Multi-factorial examination of amplicon sequencing workflows from sample preparation to bioinformatic analysis

• Published in: BMC microbiology Vol. 23; no. 1; p. 107
• Main Authors: De Wolfe, Travis J, Wright, Erik S
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 19.04.2023; BioMed Central; BMC
• Subjects: 16S rRNA; Accuracy; Analysis; Bacteria - genetics; Best practice; Community; Composition; Computational biology; Computational Biology - methods; DADA2; DNA sequencing; Ecology; Elongation; Environmental health; Evaluation; Fidelity; High-Throughput Nucleotide Sequencing - methods; Humans; Laboratories; Microbiomes; Microbiota; Microbiota (Symbiotic organisms); Microbiota - genetics; Microorganisms; Mothur; Nucleotide sequencing; Optimization; PCR artifacts; PCR biases; Polymerase chain reaction; QIIME2; Reproducibility; Reproducibility of Results; RNA; RNA, Ribosomal, 16S - genetics; Sample preparation; Sequence Analysis, DNA - methods; Soil isolates; Testing; Workflow; Canada; Polymerase; 16S rRNA; DADA2; Mothur; PCR errors; QIIME2; Sequencing errors; PCR biases; PCR artifacts; Chimera
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-023-02851-8

The development of sequencing technologies to evaluate bacterial microbiota composition has allowed new insights into the importance of microbial ecology. However, the variety of methodologies used among amplicon sequencing workflows leads to uncertainty about best practices as well as reproducibility and replicability among microbiome studies. Using a bacterial mock community composed of 37 soil isolates, we performed a comprehensive methodological evaluation of workflows, each with a different combination of methodological factors spanning sample preparation to bioinformatic analysis to define sources of artifacts that affect coverage, accuracy, and biases in the resulting compositional profiles. Of the workflows examined, those using the V4-V4 primer set enabled the highest level of concordance between the original mock community and resulting microbiome sequence composition. Use of a high-fidelity polymerase, or a lower-fidelity polymerase with an increased PCR elongation time, limited chimera formation. Bioinformatic pipelines presented a trade-off between the fraction of distinct community members identified (coverage) and fraction of correct sequences (accuracy). DADA2 and QIIME2 assembled V4-V4 reads amplified by Taq polymerase resulted in the highest accuracy (100%) but had a coverage of only 52%. Using mothur to assemble and denoise V4-V4 reads resulted in a coverage of 75%, albeit with marginally lower accuracy (99.5%). Optimization of microbiome workflows is critical for accuracy and to support reproducibility and replicability among microbiome studies. These considerations will help reveal the guiding principles of microbial ecology and impact the translation of microbiome research to human and environmental health.