Boryana Garcia Doyle

All Publications

• Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR. Nature protocols Doyle, B., Reynolds, G. Z., Dvorak, M., Maghini, D. G., Natarajan, A., Bhatt, A. S. 2025

  Abstract

  Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.

  View details for DOI 10.1038/s41596-025-01165-5

  View details for PubMedID 40389632

  View details for PubMedCentralID 6750738

• Replicative selfish genetic elements are driving rapid pathogenic adaptation of Enterococcus faecium. bioRxiv : the preprint server for biology Grieshop, M. P., Behr, A. A., Bowden, S., Lin, J. D., Molari, M., Reynolds, G. Z., Brooks, E. F., Doyle, B., Rodriguez-Nava, G., Salinas, J. L., Banaei, N., Bhatt, A. S. 2025

  Abstract

  Understanding how healthcare-associated pathogens adapt in clinical environments can inform strategies to reduce their burden. Here, we investigate the hypothesis that insertion sequences (IS), prokaryotic transposable elements, are a dominant mediator of rapid genomic evolution in healthcare-associated pathogens. Among 28,207 publicly available pathogen genomes, we find high copy numbers of the replicative ISL3 family in healthcare-associated Enterococcus faecium, Streptococcus pneumoniae and Staphylococcus aureus. In E. faecium, the ESKAPE pathogen with the highest IS density, we find that ISL3 proliferation has increased in the last 30 years. To enable better identification of structural variants, we long read-sequenced a new, single hospital collection of 282 Enterococcal infection isolates collected over three years. In these samples, we observed extensive, ongoing structural variation of the E. faecium genome, largely mediated by active replicative ISL3 elements. To determine if ISL3 is actively replicating in clinical timescales in its natural, gut microbiome reservoir, we long read-sequenced a collection of 28 longitudinal stool samples from patients undergoing hematopoietic cell transplantation, whose gut microbiomes were dominated by E. faecium. We found up to six structural variants of a given E. faecium strain within a single stool sample. Examining longitudinal samples from one individual in further detail, we find ISL3 elements can replicate and move to specific positions with profound regulatory effects on neighboring gene expression. In particular, we identify an ISL3 element that upon insertion replaces an imperfect -35 promoter sequence at a folT gene locus with a perfect -35 sequence, which leads to substantial upregulation of expression of folT, driving highly effective folate scavenging. As a known folate auxotroph, E. faecium depends on other members of the microbiota or diet to supply folate. Enhanced folate scavenging may enable E. faecium to thrive in the setting of microbiome collapse that is common in HCT and other critically ill patients. Together, ISL3 expansion has enabled E. faecium to rapidly evolve in healthcare settings, and this likely contributes to its metabolic fitness and may strongly influence its ongoing trajectory of genomic evolution.

  View details for DOI 10.1101/2025.03.16.643550

  View details for PubMedID 40161577

  View details for PubMedCentralID PMC11952509

• Author Correction: Quantifying bias introduced by sample collection in relative and absolute microbiome measurements. Nature biotechnology Maghini, D. G., Dvorak, M., Dahlen, A., Roos, M., Doyle, B., Kuersten, S., Bhatt, A. S. 2024

  View details for DOI 10.1038/s41587-024-02341-w

  View details for PubMedID 39048712

• Quantifying bias introduced by sample collection in relative and absolute microbiome measurements. Nature biotechnology Maghini, D. G., Dvorak, M., Dahlen, A., Roos, M., Kuersten, S., Bhatt, A. S. 2023

  Abstract

  To gain insight into the accuracy of microbial measurements, it is important to evaluate sources of bias related to sample condition, preservative method and bioinformatic analyses. There is increasing evidence that measurement of the total count and concentration of microbes in the gut, or 'absolute abundance', provides a richer source of information than relative abundance and can correct some conclusions drawn from relative abundance data. However, little is known about how preservative choice can affect these measurements. In this study, we investigated how two common preservatives and short-term storage conditions impact relative and absolute microbial measurements. OMNIgene GUT OMR-200 yields lower metagenomic taxonomic variation between different storage temperatures, whereas Zymo DNA/RNA Shield yields lower metatranscriptomic taxonomic variation. Absolute abundance quantification reveals two different causes of variable Bacteroidetes:Firmicutes ratios across preservatives. Based on these results, we recommend OMNIgene GUT OMR-200 preservative for field studies and Zymo DNA/RNA Shield for metatranscriptomics studies, and we strongly encourage absolute quantification for microbial measurements.

  View details for DOI 10.1038/s41587-023-01754-3

  View details for PubMedID 37106038

  View details for PubMedCentralID 5069758