Microbial Profiling using conserved regions of genomes has quickly become a popular tool to identify bacteria & fungi present in mixed microbial communities. Due to a range of applications within healthcare, agriculture, industry & commercial sectors, there is a large scale interest in this area including research questions such as: What is the bacteria that has caused a skin infection in a hospital patient?  What bacteria will help make the best fertiliser to make plants grow?  What probiotics have a positive impact on health?  What microbes are present in my gut and do microbes in your gut contribute to psychological disorders?

AGRF has analysed the success of amplicon sequencing using Illumina’s MiSeq paired end chemistry over hundreds of runs. We review the reproducibility of sequencing identical samples over time and comment on what aspects of lab operation impact sequencing quality most, including the effect of sequencing multiple amplicon targets in the same sequencing run, what biases are generated when you sequence low biomass samples, how altering the number of PCR cycles changes the profile, what influences the generation of chimeras during the sequencing run, and if this correlates with the amount of OTU’s identified.

In doing so, we identify several critical parameters for amplicon-based diversity profiling of microbial communities, and demonstrate how optimising these parameters gives consistent high-quality outputs that accurately reflect the actual microbial communities.