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.
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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