A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

Shaffer, J. P.; Marotz, C.; Belda-Ferre, P.; Martino, C.; Wandro, S.; Estaki, M.; Salido, R. A.; Carpenter, C. S.; Zaramela, L. S.; Minich, J. J.; Bryant, M. B.; Sanders, K.; Fraraccio, S.; Ackermann, G.; Humphrey, G.; Swafford, A. D.; Miller-Montgomery, S.; Knight, R.

doi:10.1101/2020.11.13.370387

bioRxiv · 10.1101/2020.11.13.370387

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

Shaffer, J. P.·Marotz, C.·Belda-Ferre, P.·Martino, C.·Wandro, S.·Estaki, M.·Salido, R. A.·Carpenter, C. S.·Zaramela, L. S.·Minich, J. J.·Bryant, M. B.·Sanders, K.·Fraraccio, S.·Ackermann, G.·Humphrey, G.·Swafford, A. D.·Miller-Montgomery, S.·Knight, R.

View original source ↗Export metadata ↓

Abstract

One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX). Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

Source connections

Published article ↗

Explore related subjects

ecology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Shaffer, J. P., Marotz, C., Belda-Ferre, P., Martino, C., Wandro, S., Estaki, M., Salido, R. A., Carpenter, C. S., Zaramela, L. S., Minich, J. J., Bryant, M. B., Sanders, K., Fraraccio, S., Ackermann, G., Humphrey, G., Swafford, A. D., Miller-Montgomery, S., Knight, R.. 2020-11-14. A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities. https://doi.org/10.1101/2020.11.13.370387

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Beyond Single-Metric Assessments: Uncovering Masked Butterfly Declines via Multi-Scalar Analysis in Central Alberta

1. This study analyzed 21 years (2000-2025) of butterfly count data from Central Alberta, integrated with intensive 5-year (2021-2025) high-resolution intra-seasonal sampling. 2. Long-term macro-scale analysis revealed a significant decline in Shannon Diversity, a change that remained obscured when relying solely on traditional metrics of species richness and evenness. 3. This diversity decline was primarily driven by the severe, long-term collapse of the native Common Ringlet (Coenonympha tullia). 4. Four other dominant species--Cabbage White (Pieris rapae), Clouded Sulphur (Colias eriphyle), European Skipper (Thymelicus lineola), and Common Wood Nymph (Cercyonis pegala)--maintained long-term population stability, though their abundances were significantly constrained by extreme winter minimum temperatures and rapid spring warming. 5. High-resolution intra-seasonal analysis (2021-2025) demonstrated that community indices and species-specific abundances were strongly limited by daily weather, particularly wind velocity and temperature. 6. These findings illustrate that while traditional metrics like richness and evenness are fundamental to community ecology, they provide incomplete insights when applied in isolation; they are most effective when utilized as part of a complementary, multi-scalar framework. 7. This study highlights the necessity of coupling multi-decadal historical datasets with high-frequency, fine-scale sampling to accurately identify the mechanisms of community turnover that simpler metrics may overlook. 8. The results underscore the critical importance of standardized citizen science monitoring in quantifying environmental impacts and establishing conservation priorities for terrestrial insect groups.

ecology↗

bioRxiv2026

From concentration to export: resource contrasts and bee traits shape pollinator spillover to crops

Floral plantings can either concentrate bees or export them to adjacent crops, yet the ecological conditions influencing these outcomes remain unclear. Here, we develop a mathematical model as proof of concept for our previous integrative hypothesis: concentrator and exporter outcomes can arise as alternative, context-dependent outcomes of the same underlying resource-selection process. Using bees as a model and focusing specifically on spillover from floral plantings to crops, we identified resource-specific thresholds separating concentration- and export-favoring conditions. Our model translates differences in relative patch attractiveness into context-dependent concentration and export outcomes and generates resource-specific, testable predictions about the conditions favoring pollinator movement into crops. In our simulations, the concentrator-exporter transition occurred at a lower flowering-intensity contrast than at pollen or nectar contrasts, which suggests that flowering intensity may provide an initial cue for bee movement, whereas nectar and pollen rewards refine or sustain bee responses once crops are perceived as attractive. Spillover thresholds differed among resource contrasts, whereas response steepness varied across bee-trait and community scenarios. Under the model's trait-sensitivity formulation, predicted spillover probability responded more strongly to flowering contrast for specialists than for generalists; colony size amplified this response, whereas bee richness dampened it. Together, these patterns show how flowering and resource contrasts interact with bee traits and community context to shape predicted spillover. Our results confirm that the concentrator and exporter hypotheses can be understood as context-dependent outcomes of the same ecological process rather than as mutually exclusive alternatives. Experimental tests of the predicted thresholds conducted in the field could reveal when and where floral plantings are most likely to promote bee spillover to crops, potentially supporting crop pollination.

ecology↗

bioRxiv2026

A Computational Re-evaluation of Spatial Trials for Zoonotic Tuberculosis Control: Model Misspecification, Diagnostic Miss-classification, and the Illusion of Wildlife Culling Efficacy

1. Wildlife reservoir management frequently relies on the Randomised Badger Culling Trial's (RBCT) trade-off hypothesis, which posits that reductions in cattle herd infections are offset by a perturbation effect driven by disrupted host dispersal. This paper evaluates the computational and epidemiological robustness of this historical trial, which serves as the foundational empirical experiment guiding zoonotic tuberculosis (Mycobacterium bovis) control policies. 2. Using generalized linear mixed models with a generalized Poisson error distribution to explicitly address historical data overdispersion, this study contrasts traditional parametric inference against exact cluster-constrained permutation tests across distinct operational definitions of disease incidence. 3. Non-parametric diagnostics reveal that previously reported treatment and perturbation effects render as statistical artifacts under exact non-parametric permutation. Inside culling zones, parametric significance fails to withstand exact permutation verification due to extreme data leverage in localized cluster blocks. 4. Crucially, when diagnostic misclassification biases are eliminated by analysing total reactor datasets, all apparent culling effects disappear, and information criteria overwhelmingly favour nested null architectures. Unconfirmed reactors likely represent true biological infections missed by low-sensitivity post-mortem macro-necropsy, proving that host removal tracks observation noise rather than genuine zoonotic transmission pathways. 5. Finally, empirical scaling conducted in this study identifies a novel mathematical saturation effect, demonstrating that this sub-linear scaling is an operational artifact of unmodelled herd-level disease recurrence over time. 6. Policy implications. Because current zoonotic tuberculosis intervention frameworks are built upon a structurally misspecified statistical model, they have driven large-scale veterinary policies resulting in substantial, unevidenced ecological and economic interventions while failing to provide genuine public health, animal health, or disease control benefits.

ecology↗