We are a core facility that receives samples from multiple labs. We have one lab in particular that submits which are ran on a 3730XL. We are trying to determine what is happening with these particular results. The first time that we run their plate the results don't look particularly good but if we put that same plate back into the ABI we see a much better signal. I've spoken with another facility who thought it was due to some contamination. I don't really know where this would be coming from as we run the bigdye reaction and cleanseq on all the plates and it's only seen with this particular lab's plates. I would really like to try to help troubleshoot this issue for them. I have attached images of the first and second run. Subsequent plates look pretty good on the first run but could also be improved on the second run. These are also being ran back to back.
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If the second run from the same plate is better, also the signal is declining with reads length, than it is salts being injected instead of labelled DNA fragments on the first run.
I would suggest to use 200ul of 70% ethanol during the ethanol wash stage (if using ethanol precipitation in plates for sequencing reaction products purification). If doing precipitation in 1.5 ml tubes - than use 1ml of 70% ethanol on the wash step. -
Based on the fact that signal quality improves upon re-running the same plate, the issue is most consistent with sample-related inhibitory factors (e.g., high salt, EDTA, or residual ethanol) originating from the submitting laboratory rather than an instrument or reagent failure. The first run likely reduces these inhibitory components, resulting in improved signal quality in subsequent runs.Originally posted by jblain_3 View PostComment
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