Target DNA Capture

[Lj Lee]

'm still new in molecular biology. Do anyone has any idea to manually do the target capture of DNA manually prior to the next generation sequencing? The cost of sending my samples to let the company do it is still high. So plan to do it by ourselves. It can be done by using oligo probes that coated with magnetic beads or PCR? If PCR I have to amplify the whole genes as I need the whole exons and introns.

Thanks if you have any idea to share with me

[Heisman]

If you're doing entire genes PCR will be annoying if you have a lot of genes. Right now you can go to earray.chem.agilent.com and read up there, upload a list of target sites, and get a quote to see what the reagents will cost. That would be using the Agilent SureSelect platform.

[Lj Lee]

Thanks Heisman,

Do you think that it's possible that I do it all manually? i.e. synthesize my own oligo probes that coated with biotin or amino-group instead of using SureSelect or other capture platforms?

[Heisman]

There are papers where people have done that, but I have not done it myself so I can't offer any advice regarding what to do. You can try to find those papers and if you can't I'll see if I can dig them up (it's been awhile).

[Chipper]

What is the size of your target region and how many samples are you planning to sequence?

[Lj Lee]

around 10kbp for each genes, actually have a list of genes that I wish to capture. Plan to capture all these genes from around 21 to 36 samples. After this step hopping that each captured genes from each samples can be tagged or barcoded so that I can pool them together for NGS.

[JPC]

The Nimblegen titanium array will capture 385K and works out quite cheap if you have access to the array equipment. If that's no good you could try Agilent Haloplex, I don't think this requires any specialist equipment.

If you're thinking about trying to replicate what exome capture kits do it may be possible for a small number of genes but I would say it's unlikely to be economical. The exome capture kits have precise balance of probes to try and give a uniform capture result. If you buy in a load of probes and mix them together evenly you will probably find your capture is unbalanced......optimising will be a laborious and expensive process.

[rnal]

I think the exom capture kit and the DNA shearing instrument(Covaris) will be the most cost .

[hbn]

I have a related question: I would like to do target enrichment using the SureSelect system for a non-model species.
My question is; what software to use for this? Agilent advises me to use eArrayXD (local software) but I can not find this software anywhere...
Does someone have experience with this? I would appreciate any advice...