Full-factorial examination of high-throughput microbiome sequencing workflows from sample preparation to bioinformatic analysis

The development of sequencing technologies to evaluate bacterial microbiota composition has allowed insight into the role of the microbiome in human health. However, the variety of methodologies used to prepare and analyze samples for microbiota composition can introduce artifacts, including errors and biases. These artifacts alter our perception of bacterial diversity and our final interpretation of microbiota differences among samples. Using a mock bacterial microbiota of known composition and abundance, we performed a translational bioinformatic pipeline evaluation of various PCR conditions, amplicon library preparation methods, and bioinformatic analyses to gain insight into methodological sources of artifacts.