Questions on sequence capture

[kokslim]

Hi everyone,

I am a newbie and have several questions about sequence capture. Would really appreciate your thoughts:

1. I am thinking of capturing the whole exome and use the concentrated exome sample for other different applications than sequencing. I need to do targeted region analysis without using PCR. Do you think this is possible?

2. How much does it cost to do sequence capture?

3. If I can have probably about 300ug of genomic DNA for the capture, how much of sample (in ug) can I get after sequence capture?

Thank you!!

KS

[suludana]

Agilent Prices for exome capture (SureSelect Human All Exon Plus):

- 5 exomes: 6349 euros
-10 exomes: 11005 euros
- 25 exomes: 25396 euros
-50 exomes: 46559 euros


You could consult the manual online to see the required material and the obtained captured sample.

I used SureSelect for another application (capture of 5Mb of sequence) and all it was perfect.

[GW_OK]

Sureselect only needs 500ng of DNA going into the capture. And when you say no PCR do you mean at all or what? You can't really do this without PCR...
Sequence capture adds about $1200 to your standard library prep costs.

[kokslim]

Thanks for your response, Suludana and GW_OK.

Please correct me if I am wrong:
I am hoping to capture certain sequence to do some biochemical analysis, and so I need a method to capture the sequence of interest and concentrate them for the subsequent analyses. I thought that I could add in the RNA library for hybridization, pulled down the sequences that I want and then release them and keep the DNA sequence for subsequent analyses. Hence I can't use PCR for this purpose and I am not going to do sequencing. Do you guys think this will work?

Many thanks.

KS

[ECO]

How big is the region you want to capture?

[GW_OK]

Well, you're going to have to turn the ssDNA that's caught by the RNA into dsDNA somehow. I'm not entirely sure how to do it without at least a round or two of PCR off ligated adapters.
It's an interesting problem.

[kokslim]

Hi Eco, for a start, I am thinking of targeting (1) all exons or (2) all promoter regions.

GW_OK, I actually would prefer keeping the ssDNA as single-stranded form (if possible).

[kokslim]

Hi GW_OK and everyone, do you think what I describe here makes sense at all? I am too new to this field, so please feel free to tell me if this is totally wrong in concept. Thanks.

[krobison]

If I am parsing your text correctly, you want to analyze the chemistry of the input DNA/RNA. All of the capture protocols so far are working with DNA converted to libraries (insert RNA converted to DNA upstream of that for RNA) and then PCR amplified. If you really want to analyze the original DNA, you are working in an interesting but very untested field.

With 300ug of input DNA, you do have a lot so perhaps you could work out what would be a reasonable ratio of capture probes to DNA/RNA & then a way to destroy/remove the capture probes. For RNA in particular that would be tricky since the in solution probes are typically RNA.

I'd suggest you try your application first with a single oligo capture probe & get it optimized -- as I said, this sounds like it is out in front of the technology & you'd better be prepared to forge new ground.

[kokslim]

Thanks for your reply, Krobison. That's what I am thinking, but it sounds like things are going to be pretty challenging and risky!