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  • 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.

  • #2
    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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    • #3
      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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