Fig. 2

Sample preparation significantly impacts coverage and accuracy of amplicon sequencing workflows prior to bioinformatic processing. The workflow with the highest coverage in this study could recall 83% of the original mock community members from the raw reads using the V4-V4 region of the 16S rRNA marker gene while the workflow with the highest accuracy could only recall approximately 13% of the original mock community members. A The V4-V4 primer set was associated with the highest accuracy and coverage. Raw reads corresponding with KAPA HiFi had higher accuracy than both iTaq and SsoAdvanced, and accuracy was maximized when KAPA HiFi was paired with the V4-V4 primer set. 1-step PCR had the best overall coverage but provided inferior accuracy. B The workflow with the highest coverage in this study was amplified with KAPA HiFi and 1-step PCR while the workflow with the highest accuracy was amplified with KAPA HiFi and 2-step PCR