Estimating genome size and coverage

[newbie25]

Hi everyone,

I'm new to sequencing and am wondering if I have estimated my genome size and therefore expected coverage for 454 sequencing correctly.

From flow cytometry I know that my plant is 25pg/2c=12.5pg/c
if 1pg=978Mb
then 978*12.5=12225Mb.
This is what I believe my genome size to be.

Therefore if I did 1/8th of a plate with an estimated throughput of 30-55Mb, my expected coverage is then:
30/12225-55/12225 = 0.00245x-0.0045X coverage.

This seems very small and I am wondering if I did this all wrong??
Thanks in advance for your help!

[westerman]

Your math seems to be correct. Basically you have a very large genome of 12 Gbases (not too atypical for a plant but certainly much larger than arabidopsis or rice) being sequenced on 1/8th of the smallest 2nd gen sequencer. Your coverage is going to be small.

The 454 is useful for transcriptome, RNA-seq and coverage of small genomes (think bacterial size) but will be expensive trying to sequence a large genome on it. You should use the SOLiD or one of the Illumina machines and an entire plate for large-genome whole genome sequencing projects. A SOLiD-4 full plate mate-pair project (two runs) would produce somewhere around 40-50 Gbases. Short reads, of course, but you are getting closer to the 'X' coverage you would need for whole-genome sequencing.

A concern in your calculations is the effect of ploidy -- are you sure that you are 12.5pg per 2-C? Or is the C-level higher?

Even if the C-level is higher (say a hexaploid like wheat) then your calculations are only increase the coverage level by 2 or 3 or 4 or some other low number. 1/8th of a 454 plate just does not go very far.

Of course talk to me 5 years in the past and tell me that 50Mbases is "nothing". Oh my ... how times change. :-)

[newbie25]

Thanks!
I appreciate the advice, I'll look into this.