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Hi, I'm writing my thesis and I'm trying to figure out what is the probability of sequencing a read once you put it into the 454. I know that you will loose some reads at emPCR step and then in the sequencing. So the question is if I feed the 454 with 1 million reads how many will probably be sequenced?
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Hi aleferna,
Copying from a paper "Using only one nanogram of fragmented DNA, we prepared ... 53.6 millions Y library molecules, which is sufficient for 10 Titanium runs (4 million enrichment beads to yield 1 million high quality reads per run) for Roche 454 Titanium sequencing".
If you have 1M library molecules, beads recovery around 70 ~ 90%, one fourth beads being sequenced, you might end up around 200,000 reads.
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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