Bacterial DNA enrichment for low-inoculum fracture-related infection diagnostic using high-throughput sequencing.

One of the main barriers for the implementation of metagenomic sequencing in routine diagnosis of infectious diseases is the presence of host DNA. While several enrichment methods are likely to overcome this issue, their effectiveness for specimens such as bone in the case of chronic infections remains to be determined. We compared the relevance of two methods for bacterial DNA enrichment when compared to a reference protocol during pretreatment of bone samples from fracture-related infections before HTS by both Illumina Miseq and Oxford Nanopore Technology (ONT). The bacterial/human DNA ratio was higher for either protocols than the reference technique (p = 0.00012), without any significant difference between them. HTS sensitivity over culture ranged from 21.7 % to 85 %. The ability of the studied protocols to improve the bacterial/human DNA ratio depends on the sequencing technique employed. In this context, there is room for improvement in enhancing the sensitivity of HTS for diagnostic purpose.

Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses.

Background: Recent advances in microbiome sequencing techniques have provided new insights into the role of the microbiome on human health with potential diagnostic implications. However, these developments are often hampered by the presence of a large amount of human DNA interfering with the analysis of the bacterial content. Nowadays, extensive scientific literature focuses on eukaryotic DNA depletion methods, which successfully remove host DNA in microbiome studies, even if a precise assessment of the impact on bacterial DNA is often missing. Methods: Here, we have investigated a saponin-based DNA isolation protocol commonly applied to different biological matrices to deplete the released host DNA. Results: The bacterial DNA obtained was used to assess the relative abundance of bacterial and human DNA, revealing that the inclusion of 2.5% wt/vol saponin allowed the depletion of most of the host's DNA in favor of bacterial DNA enrichment. However, shotgun metagenomic sequencing showed inaccurate microbial profiles of the DNA samples, highlighting an erroneous increase in Gram-positive DNA. Even the application of 0.0125% wt/vol saponin altered the bacterial profile by depleting Gram-negative bacteria, resulting in an overall increase of Gram-positive bacterial DNA. Conclusion: The application of the saponin-based protocol drastically changes the detection of the microbial composition of human-related biological specimens. In this context, we revealed that saponin targets not only host cells but also specific bacterial cells, thus inducing a drastic reduction in the profiling of Gram-negative bacterial DNA.

Host DNA depletion on frozen human respiratory samples enables successful metagenomic sequencing for microbiome studies.

Background: Most respiratory microbiome studies have focused on amplicon rather than metagenomics sequencing due to high host DNA content. We evaluated efficacy of five host DNA depletion methods on previously frozen human bronchoalveolar lavage (BAL), nasal swabs, and sputum prior to metagenomic sequencing. Results: Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum and BAL samples had 94.1%, 99.2%, and 99.7% host-reads. The effect of host depletion differed by sample type. Most treatment methods increased microbial reads, species richness and predicted functional richness; the increase in species and predicted functional richness was mediated by higher effective sequencing depth. For BAL and nasal samples, most methods did not change Morisita-Horn dissimilarity suggesting limited bias introduced by host depletion. Conclusions: Metagenomics sequencing without host depletion will underestimate microbial diversity of most respiratory samples due to shallow effective sequencing depth and is not recommended. Optimal host depletion methods vary by sample type.

Host DNA depletion methods and genome-centric metagenomics of bovine hindmilk microbiome.

