library: control signal ratio

gallus · 08-03-2010, 11:35 AM

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.

flxlex · 08-10-2010, 11:57 PM

Quote:

Originally Posted by gallus

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?

gallus · 08-11-2010, 06:02 AM

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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08-03-2010, 11:35 AM	#1
gallus Junior Member Location: USA Join Date: Aug 2010 Posts: 4	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.

08-11-2010, 06:02 AM	#3
gallus Junior Member Location: USA Join Date: Aug 2010 Posts: 4	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.