Hi all, I would like to ask your opinion about inconsistent results regarding the abundance of certain bacteria between sequence data and qPCR using the same DNA samples.
I have 2 treatments (A and B) and I want to know the effect of those treatments in the composition of the microbiota. In a first instance we did qPCR for certain bacterias and observed differences in the samples before and after the treatments with Wilcoxon signes rank test (paired test) p<0,05) so we went on sequencing the same DNA samples.
The sequencing data was analyzed using proportion ( tested also with Wilcoxon signes rank test) and using the DESeq2 (differential abundance).
The sequence data shows significative difference with the proportion analysis for treatment A but not forB (p-value=0.2754 ) and for treatment B but not A with DESeq2 ( Doing the analysis at OTU level, all the OTUs of treatment A had p-values >0.2).
What can be the causes of this inconsistency between the analysis done with sequencing data and qPCR?
and
What is the most reliable result, qPCR or sequence data?
thanks, Cheers.
PS: We did pair end sequencing 300x2 with primers for the V3-V4 region with 70.000 sequence per sample ( 82 samples).
The primers used for the qPCR were checked with BLAST and their specific for the bacterias of interest.
I have 2 treatments (A and B) and I want to know the effect of those treatments in the composition of the microbiota. In a first instance we did qPCR for certain bacterias and observed differences in the samples before and after the treatments with Wilcoxon signes rank test (paired test) p<0,05) so we went on sequencing the same DNA samples.
The sequencing data was analyzed using proportion ( tested also with Wilcoxon signes rank test) and using the DESeq2 (differential abundance).
The sequence data shows significative difference with the proportion analysis for treatment A but not forB (p-value=0.2754 ) and for treatment B but not A with DESeq2 ( Doing the analysis at OTU level, all the OTUs of treatment A had p-values >0.2).
What can be the causes of this inconsistency between the analysis done with sequencing data and qPCR?
and
What is the most reliable result, qPCR or sequence data?
thanks, Cheers.
PS: We did pair end sequencing 300x2 with primers for the V3-V4 region with 70.000 sequence per sample ( 82 samples).
The primers used for the qPCR were checked with BLAST and their specific for the bacterias of interest.
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