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Old 08-03-2010, 10:35 AM   #1
gallus
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Default library: control signal ratio

i've been poking around with the last 60 or so 454 runs that we've done at our core facility. i saw a pattern that i wanted to share and see if anyone else sees a similar pattern.
we've had pretty good success this year with Ti but we had a few notable runs where we got read distributions such that the left tail was very long (for amplicons). One of these runs was essentially a rerun of a previous Ti amplicon. The first run produced over 400 Mb and the rerun produced about 150 Mb.
when i looked at the signal for the library key divided by the signal of the signal for the control key, the runs that looked good had a ratio of ~1 to 1.7. For runs that didn't work well, the ratio was invariably above 2.0. For reference, our cpb for Ti amplicons is usually 1 and the % + is usually 9-11%.
I'm wondering if you could check this signal ratio with qPCR before burning through reagents. Thoughts/comments much appreciated.
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Old 08-10-2010, 10:57 PM   #2
flxlex
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Quote:
Originally Posted by gallus View Post
when i looked at the signal for the library key divided by the signal of the signal for the control key, the runs that looked good had a ratio of ~1 to 1.7. For runs that didn't work well, the ratio was invariably above 2.0. For reference, our cpb for Ti amplicons is usually 1 and the % + is usually 9-11%.
Interesting, but... what do you mean exactly with 'signal' here?
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Old 08-11-2010, 05:02 AM   #3
gallus
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the signal is the intensity of a given nucleotide on a bead. Typically key (CATG/ATGC and the like) signals are 400-500 and the library (GACT/TCAG) is above that by about 1.5-2X. the keySignalperBase in the 454RuntimeMetrics where you can get the exact numbers. You can also take a look at histograms of the signals under the Signals tab in GSRun Browser as well. I typically use awk ($ awk 'date|region|key' 454RuntimeMetrics.txt > output.txt) to summarize.
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