Abstract
Next-generation sequencing approaches used to characterize microbial communities are subject to technical caveats that can lead to major distortion of acquired data. Determining the optimal sample handling protocol is essential to minimize the bias for different sample types. Using a mock community composed of 22 bacterial strains of even concentration, we studied a combination of handling conditions to determine the optimal conditions for swab material. Examining a combination of effects simulates the reality of handling environmental samples and may thus provide a better foundation for the standardization of protocols. We found that the choice of storage buffer and extraction kit affects the detected bacterial composition, while different 16S rRNA amplification methods only had a minor effect. All bacterial genera present in the mock community were identified with minimal levels of contamination independent of the choice of sample processing. Despite this, the observed bacterial profile for all tested conditions were significantly different from the expected abundance. This highlights the need for proper validation and standardization for each sample type using a mock community and blank control samples, to assess the bias in the protocol and reduce variation across the datasets.
Highlights
Microorganisms colonize various anatomical sites and play a crucial role in the balance of health and disease
After rarefying to 95,870 sequences per sample, de novo Operational taxonomic units (OTUs) picking returned 228 OTUs, of which 19 OTUs corresponding to the mock community make up more than 99% of the pooled community
Since the results indicate that these two applied high-fidelity polymerases do not significantly impact the observed microbial diversity, we pooled the data from the two polymerases for identical sample for the analyses of buffer and extraction kit choice
Summary
Microorganisms colonize various anatomical sites and play a crucial role in the balance of health and disease. The advent of cultivation-independent molecular approaches, such as 16S rRNA amplicon sequencing, has allowed for a better understanding of the microbes that inhabit different biological niches These powerful tools are not without important technical caveats that can lead to a distortion in the acquired data[2]. Such limitations have been well documented, and include sample collection, storage buffer, DNA extraction, amplification primers and methods, sequencing technology, and analysis techniques[3,4]. Only focus on the effect of a single technical variation instead of examining the effect of different combination of storage buffer, DNA extraction kit, and amplification methods[2]. Studying a combination of effects mirrors the reality of sample handling more closely and may provide a better foundation for the standardization of sampling handling protocols prior to microbial analysis
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