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Old 12-01-2010, 11:18 AM   #1
splicemaster
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Question Capillary DNA sequencing

hello all,

So my question is: is there a particular type of library i need to construct if i am to use capillary sequencing?

This is a question for an HW of mine, but I want to emphasize that I DONT WANT you to give me the answer!

I would just like to be pointed in the right direction, in terms of the theory behind building the appropriate library

Thank you
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Old 12-01-2010, 01:57 PM   #2
splicemaster
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hello all,

So I came to the conclusion that I should use a combination of cosmid and BAC libraries, as was used in the pibmed article 16372010

my issue now is how to use the following equation:

Genome coverage = (total # of clones - # of non-contributing clones) X
average insert size/haploid genome size

according to the poisson distribution, the probability of obtaining one or more clones per locus is a function of library size

in my case, I need 8fold coverage which would be 99.97%

btw, the genome im working with is 20Mb

i knw that in terms of cloning capacity, cosmids can take about 40 kb

and BAC can take up to 300kb

but I cant seem to put it all together

Common seqanswer community! help a newbie out

thank you
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Old 12-02-2010, 08:19 AM   #3
epibio
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I'd actually recommend using fosmids, rather than BACs or cosmids, but the principle is the same.Here's how to do the calculation (for more details, download the PDF from the first link on this page.)

Determining the Approximate Number of Clones for a Complete Fosmid Library:

Using the following formula, determine the number of fosmid clones required to reasonably ensure that any given DNA sequence is contained within the library.
N = ln (1-P ) / ln (1-f )
Where P is the desired probability (expressed as a fraction); f is the proportion of the genome contained in a single clone; and N is the required number of fosmid clones.

For example, the number of clones required to ensure a 99% probability of a given DNA sequence of E. coli (genome = 4.7 Mb) being contained within a fosmid library composed of 40-kb inserts is:
N = ln (1 – 0.99) / ln (1 – [4 x 10^4 bases / 4.7 x 10^6 bases]) = –4.61 / –0.01 = 461 clones

Hope that helps.
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Old 12-02-2010, 08:30 AM   #4
splicemaster
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much appreciated, helps a lot!

your company's site is prefect

Cheers,
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