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Old 02-01-2016, 06:10 AM   #1
Location: OP Kansas

Join Date: Mar 2014
Posts: 53
Default Sample input vs genome length or both?

Hellos group,

I have a technical question, I'm working with a viral (surprise!) genome. It is approx 200kbp in length and is a DNA virus. We will be sequencing this on the Ion PGM and I have a question about library prep.

We will be using the enzymatic shearing protocol which is optimized for e.coli which is 4.6 - 5.6 Mbp so I need to optimize the shear times. So my question is which is the more important factor to start tweaking, the amount of time spent in the reaction or the amount of enzyme used?

Has anyone had any experience with this type of question I would like to know some of the pitfalls before I start burning through sample preps like a mad man.
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Old 02-02-2016, 05:55 AM   #2

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Is the enzymatic fragmentation done with transposomes or "randomly" cutting dsDNA endonucleases?

I'd imagine the enzyme concentration having a higher impact than adjusting incubation times. See these two dsDNA shearases:

Not sure what the difference between the "plus" and the "normal" version is (probably old and newer version), but in the "plus" one the sheared DNA exhibits clearer shifts with increasing units of enzyme added. Although that makes the price per reaction also higher.

We also had this optimization in our lab, but I can't find the gel picture atm.

Although these reactions are mostly optimized on humans or E.coli as you say, the DNA it is usually tested on is rarely bigger than the size of your intact viral genome. This heavily depends on the DNA prep. I wouldn't expect a drastically different shearing outcome...
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Old 02-02-2016, 06:07 AM   #3
Location: OP Kansas

Join Date: Mar 2014
Posts: 53

Thank you lorendarith,

These links are helpful and will give me a narrow window to try
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library preparation, pgm, viral ngs

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