mitochondrial genome

[bbsinfo]

Hello, everyone, I am new here. I need some suggestion or thoughts on this question I have.
I plan to sequence mitochondrial genome of a plant. So I need to figure out a way to design primers to isolate the whole mitochondrial genome out and use NGS technique to sequence the mitochondrial genome I am interested?

Any expert here on Primer design?

Am I thinking on the right track?

Many thanks

[cascoamarillo]

Hi,

If you are planning to use a NGS platform, why don't you run a WGS in a 454 (eg) and then you get an assembly with a ref mitochondrial seq?

I heard of people doing this for chloroplast genome.

[bbsinfo]

Do you mind telling me what is WGS?

[cascoamarillo]

Sorry. I was referring to the Whole Genomic Shotgun sequencing; I mean, you extract the whole genome (nuclear, mt, chloroplast), fragment the dna with a shearing method and continue with the NGS platform instructions (adaptor ligation, amplification, etc...).

[bbsinfo]

Long-range-PCR

Hello, Can anyone tell me whether there is any difference in designing primers for long-range PCR compared to designing primers for ordinary PCR?
Many thanks.

[TonyBrooks]

There are kits out there specifically for isolating mtDNA (http://www.abcam.com/Mitochondrial-D...t-ab65321.html). I have no experience of them so couldn't vouch for how good they are, but it would be interesting to see how much genomic DNA contamination there is when sequenced (compared to what the companies claim, that is). The protocol also looks a bit fiddly, but estimated yield is >5µg, so that's way more than enough to whack on an NGS.

[yaximik]

I would rather suggest to use sequence capture by Nimblegen or Agilent, see info on their sites), they use proprietary soft to design vapture oligos. This will give you a very good coverage of captured sequences with a lot of room for multiplexing if you have multiple samples.

[TonyBrooks]

I was thinking that too, but I wonder whether the capture probes would be too hard to design for mtDNA after all the masking filters are applied.

[yaximik]

I would ask them through their salesreps, guys at both are very helpful on initial stages before you have to pay for actual services. I doubt that mtDNA has many repetitive sequences, most of it is coding, no introns. Perhaps CR may give some problems, but it is small and may well fit within boundaries of adjoining sequences.

[hinkwok]

Hello all. I'm exploring ways to sequence Epstein-Barr virus genome (~170kb episomal dsDNA) in a mixture of human DNA. I'm thinking the situation might be similar to that of sequence mtDNA/chloroplast DNA in total DNA. We tried PCR + Illumina GA method and it worked well. But for some of our samples we don't have enough DNA for all PCR reactions. We are thinking abt the WGA + Nimblegen technology before going for NGS. Can anybody tell me what are there pitfalls of such approach? Thanks a lot.