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-   -   No usable reads (http://seqanswers.com/forums/showthread.php?t=82338)

ferjusa 05-10-2018 12:16 PM

No usable reads
 
Hi everybody!
I have a problem with my last run (chip 316) on Ion torrent.
I had only a 31% of loading so I thought it was a charge problem.
But in addition I have 0% of usable reads and 3% of live ISPs.
What's the problem? Could it be the dilution factor of my library (64 barcodes) before the emulsion pcr? or it's an upstream problem when preparing libraries?
Many thanks for who can help me.
Isabella

r.rosati 05-10-2018 12:53 PM

Ciao Isabella!
Some troubleshooting information can be obtained from the report.
How did the consensus keys look?
Were the test fragments OK (according to the % 50AQ17 and histogram)?
If you want, share the PDF run report, it'd be easier to troubleshoot.

ferjusa 05-21-2018 12:36 PM

1 Attachment(s)
Ciaooooo!
Here I attached the PDF run report...horrible!:(

Could the problem be the dilution factor of my library (64 barcodes) before the emulsion pcr? or maybe the chip loading?

Please help!

Grazie mille

r.rosati 05-21-2018 03:11 PM

Ciao Isabella!
I see that the consensus key is very low... I would think that you had an issue with either the polymerase or the sequencing primer... or with the run itself.
You do have a huge % of "no template" ISPs and very low policlonality. You mention the dilution factor of your libraries, do you suspect having used too few DNA at the emulsion PCR step? But then you should have likely removed the empty beads at the ES purification step, having very few ISP left, and you'd see a lot of test fragment ISPs in the run. (Do you by chance save those 2ul of ISPs for the Ion Sphere Quality Control Kit on the Qubit?)

r.rosati 05-21-2018 03:19 PM

P.S.: There's a useful document from the now-defunct IonCommunity called "How to assess a PGM sequencing run report". Do you have it? I don't know where Thermo would be storing it nowadays, if it wasn't lost forever in the pyre.


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