Hi, My lab recently purchased and set up a Ion S5 XL system and after the initial set up and verification runs we began running 400bp fusion primer samples using OneTouch 2 (there was about 2 months down time inbetween set up/verification and normal usage). However, we continuously kept getting low usable reads/very short reads for our samples.
Initially we assumed it was a problem with the pooling volumes and increased the volume we were taking for OT2 (upto 36ul). However, we still had low usable reads. We noticed that some of the reagent cartridges were expired and then contacted thermo to find out whether this was causing the problem. We were assured that this was unlikely and after they checked out run output, they suggested the problem was with our library preparation process (too many primer dimers appearing even after purification).
So we changed our library process to reduce the dimers and proceeded with double purification for our fusion primer libraries. However, still no improvement. We went even further and purified our pooled samples as well hoping to see an improvement but no luck
We finally requested an FAS for our site and after testing they concluded again that our library process was giving the problem. However, even after multiple changes to our purification process and library process we are still seeing very low usable reads.
Has anyone come across this problem before? Please help
P.S: I am attaching some of run summary files and the verification run we did with the FAS.
Initially we assumed it was a problem with the pooling volumes and increased the volume we were taking for OT2 (upto 36ul). However, we still had low usable reads. We noticed that some of the reagent cartridges were expired and then contacted thermo to find out whether this was causing the problem. We were assured that this was unlikely and after they checked out run output, they suggested the problem was with our library preparation process (too many primer dimers appearing even after purification).
So we changed our library process to reduce the dimers and proceeded with double purification for our fusion primer libraries. However, still no improvement. We went even further and purified our pooled samples as well hoping to see an improvement but no luck
We finally requested an FAS for our site and after testing they concluded again that our library process was giving the problem. However, even after multiple changes to our purification process and library process we are still seeing very low usable reads.
Has anyone come across this problem before? Please help
P.S: I am attaching some of run summary files and the verification run we did with the FAS.
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