I have a experiment design question. I have x number of samples that I do 16S PCR on and sequence on the 454. Alas, as is always the case some samples don’t give as many reads as others, and I’m missing replicates for a proper analysis.
Now I can do another PCR on the same genomic DNA from my missing samples, and rerun these on the 454 (just these samples, not all again). Now the questions:
- Would you do that at all? Theoretically with the same PCR and primer conditions the result should be the same, at least somewhat.
- At what stage would you combine the data from the two sequencing runs? From the sff file onwards, or after all the sequence editing steps like alignment and so on?
- If sample A has only 200 reads in the first 454 run, and in the second it has 1800 reads, would you combine the two to get 2000 reads in a single analysis?