33 AM   #8
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by GenoMax View Post
Hi Phillip,

Thank you for the useful information.

Can you clarify what do you mean by just enough to allow titration? You don't let the MiSeq run finish (I assume you are using 50x8x8 run)? You run the "pool of pools" that is going to be loaded on NovaSeq and not individual customer pools, correct?

AFAIK iSeq reagent costs are very similar to MiSeq so other than the possibility of having a platform with patterned wells it does not appear to have any major advantage. Illumina is also not discounting iSeq reagents for the foreseeable future.
We just create a pool using 1 ul of each library to be tested. But when we analyze the results we are looking at relative clusters/ul from libraries of the same type. That info is used to make a pool and that pool is actually quantitated by qPCR.

We sell fractional MiSeq runs -- down to 1% of a run. So many of our MiSeq runs have samples from many labs on it. So we can use 5-10% of the run for titration of samples. Most often these would be MiSeq 500 or 300 cycle runs.

---Begin tangential rant:
I like having a little 2x250 information for an RNAseq library -- you can catch cases where there is high ribosomal contamination or something else going wrong. And I almost invariably get asked by people processing the data afterward "what is the insert size of the library?" Which drives me nuts because (1) they don't want to know the insert size of the library, they want to know the insert sizes of the library molecules that clustered and (2) why ask me? They have paired-end data, all they need to do is map 100K reads to 28S rRNA for their species and do the calculation themselves. Probably more importantly, why do RNAseq packages ask for the average insert size of a library? Refer to points 1 and 2 above programmers!
Anyway, since we can pair merge the read pairs, adapter trim them and run them through fastqc. Then you can actually read the average insert size off the fastqc "read length" chart. As long as your inserts aren't too big. But generally RNAseq library inserts are short enough for this to work.
---End tangential rant.

Anyway, MiSeq titration still sucks compared to using one NovaSeq run to titrate another. But that isn't often an option. And the MiSeq method seems to work as well as qPCR does (not great) and isn't as laborious. We just have a little ABI Step-One -- 48 wells total. So it isn't really ideal for processing large numbers of samples. The standards eat up enough of the plate that we can only fit 11 samples per run if we do them in triplicate.

The main limitation of the iSeq is that it doesn't, as far as I know, have a 500 cycle kit. Most amplicon experiments people do use the 500 cycle kit. If it did have a 500 cycle kit I would seriously consider trying to obtain an instrument. Well, if is shown to actually provide better titration info than the MiSeq. Which it might not.

--
Phillip

Last edited by pmiguel; 03-15-2018 at 08:37 AM. Reason: Added some more information
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