Bovine mastitis is a multi-etiological and complex disease, resulting in serious economic consequences for dairy farmers and industry. In recent years, the microbiological evaluation of raw milk has been investigated in-depth using next-generation sequencing approaches such as metataxonomic analysis. Despite this, host DNA is a major concern in the shotgun metagenomic sequencing of microbial communities in milk samples, and it represents a big challenge. In this study, we aimed to evaluate different methods for host DNA depletion and/or microbial DNA enrichment and assess the use of PCR-based whole genome amplification in milk samples with high somatic cell count (SCC) by using short- and long-read sequencing technologies. Our results evidenced that DNA extraction performed differently in terms of host DNA removal, impacting metagenome composition and functional profiles.. Moreover, the ratio of SCC/bacteria ultimately impacts microbial DNA yield, and samples with low SCC (SCC below 100,000 cells/mL) are the most problematic. When milk samples with high SCC (SCC above 200,000 cells/mL) underwent multiple-displacement amplification (MDA), we successfully recovered high-quality metagenome-assembled genomes (MAGs), and long-read sequencing was feasible even for samples with low DNA concentration. By associating MDA and short-read sequencing, we recovered two times more MAGs than in untreated samples, and an ongoing co-infection not reported by traditional methods was detected for mastitis pathogen. Overall, this new approach will improve the detection of mastitis-associated microorganisms and make it possible to examine host-microbiome interactions in bovine mastitis.IMPORTANCENext-generation sequencing technologies have been widely used to gain new insights into the diversity of the microbial community of milk samples and dairy products for different purposes such as microbial safety, profiling of starter cultures, and host-microbiome interactions. Milk is a complex food matrix, and additionally, the presence of host nucleic acid sequences is considered a contaminant in untargeted high-throughput sequencing studies. Therefore, genomic-centric metagenomic studies of milk samples focusing on the health-disease status in dairy cattle are still scarce, which makes it difficult to evaluate the microbial ecophysiology of bovine hindmilk. This study provides an alternative method for genome-centric metagenome studies applied to hindmilk samples with high somatic cell content, which is indispensable to examining host-microbiome interactions in bovine mastitis.

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

Purpose: The low microbial abundance on the ocular surface results in challenges in the characterization of its microbiome. The purpose of this study was to reveal factors introducing bias in the pipeline from sample collection to data analysis of low-abundant microbiomes. Methods: Lower conjunctiva and lower lid swabs were collected from six participants using either standard cotton or flocked nylon swabs. Microbial DNA was isolated with two different kits (with or without prior host DNA depletion and mechanical lysis), followed by whole-metagenome shotgun sequencing with a high sequencing depth set at 60 million reads per sample. The relative microbial compositions were generated using the two different tools MetaPhlan3 and Kraken2. Results: The total amount of extracted DNA was increased by using nylon flocked swabs on the lower conjunctiva. In total, 269 microbial species were detected. The most abundant bacterial phyla were Actinobacteria, Firmicutes and Proteobacteria. Depending on the DNA extraction kit and tool used for profiling, the microbial composition and the relative abundance of viruses varied. Conclusion: The microbial composition on the ocular surface is not dependent on the swab type, but on the DNA extraction method and profiling tool. These factors have to be considered in further studies about the ocular surface microbiome and other sparsely colonized microbiomes in order to improve data reproducibility. Understanding challenges and biases in the characterization of the ocular surface microbiome may set the basis for microbiome-altering interventions for treatment of ocular surface associated diseases.

Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.

A new and efficient enrichment method for metagenomic sequencing of Monkeypox virus.

BACKGROUND: The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. RESULTS: A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. CONCLUSIONS: Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.

Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum.

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients' sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human ß-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications.

The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some ß-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

High Sensitivity of Shotgun Metagenomic Sequencing in Colon Tissue Biopsy by Host DNA Depletion.

The high host genetic background of tissue biopsies hinders the application of shotgun metagenomic sequencing in characterizing the tissue microbiota. We proposed an optimized method that removed host DNA from colon biopsies and examined the effect on metagenomic analysis. Human or mouse colon biopsies were divided into two groups, with one group undergoing host DNA depletion and the other serving as the control. Host DNAs were removed through differential lysis of mammalian and bacterial cells before sequencing. The impact of host DNA depletion on microbiota was compared based on phylogenetic diversity analyses and regression analyses. Removing host DNA enhanced bacterial sequencing depth and improved species discovery, increasing bacterial reads by 2.46 ± 0.20 fold while reducing host reads by 6.80% ± 1.06%. Moreover, 3.40 times more of bacterial species were detected after host DNA depletion. This was confirmed from mouse colon tissues, increasing bacterial reads by 5.46 ± 0.42 fold while decreasing host reads by 10.2% ± 0.83%. Similarly, significantly more species were detected in the mouse colon tissue upon host DNA depletion (P < 0.001). Furthermore, an increased microbial richness was evident in the host DNA-depleted samples compared with non-depleted controls in human colon biopsies and mouse colon tissues (P < 0.001). Our optimized method of host DNA depletion improved the sensitivity of shotgun metagenomic sequencing in bacterial detection in the biopsy, which may yield a more accurate taxonomic profile of the tissue microbiota and identify bacteria that are important for disease initiation or progression.

