mothur and real-time PCR

Integrating mothur with other programs
1

I have been looking into the gut bacterial communities of ants and using 454 or Miseq 16S I get pretty good insights however I dont get any information about the actual bacterial levels as during the library prep everything gets normalized

I was wondering:
what if I perform a Real-time PCR using the 16S gene as target gene and an insect gene to normalize and get an idea about the bacterial levels in my samples
then I could have a rarefied OTU table and adjust the values of the OTU reads using the values from the 16S-Real-time PCR

for example:
if I have 1000reads pseudomonas and 3000reads wolbachia in sample A but 2000reads pseudomonas and 2000reads wolbachia in sample B and the Real-time PCR is showing that sample A has 3x times higher bacterial levels than sample B
then the final (normalized with the Real-time PCR values) table would be: for sample A: 3x1000reads pseudomonas and 3x3000reads wolbachia in sample A
and for sample B: 1x2000reads pseudomonas and 1x2000reads wolbachia in sample B

does that sound right to you or is it too arbitrary?

any better suggestions?

2

I’m somewhat leery of merging qPCR and relative abundance data. They both get at different things. If you want absolute numbers, I’d probably do qPCR for those specific populations that you are interested in.

Pat

3

Hi,

I believe this CopyRighter program is designed to address the issue of incorporating real-time PCR results with read counts data of bacterial communities.

However I haven’t used it yet, still trying to figure out a way to convert mothur output to its acceptable input format. I might have to redo classify.seqs with greengenes database to make it compatible.

D