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Old 08-25-2016, 02:36 AM   #1
AP38
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Default Estimating the number of ddRAD fragments

Hi all,

I am wondering if there is an easy way to estimate the number of fragments resulting from a double digest reaction (in my case ddRAD). For a single digest, one can do the following:

((proportion of cut sites)^(enzyme cut frequency)) x (genome size) = number of RAD fragments

For instance, with ecoRI (6 cutter) and a proportion of 0.25 across a genome of 2.5Gbs:

(0.25)^6 x 2,500,000,000 = 610,351.6 RAD fragments


Is there a similar way for calculate this for a double digest reaction? One could use simRAD in R or even use grep or cat commands on the reference fasta file (cat ref.fasta | tr -d "\n" | grep -o -E “enz1sequence.{200,400}enzyme2sequence” | wc -l).

However, I would like to know if there is an easier solution to this such as the one described above for single digest.

Thanks!
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Old 08-25-2016, 03:09 AM   #2
nucacidhunter
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Calculation method that you have described is incorrect because it assumes that nucleotide sequences in genomes are random and ignores the repetitive content. Two correct methods:

1- Bioinformatically if there is a reference as you have mentioned
2- Empirical (most accurate)
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Old 08-25-2016, 03:37 AM   #3
AP38
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Thanks nucacidhunter,

You are right about the calculation method. It is not the best because of the point you made but should still give a rough estimate. The empirical method is definitely the most accurate way but I would prefer to have a precise in silico estimate prior to jumping into lab work.

Would you have a good bioinformatic method to propose?

Thanks!
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Old 08-25-2016, 04:18 AM   #4
nucacidhunter
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I know that there are some open source software that gives the number and size of the in silico digest fragments. Hopefully someone in the forum can give the names. I found the following thesis interesting in this regard:
http://www.masterbioinformatica.com/...Z_FERNANDO.pdf
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