Evaluation of Host Depletion and Extraction Methods for Shotgun Metagenomic Analysis of Bovine Vaginal Samples

Ong, Chian Teng; Boe-Hansen, G.; Ross, Elizabeth M.; Blackall, P. J.; Hayes, B. J.; Lew-Tabor, A.E.

doi:10.1128/spectrum.00412-21

Cited by 4 publications

(3 citation statements)

References 107 publications

(108 reference statements)

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“…Further work is required to define the balance of Gram-negative and Gram-positive genera in this matrix. Differences in the recovery of Gram positive organisms based on extraction method have also been reported for other matrices [24].…”

Section: Discussionmentioning

confidence: 72%

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast

Flint

Laidlaw

et al. 2022

BMC Microbiol

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Background Sequence-based methods for the detection of bacteria such as 16S rRNA amplicon sequencing and metagenomics can provide a comprehensive view of the bacterial microbiome of food. These methods rely on the detection of gene sequences to indicate the presence of viable bacteria. This indirect form of detection can be prone to experimental artefacts. Sample handling and processing are key sources of variation that require standard approaches. Extracting sufficient quantities of high quality DNA from food matrices is challenging because target bacterial species are usually minor components of the microbiota and foods contain an array of compounds that are inhibitory to downstream DNA applications. Here, three DNA extraction methods are compared for their ability to extract high quality bacterial DNA from retail chicken breast rinses, with or without enrichment. Method performance was assessed by comparing ease of use, DNA yield, DNA quality, PCR amplicon yield, and the detection of bacterial taxa by 16S rRNA amplicon sequencing. Results All three DNA extraction methods yielded DNA of sufficient quantity and quality to perform quantitative PCR and 16S rRNA amplicon sequencing. The extraction methods differed in ease of use, with the two commercial kits (PowerFood, PowerSoil) offering considerable time and cost savings over a hybrid method that used laboratory reagents for lysis and commercial column based kits for further purification. Bacterial richness as determined by 16S rRNA amplicon sequencing was similar across the three DNA extraction methods. However, differences were noted in the relative abundance of bacterial taxa, with significantly higher abundance of Gram-positive genera detected in the DNA samples prepared using the PowerFood DNA extraction kit. Conclusion The choice of DNA extraction method can affect the detection of bacterial taxa by 16S rRNA amplicon sequencing in chicken meat rinses. Investigators should be aware of this procedural bias and select methods that are fit for the purposes of their investigation.

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Section: Discussionmentioning

confidence: 72%

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast

Flint

Laidlaw

et al. 2022

BMC Microbiol

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“…However, when we employed phylogeny-aware metrics (Unweighted UniFrac), we saw signi cant differences in microbial composition by extraction method and by dog, suggesting that some host depletion methods bias microbial community pro les through preferential lysis of speci c bacterial clades. Importantly, Bacteremia and DNA Microbiome have been identi ed as accurate and effective DNA extraction methods in other high-host, low-microbial biomass substrates (i.e., nasal swabs, vaginal swabs, urine, biopsies) [18,[69][70][71].…”

Section: Discussionmentioning

confidence: 99%

Evaluating urine volume and host depletion methods to enable genome-resolved metagenomics of the urobiome

Lewis,

Scott,

Madden

et al. 2024

Preprint

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Background: The gut microbiome has emerged as a clear player in health and disease, in part by mediating host response to environment and lifestyle. The urobiome (microbiota of the urinary tract) likely functions similarly. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. Here, to begin addressing these challenges, we evaluate urine sample volume (100 ml – 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into aliquots. One set of samples was spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S rRNA gene and shotgun metagenomic sequencing. We then assembled metagenome assembled genomes (MAGs) and compared microbial composition and diversity across groups. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. Results: In relation to urine sample volume, ³ 3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S rRNA sequencing data and shotgun metagenomic sequencing data, and maximized MAG recovery while effectively depleting host DNA in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. Conclusions: This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings provide guidelines for studying the urobiome in relation to sample volume and host depletion, and lay the foundation for future evaluation of urobiome function in relation to health and disease.

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“…To efficiently perform mNGS and improve microbial DNA detection in different samples, a host depletion approach is crucial. A variety of approaches have been used to overcome host contamination, including capture probes for subtractive hybridization [ 21 , 81 ], CRISPR-Cas9 cleavage on target sequence [ 82 ] and ribonuclease (RNase) H-based depletion methods [ 83 ], as well as nanopore adaptive sequencing [ 84 ]. In the case of RNA libraries, DNase I treatment should be performed after extraction to remove residual human background DNA [ 85 ].…”

Section: Assay Descriptionmentioning

confidence: 99%

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

Kan,

Tsang,

Pei

et al. 2024

IJMS

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Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives.

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Evaluation of Host Depletion and Extraction Methods for Shotgun Metagenomic Analysis of Bovine Vaginal Samples

Cited by 4 publications

References 107 publications

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast

Evaluating urine volume and host depletion methods to enable genome-resolved metagenomics of the urobiome

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

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