Hello, sorry if this was not the best subforum for this question, but I had a tough time trying to decide where to post this.
Simple question, in both the 454 and MiSeq SOPs, there is mention of “a mock community composed of genomic DNA from 21 bacterial and archaeal strains”. Is it possible to get more information about the identity of these species and how exactly this community is generated in the laboratory? Or a reference if this is published? I tried looking through some papers on Pubmed but was not readily able to find this information.
Question#1: how important is to have these positive controls with known bacterial species in every run of 454 pyrosequencing (experiment)?
Question#2: Should I also make my own mix of bacterial genomes or should I only mix those listed in PLoS ONE paper for 454 SOP? Or is there a way to use filter.seqs command (filter from my workflow) on HMP_MOCK.v35.align file and then somehow compare HMP_MOCK.v35.align.filter back to the original alignment file?
Question#3: Dear Pat, can you please provide some more details regarding sample prep for mock community? PLoS ONE paper only lists species and accession numbers, but does not tell neither where those DNA samples were purchased nor what were the DNA concentrations used for pyrosequencing. It seems to be important to include in 454 SOP on wiki page.
Question#1: how important is to have these positive controls with known bacterial species in every run of 454 pyrosequencing (experiment)?
Well, we think it’s important and easy enough to use one mock community per 96 samples. We also have a “real” sample that we put on every run to quantify the drift between runs. If you are doing a bunch of small runs, I’d think the latter would be very important.
Question#2: Should I also make my own mix of bacterial genomes or should I only mix those listed in PLoS ONE paper for 454 SOP? Or is there a way to use filter.seqs command (filter from my workflow) on HMP_MOCK.v35.align file and then somehow compare HMP_MOCK.v35.align.filter back to the original alignment file?
You can make your own and in fact it would probably be much easier that way. If you’re doing soil pick soil bugs, if you’re doing marine, do marine bugs, etc. The only requirement is that you know the real sequence. You could even use plasmids from old clone libraries as input.
Question#3: Dear Pat, can you please provide some more details regarding sample prep for mock community? PLoS ONE paper only lists species and accession numbers, but does not tell neither where those DNA samples were purchased nor what were the DNA concentrations used for pyrosequencing. It seems to be important to include in 454 SOP on wiki page.
The SOP is meant as a bioinformatics SOP, not really a wet-lab SOP. The Mock was set up by Sarah Highlander at Baylor College of Medicine. The input to the communities was meant to be uniform abundances across the samples.