individual versus universal primers in 454 pyrosequencing
Hello,
I am new to pyrosequencing so I hope my questions make sense. We have mouse colon samples from mice that were subjected to control or treatment diet and that were analyzed with individual primers for firmicutes, proteobacteria and bacteroidetes. The analysis excel file we received has basically three columns for each sample (i.e. control.1.firm, control.1.bacteroid, control.1.proteobacteria, etc.). Obviously the same applies to raw data which I am trying to re-analyze using QIIME. At the phylum level, we obviously get 100% of firmicutes for sample C.1.firmicutes, 100% of bacteroidetes for sample C.1.bacteroidetes, and so on. But for some samples, especially in the Protebacteria samples we pick up other phylum in addition to proteobacteria (which is now less than 100%). I was told that this is an issue of primer selectivity. At other levels (family, order, genus, etc), we also get some overlap and differences within the sample sample that was analyzed using the three different primers.
Ok, so our goal is to be able to detect differences in the abundance of the three phyla as well as differences in abundance of families, genera and species, if possible. However, I don't know if combining data from the 3 primers sets will create overlapping. I also don't know how to get abundances of each phyla if we have three separate assays for each phylum.
Thanks for any advice you can provide!
I am new to pyrosequencing so I hope my questions make sense. We have mouse colon samples from mice that were subjected to control or treatment diet and that were analyzed with individual primers for firmicutes, proteobacteria and bacteroidetes. The analysis excel file we received has basically three columns for each sample (i.e. control.1.firm, control.1.bacteroid, control.1.proteobacteria, etc.). Obviously the same applies to raw data which I am trying to re-analyze using QIIME. At the phylum level, we obviously get 100% of firmicutes for sample C.1.firmicutes, 100% of bacteroidetes for sample C.1.bacteroidetes, and so on. But for some samples, especially in the Protebacteria samples we pick up other phylum in addition to proteobacteria (which is now less than 100%). I was told that this is an issue of primer selectivity. At other levels (family, order, genus, etc), we also get some overlap and differences within the sample sample that was analyzed using the three different primers.
Ok, so our goal is to be able to detect differences in the abundance of the three phyla as well as differences in abundance of families, genera and species, if possible. However, I don't know if combining data from the 3 primers sets will create overlapping. I also don't know how to get abundances of each phyla if we have three separate assays for each phylum.
Thanks for any advice you can provide!
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