Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics

Donnelly, Ann; Narayanan, Nithya; Birer-Williams, Caroline; Wolfe, Travis J. De; Chu, Rosalie K.; Anderton, Christopher R.; Wright, Erik S.

doi:10.1128/aem.00922-22

Cited by 1 publication

(1 citation statement)

References 34 publications

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“…Purified bacterial isolates ( n = 37) from soil with near full-length 16S rRNA gene sequences determined by Sanger sequencing (GenBank MN186620-MN186656) were inoculated on individual Luria–Bertani agar plates and incubated overnight (28 °C) (Supplemental Fig. 4 ; Supplemental Table 1 ) [ 57 ]. A single colony of each isolate was transferred to individual 0.2 mL thin-wall PCR tubes containing 100 μL sterile DNA grade water and sonicated at 50% amplitude for 60 s using a sonic dismembrator with cup horn attachment [ 58 ].…”

Section: Methodsmentioning

confidence: 99%

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

Wolfe

Wright

2023

BMC Microbiol

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Background 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. Results 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%). Conclusions 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.

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Section: Methodsmentioning

confidence: 99%