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-   -   iSeq, High Q30% but Low PF% (http://seqanswers.com/forums/showthread.php?t=87526)

ShyN 02-05-2019 04:06 PM

iSeq, High Q30% but Low PF%
 
So there's not a lot of data for iSeq since its pretty new - but if anyone has had this issue on MiSeq or in general sequencing please help!

We did an iSeq run today and the results had high Q30% (94%97%) but a VERY low PF% (19%)

What could cause this kind of variation? I've found answers for the other way around but not this.

We're considering the demultiplexing having gone wrong and we're working to correct that but if anyone else has experienced this error - what was the reason, and if it was demultiplexing how did you resolve or get a higher PF%? Is that something we can calculate as we can't get that data from the machine after our data work.

misterc 02-06-2019 09:42 AM

Load up the run with SAV and see what the reported occupancy is. If it's below 90%, you've got too little library in there and need to increase the loading concentration. If it's 96% or higher, you've way over-loaded library and need to do the opposite.

ShyN 02-06-2019 01:54 PM

For some reason, we can't see the reported occupancy on SAV for iSeq but we can for NovaSeq

Ahutch08 03-09-2019 03:06 PM

You need to load the run into BaseSpace to see SAV metrics for iSeq. Ability to view SAV metrics on instrument is coming in later software update.

Did you determine whether your input library concentration was too low or too high?

kbp 03-09-2019 07:51 PM

Used iSeq/MiSeq Cartridges
 
Hi ShyN and/or others,

Any chance you have a spent iSeq and/or MiSeq cartridge(s) that my colleagues and I could have or borrow, perhaps in exchange for a beer? We are in the Bay Area and could come by to pick up.

Thanks!

misterc 03-10-2019 08:03 PM

Quote:

Originally Posted by Ahutch08 (Post 224537)
You need to load the run into BaseSpace to see SAV metrics for iSeq. Ability to view SAV metrics on instrument is coming in later software update.

Did you determine whether your input library concentration was too low or too high?

SAV 2.4.7 (now released) now gives the %occupancy without having to deal with BS.

ShyN 03-13-2019 02:56 PM

Quote:

Originally Posted by kbp (Post 224538)
Hi ShyN and/or others,

Any chance you have a spent iSeq and/or MiSeq cartridge(s) that my colleagues and I could have or borrow, perhaps in exchange for a beer? We are in the Bay Area and could come by to pick up.

Thanks!

Hi there - yes! We have a couple used cartridges that we give you. I've sent you an email via SeqAnswers.

kbp 03-13-2019 09:52 PM

Used iSeq/MiSeq Cartridges
 
Thanks Shy! Feel free to ping me at kbpa00@gmail.com. Thanks!!!

ShyN 03-21-2019 08:05 AM

Quote:

Originally Posted by Ahutch08 (Post 224537)
You need to load the run into BaseSpace to see SAV metrics for iSeq. Ability to view SAV metrics on instrument is coming in later software update.

Did you determine whether your input library concentration was too low or too high?


We're going to try to so BaseSpace now - we used a local application before but I think its time we give base space a chance.

We ended up concluding that our loading concentration was wayyy too low. So out PF was low because of this. Our %Q30 was high because out of the samples that passed (low as they were), most of them were useable.

Thanks everyone for your input!

ShyN 03-21-2019 04:24 PM

Quote:

Originally Posted by misterc (Post 223768)
Load up the run with SAV and see what the reported occupancy is. If it's below 90%, you've got too little library in there and need to increase the loading concentration. If it's 96% or higher, you've way over-loaded library and need to do the opposite.

Hi, can you tell me where you found this data? Thanks!

misterc 03-25-2019 08:38 AM

It's more empirically driven. We've been running NovaSeq (also patterned flow cell with this occupancy metric generated by Illumina) for a couple years now. iSeq performs very, very similarly.

a.boese 04-01-2019 06:39 AM

I recently started using the iSeq and I had the same problem. I felt that my library prep concentration was too high and I got these results by changing the concentrations (I was using PCR products fragmented with the NEBNext Ultra II FS library prep kit):

200pM % PF 39.26% AVG %Q30 82.64%
100pM % PF 67.54%; AVG %Q30 89.15%
50pM %PF 73.92%; AVG %Q30 91.81%

ShyN 04-23-2019 08:36 AM

Quote:

Originally Posted by a.boese (Post 225117)
I recently started using the iSeq and I had the same problem. I felt that my library prep concentration was too high and I got these results by changing the concentrations (I was using PCR products fragmented with the NEBNext Ultra II FS library prep kit):

200pM % PF 39.26% AVG %Q30 82.64%
100pM % PF 67.54%; AVG %Q30 89.15%
50pM %PF 73.92%; AVG %Q30 91.81%

We had some more issues with PF and they were corrected with the loading concentration being changed (raised or lowered depending on the % occupancy). We readily have 70% to 80% PF and the %Q30 have been in the 90th percentile.

The problem really ended up being miscalculation of our starting concentrations via qPCR due to an equation error.

50pM is the perfect loading concentration! Glad you (and I) were able to figure it out

yoderto 04-24-2019 12:45 PM

Hey ShyN-- We've had some yield issues we believe are related to loading concentration. I'm just curious, what plexities have you been running on your iSeq? Thanks!


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