Metagenomic Sequencing for Microbial DNA in Human Samples: Emerging Technological Advances.

Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a complex sample. This technology has revolutionised multiple areas from microbiome research to clinical diagnoses. However, one of the major challenges of a metagenomic study is the overwhelming non-microbial DNA present in most of the host-derived specimens, which can inundate the microbial signals and reduce the sensitivity of microorganism detection. Various host DNA depletion methods to facilitate metagenomic sequencing have been developed and have received considerable attention in this context. In this review, we present an overview of current host DNA depletion approaches along with explanations of their underlying principles, advantages and disadvantages. We also discuss their applications in laboratory microbiome research and clinical diagnoses and, finally, we envisage the direction of the further perfection of metagenomic sequencing in samples with overabundant host DNA.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Introduction: Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Methods: Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. Results: The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Discussion: Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

A novel cell permeability assay for macromolecules.

BACKGROUND: Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists. RESULTS: We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples. CONCLUSION: The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Detection of Antimicrobial Resistance Genes in the Milk Production Environment: Impact of Host DNA and Sequencing Depth.

Over the past decades, antimicrobial resistance (AMR) has been recognized as one of the most serious threats to public health. Although originally considered a problem to human health, the emerging crisis of AMR requires a "One Health" approach, considering human, animal, and environmental reservoirs. In this regard, the extensive use of antibiotics in the livestock production systems to treat mastitis and other bacterial diseases can lead to the presence of AMR genes in bacteria that contaminate or naturally occur in milk and dairy products, thereby introducing them into the food chain. The recent development of high-throughput next-generation sequencing (NGS) technologies is improving the fast characterization of microbial communities and their functional capabilities. In this context, whole metagenome sequencing (WMS), also called shotgun metagenomic sequencing, allows the generation of a vast amount of data which can be interrogated to generate the desired evidence, including the resistome. However, the amount of host DNA poses a major challenge to metagenome analysis. Given the current absence of literature concerning the application of WMS on milk to detect the presence of AMR genes, in the present study, we evaluated the effect of different sequencing depths, host DNA depletion methods and matrices to characterize the resistome of a milk production environment. WMS was conducted on three aliquots of bulk tank milk and three aliquots of the in-line milk filter collected from a single dairy farm; a fourth aliquot of milk and milk filter was bioinformatically subsampled. Two commercially available host DNA depletion methods were applied, and metagenomic DNA was sequenced to two different sequencing depth. Milk filters proved to be the most suitable matrices to evaluate the presence of AMR genes; besides, the pre-extraction host DNA depletion method was the most efficient approach to remove host reads. To our knowledge, this is the first study to evaluate the limitations posed by the host DNA in investigating the milk resistome with a WMS approach, confirming the circulation of AMR genes in the milk production environment.

Host DNA depletion efficiency of microbiome DNA enrichment methods in infected tissue samples.

Shotgun metagenomic sequencing or metagenomic whole genome sequencing is a genome-wide sequencing approach to explore bacterial communities directly from their habitat or sites of infection. However, host DNA contamination in metagenomic sequencing overwhelm low biomass of microbial signals and decrease sensitivity for microbial detection. In this study, we evaluated the host DNA depletion efficiency of four different microbiome DNA enrichment methods (NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit and Zymo HostZERO microbial DNA kit) in diabetic foot infection (DFI) tissue samples using quantitative real-time PCR and their effect on bacterial community composition by 16S ribosomal RNA amplicon sequencing. The host DNA depletion ratio (18S/16S rRNA), the percentage of bacterial DNA component and the microbial community profile of DFI were compared before (control) and after each microbiome DNA enrichment method. There was a significant difference in the 18S/16S rRNA ratio among different microbiome DNA enrichment methods (p <.001). QIAamp and HostZERO method reduced 18S/16S rRNA ratio by 32 and 57 fold than control method respectively. The percentage of bacterial DNA component increased more than ten-fold in QiaAmp (71.0 ± 2.7%) and HostZERO (79.9 ± 3.1%) method respectively than those in control method without host DNA depletion (6.7 ± 0.1%). It demonstrated the host DNA contamination was efficiently depleted and bacterial DNA was effectively enriched in HostZERO and QIAamp methods, attesting to the efficacy of these two methods in shotgun metagenomic sequencing studies. Overall, bacterial community composition of DFI samples was similar between control and microbiome enriched DNA samples.