Mitochondrial genome sequencing

[oryctolagus]

I would like to ask for some help with my project. My aim is to sequence several mitochondrial genomes. I decided to amplify every genome in 2 or 3 amplicons (which would be about 8-10kbp), and then sequence them on both strands using 454 technology.
I’m sorry for being very basic. But I’m a little bit confused. I decided to use NGS because overall it would be cheaper than Sanger sequencing (at least I hope so).

So this is how I understand the workflow: I amplify my genomes in 3 amplicons, then I nebulize my products in order to get fragments which would be appropriate for sequencing. Then I add adapters and MIDs (different MIDs for different genomes) and pool it together.

How do I know what surface of a plate I need in order to sequence my data with reasonable coverage?

In my lab, I’m able to amplify my DNA only, and I’m wondering if every service provider is able to do the rest.

Many thanks

[TonyBrooks]

For each mtDNA you would need 60X depth to be ultra confident in your sequencing, which assuming a mtDNA size of 17kb would mean you'll need a little over 1 Mb to sequence the mitochondrial chromosome.

Assuming a standard 454 Titanium run, minimum ouput is as follows (per region)

LV run (2 region) 180Mb
LV/MV run (4 region) 60Mb
SV/MV run (8 region) 30Mb
SV run (16 region) 10Mb

So you could theoretically get 10 samples per SV region.

We're a core lab and routinely prep libraries and sequence for people, but we aren't able to offer partial plates as we just don't have the demand or throughput to do it. I'd be interested to know a service provider that does so I can send our small projects to them.

Also, have you considered using the MiSeq for this kind of work. It'll more than likely work out cheaper (even though the sequencing is massively overkill).