The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

Abstract

BackgroundCharacterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits.ResultsWe observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions.ConclusionsBias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.

Pollen DNA barcoding: current applications and future prospects

“…It is, however, currently unclear to what extent different extraction strategies are comparable. Quantitative studies on DNA metabarcoding of microbiomes have found biases towards specific taxa, depending on the DNA isolation method (Brooks et al 2015), and similar biases may occur with pollen. The development of a standardized pollen DNA isolation method would ensure comparability between studies.…”

High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

“…For example, members of the rare biosphere can be easily overlooked or masked when communities are cultivated by traditional methods (e.g., petri dish agar or nutrient broth) because of their low cell densities, low rates of growth, or resistance to cultivation (7)(8)(9). Even with culture-independent molecular methods such as metagenomics or high-throughput sequencing tools, the rare biosphere is not adequately represented (10,11).…”

Does a prenatal bacterial microbiota exist?

“…Negative controls should be implemented at all steps during the experimental procedure and the addition of prespecified and quantified bacterial mock communities to the examined samples will help to reveal biases and identify batch effects. 12,19 Subsequently, the demonstration of metabolically active and proliferating diverse bacteria within the placental or fetal tissue will be required to prove the existence of a viable, diverse and unique bacterial community that merits the term microbiota. Although microbiota research has allowed unexpected and exciting insights during the last years that undoubtedly will change our understanding of the host-microbial relationship, we should keep a healthy dose of scepticism and critically view the current evidence on the existence of a prenatal bacterial microbiota.…”