Variance of fragment sizes

minhduc · 05-17-2012, 09:37 PM

Hi every one,

In PE sequencing, is it possible to control over the fragment/insert size variance? For example, can one prepare a library so that the fragment/insert sizes do not vary much?

Thanks
MD

JPC · 05-18-2012, 12:55 AM

It depends on what you are trying to achieve. We use AmPure bead size selection here on 2ug of sheared DNA and it gives a reasonably tight range. To narrow it down I would try using more DNA.

If this isn't good enough for you I would suggest loading plenty of sheared DNA onto a 0.5% agarose gel, running it out a long way and excising a small section.

The only other thing I can think of is PCR but obviously this would only be suitable if you are sequencing a pretty tiny region.

JPC

minhduc · 05-20-2012, 10:11 PM

Hi JPC,

Thank so much for your answer. I myself do not work directly with a sequencer and hence, honestly do not understand most of what you said, but I get what you meant.

Get down to numbers. I have several short read libs with fragment size mean 200bp and standard deviation 25. If I want to get another dataset with standard deviation of say 10, is it much harder or does it require much more DNA/chemical/cost?

Cheers,
MD

JPC · 05-21-2012, 12:42 AM

A SD of 25 bp is pretty good really, we don't get better than that very often and it wouldn't really befit us to try.

If your libraries have plenty of material then trying to run them out on an agarose gel is probably a good way to go. Any genetics lab would have the equipment and skills you need and it would cost a few $ per sample at most. How much DNA you recover from this will depend on how big a slice you take out of the gel. You might want to take several slices to avoid losing library.

Here is alink to the Qiagen kit that we use for extracting DNA from agarose gel;
http://www.qiagen.com/products/dnacl...t.aspx#Tabs=t0

JC

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05-17-2012, 09:37 PM	#1
minhduc Junior Member Location: Down Under Land Join Date: Apr 2012 Posts: 5	Variance of fragment sizes Hi every one, In PE sequencing, is it possible to control over the fragment/insert size variance? For example, can one prepare a library so that the fragment/insert sizes do not vary much? Thanks MD

05-18-2012, 12:55 AM	#2
JPC Senior Member Location: Wales Join Date: May 2008 Posts: 114	It depends on what you are trying to achieve. We use AmPure bead size selection here on 2ug of sheared DNA and it gives a reasonably tight range. To narrow it down I would try using more DNA. If this isn't good enough for you I would suggest loading plenty of sheared DNA onto a 0.5% agarose gel, running it out a long way and excising a small section. The only other thing I can think of is PCR but obviously this would only be suitable if you are sequencing a pretty tiny region. JPC

05-20-2012, 10:11 PM	#3
minhduc Junior Member Location: Down Under Land Join Date: Apr 2012 Posts: 5	Hi JPC, Thank so much for your answer. I myself do not work directly with a sequencer and hence, honestly do not understand most of what you said, but I get what you meant. Get down to numbers. I have several short read libs with fragment size mean 200bp and standard deviation 25. If I want to get another dataset with standard deviation of say 10, is it much harder or does it require much more DNA/chemical/cost? Cheers, MD

05-21-2012, 12:42 AM	#4
JPC Senior Member Location: Wales Join Date: May 2008 Posts: 114	A SD of 25 bp is pretty good really, we don't get better than that very often and it wouldn't really befit us to try. If your libraries have plenty of material then trying to run them out on an agarose gel is probably a good way to go. Any genetics lab would have the equipment and skills you need and it would cost a few $ per sample at most. How much DNA you recover from this will depend on how big a slice you take out of the gel. You might want to take several slices to avoid losing library. Here is alink to the Qiagen kit that we use for extracting DNA from agarose gel; http://www.qiagen.com/products/dnacl...t.aspx#Tabs=t0 JC