In solution capture of whole chunks of genomic DNA?

CG&R · 03-30-2010, 08:27 AM

Curious if anyone tried/knows the answer.

Current Target Enrichment protocols let you shear/break your genomic DNA
to a relatively small size and then add adapters and do PCR amplification, prior to Bead-based target enrichment. This is great if you want to sequence very specifically on-target.

I am interested in a genomic region that contains many repeat sequences, and duplicated genes. Basically I want to use a bead-based enrichment step to obtain the entire region (300kb) since I want to sequence it all, however since these repeat-sequences are located on many different chromosomes I cannot target these, and will do with the region-specific genes located next to these repeat sequences.

So my question for library prep are basically:
1) Is it possible to do targetted capture directly on intact genomic DNA?
2) What would the yield be? (I prefer to have as few PCR steps as possible)
3) Would this yield be enough to do downstream size fractionating and paired-end or possibly even mate-paired sequencing?

krobison · 03-31-2010, 05:29 AM

I think one challenge with large genomic pieces is the kinetics. Big pieces find their mates much more slowly.

You might also consider a PCR approach -- 300 Kb would be 1-2 384 well plates of amplicons. Not trivial, but also not utterly infeasible.

Literature on hybridization selection goes way back -- you may want to look at some of the old papers.

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03-30-2010, 08:27 AM	#1
CG&R Junior Member Location: the Netherlands Join Date: May 2008 Posts: 5	In solution capture of whole chunks of genomic DNA? Curious if anyone tried/knows the answer. Current Target Enrichment protocols let you shear/break your genomic DNA to a relatively small size and then add adapters and do PCR amplification, prior to Bead-based target enrichment. This is great if you want to sequence very specifically on-target. I am interested in a genomic region that contains many repeat sequences, and duplicated genes. Basically I want to use a bead-based enrichment step to obtain the entire region (300kb) since I want to sequence it all, however since these repeat-sequences are located on many different chromosomes I cannot target these, and will do with the region-specific genes located next to these repeat sequences. So my question for library prep are basically: 1) Is it possible to do targetted capture directly on intact genomic DNA? 2) What would the yield be? (I prefer to have as few PCR steps as possible) 3) Would this yield be enough to do downstream size fractionating and paired-end or possibly even mate-paired sequencing?

03-31-2010, 05:29 AM	#2
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	I think one challenge with large genomic pieces is the kinetics. Big pieces find their mates much more slowly. You might also consider a PCR approach -- 300 Kb would be 1-2 384 well plates of amplicons. Not trivial, but also not utterly infeasible. Literature on hybridization selection goes way back -- you may want to look at some of the old papers.