Host DNA Content Research Articles

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

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. 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. 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. 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.

CoproID predicts the source of coprolites and paleofeces using microbiome composition and host DNA content.

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue.

A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and added varying proportions of host DNA. However, the use of a marker-gene-based abundance estimation tool (MetaPhlAn2) requires considerable depth to detect marker genes from low-abundance organisms. Here, we reanalyse the deposited data, and place the study in the broader context of low microbial biomass metagenomics. We opted for a fast and sensitive read binning tool (Kraken 2) with abundance estimates from Bracken. With this approach all organisms are detected even when the sample comprises 99 % host DNA and similarly accurate abundance estimates are provided (mean squared error 0.45 vs. 0.3 in the original study). We show that off-target genera, whether contaminants or misidentified reads, come to represent over 10 % of reads when the sample is 99 % host DNA and exceed counts of many target genera. Therefore, we applied Decontam, a contaminant detection tool, which was able to remove 61 % of off-target species and 79 % of off-target reads. We conclude that read binning tools can remain sensitive to low-abundance organisms even with high host DNA content, but even low levels of contamination pose a significant problem due to low microbial biomass. Analytical mitigations are available, such as Decontam, although steps to reduce contamination are critical.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007–0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003–70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque

Metagenomic techniques are used to analyse bacterial communities allowing both culturable and unculturable species to be represented. However, the screening of oral metagenomic samples can be hindered by high animal host DNA content. This study evaluated methods for the reduction of human DNA concentrations within oral metagenomic samples. Plaque samples were collected from 27 patients presenting with periodontal disease and treated to remove human DNA using either selective lysis of eukaryotic cells at several buffer concentrations or differential centrifugation after treatment with trypsin and/or detergents. Human and bacterial DNA levels were determined by quantitative polymerase chain reaction (qPCR). The human DNA content of plaque extracts was significantly reduced by all treatments compared with an untreated control (P < 0.05). However, differential centrifugation simultaneously reduced the bacterial DNA content unless samples were pretreated with a detergent. Observations of Gram stained samples that were processed using differential centrifugation without detergent suggest that many bacteria remain adhered to human cells. An approach that uses differential centrifugation in parallel with selective lysis is recommended to fully represent the oral microbiota in metagenomic samples, including those tightly adhered to human cells and more delicate bacteria such as Mycoplasma.

Are phytoplankton population density maxima predictable through analysis of host and viral genomic DNA content?

Phytoplankton:virus interactions are important factors in aquatic nutrient cycling and community succession. The number of viral progeny resulting from an infection of a cell critically influences the propagation of infection and concomitantly the dynamics of phytoplankton populations. Host nucleotide content may be the resource limiting viral particle assembly. We present evidence for a strong linear correlation between measured viral burst sizes and viral burst sizes predicted from the host DNA content divided by the viral genome size, across a diversity of phytoplankton:viral pairs. An analysis of genome sizes therefore supports predictions of taxon-specific phytoplankton population density thresholds beyond which viral proliferation can trim populations or terminate phytoplankton blooms. We present corollaries showing that host:virus interactions may place evolutionary pressure towards genome reduction of both phytoplankton hosts and their viruses.

Cell survival after transplantation of fresh meniscal allografts. DNA probe analysis in a goat model.

Fibrochondrocytes synthesize and maintain the extracellular matrix responsible for the distinctive material and structural properties of a normal meniscus. Viable meniscal cells are believed to be necessary for the long-term maintenance of these properties in meniscal allografts. The purpose of this study was to determine if the donor cells (fibrochondrocytes) survive after a fresh meniscal allograft transplantation. A DNA probe technique was used to clearly distinguish the DNA patterns in donor cells from the host cells in the Spanish goat. No remaining donor DNA could be demonstrated at 4 weeks in transplanted meniscal tissue; it was all of host origin. The host DNA content at 4 weeks approached or exceeded the amount present in the contralateral control meniscus. The results of this study demonstrate that viable cells in medial meniscal allografts transplanted from one animal to another do not survive. Host cells rapidly repopulate the transplanted meniscus. There is no evidence these new cells will maintain on a long-term basis the extracellular matrix of the meniscus. The evidence in this paper, that the fibrochondrocytes do not survive transplantation, suggests further justification is necessary for using grafts with living cells. Allografts with living cells have an increased expense, more complicated surgical logistics, and have a higher potential risk of disease transmission.