I'm interested in doing an Ultra deep sequencing experiment. Specifically I would like to sequence amplicons of 400 bp size with the 454 and I would like to know how many reads as a minimum people do to consider ultra deep sequencing.
thanks a lot!!!
What people consider "deep sequencing" depends on many variables so there is no simple answer. In short, it depends on the type of gene you are amplifying, the species/mutation frequency in your sample, and the total amount of samples you will combine on the run. A typical 454 run will yield about 1 million reads. You did not specify what you are analyzing, but say that you want to analyze 300 samples, each containing a very complex bacterial mixture with 1000s of bacterial species, and you want to do a 16S rRNA barcoded run. Your 1 million reads then will be divided over 300 samples, so each sample would have about 3000 reads. If your sample has a very high bacterial richness, that means you are not really sequencing very deep. At that level, you will still see new species, and you can assume you will have not seen all bacterial diversity. But if you had only 1 sample on that run, and that sample only has 100s of bacterial species, you would be sequencing very deep. Basically, you need to divide the number of expected reads by the number of samples on your run, and then see if the number of expected reads per sample is much more than the expected bacterial richness.
In general, 1 million reads for complex bacterial samples would be considered "very deep sequencing" about 5 years ago, and it might still work for your particular application, but most people would not consider this "ultra deep" nowadays.
The answer might be even different if you are doing e.g. mutation analysis, and it would depend on the frequency of that mutation, but this is not my area of expertise.
I hope this helps!
Thanks for your help, you've done a good introduction to start with deep sequencing
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