depth calculation

[sheilal]

Hi,
I wanted to ask how to calculate the expected depth coverage in 454 if I know the size of the sequenced genome?

[maubp]

Your local sequencing center should be able to tell you how much raw sequence data a plate (or half a plate, 1/4, 1/8 or even 1/16 of a plate) would give you (in base pairs), so divide this by the estimated genome size. That will will give you an upper bound on the coverage assuming uniformity of coverage, linear amplification, no contamination, etc.

If you want more practical comments, I suggest you tell the forum what kind of organism it is and the estimated genome size. The GC% might also be important...

[dina]

GC content

Why is GC content important? how to consider it?

[maubp]

Well, not directly, but extreme GC values could cause trouble at several steps.

[dina]

GC

So, what would you sugggest? I have to check the GC content of the whole genome before the sequencing? and if it is high , what can I so?

[maubp]

If you have any genes or ESTs already sequenced you should be able to guess the GC%, or even look at related organisms. If it is extreme then some background reading of the literature might be in order - but you'll probably be fine.