Mislabeled, contaminated or poorly performing samples can threaten power in methylation microarray analyses or even result in spurious associations. We describe a set of quality checks for the ...popular Illumina 450K and EPIC microarrays to identify problematic samples and demonstrate their application in publicly available datasets.
Quality checks implemented here include 17 control metrics defined by the manufacturer, a sex check to detect mislabeled sex-discordant samples, and both an identity check for fingerprinting sample donors and a measure of sample contamination based on probes querying high-frequency SNPs. These checks were tested on 80 datasets comprising 8327 samples run on the 450K microarray from the GEO repository.
Nine hundred forty samples were flagged by at least one control metric and 133 samples from 20 datasets were assigned the wrong sex. In a dataset in which a subset of samples appear contaminated with a single source of DNA, we demonstrate that our measure based on outliers among SNP probes was strongly correlated (> 0.95) with another independent measure of contamination.
A more complete examination of samples that may be mislabeled, contaminated, or have poor performance due to technical problems will improve downstream analyses and replication of findings. We demonstrate that quality control problems are prevalent in a public repository of DNA methylation data. We advocate for a more thorough quality control workflow in epigenome-wide association studies and provide a software package to perform the checks described in this work. Reproducible code and supplementary material are available at 10.5281/zenodo.1172730.
DNA methylation microarrays are popular for epigenome-wide association studies (EWAS), but spurious values complicate downstream analysis and threaten replication. Conventional cut-offs for detection ...p values for filtering out undetected probes were demonstrated in a single previous study as insufficient leading to many apparent methylation calls in samples from females in probes targeting the Y-chromosome. We present an alternative approach to calculate more accurate detection p values utilizing non-specific background fluorescence. We evaluate and compare our proposed approach of filtering observations with conventional ones by assessing the detection of Y-chromosome probes among males and females in 2755 samples from 17 studies on the 450K microarray and masking of large outliers between technical replicates and their impact downstream via an EWAS reanalysis.
In contrast to conventional approaches, ours marks most Y-chromosome probes in females as undetected while removing a median of only 0.14% of the data per sample, catches more (30% vs. 6%) of large outliers (more than 20 percentage point difference between technical replicates), and helps to identify strong associations previously obfuscated by outliers between whole blood DNA methylation and chronological age in a well-powered EWAS (n = 729).
We provide guidance for filtering both 450K and EPIC microarrays as an essential preprocessing step to reduce spurious values. An implementation (including a function compatible with objects from the popular minfi package) was added to ewastools, an R package for comprehensive quality control of DNA methylation microarrays. Scripts to reproduce all analyses are available at doi.org/10.5281/zenodo.1443561 .
Saliva is a widely used biological sample, especially in pediatric research, containing a heterogenous mixture of immune and epithelial cells. Associations of exposure or disease with saliva DNA ...methylation can be influenced by cell-type proportions. Here, we developed a saliva cell-type DNA methylation reference panel to estimate interindividual cell-type heterogeneity in whole saliva studies. Saliva was collected from 22 children (7-16 years) and sorted into immune and epithelial cells, using size exclusion filtration and magnetic bead sorting. DNA methylation was measured using the Illumina MethylationEPIC BeadChip. We assessed cell-type differences in DNA methylation profiles and tested for enriched biological pathways. Immune and epithelial cells differed at 181,577 (22.8%) DNA methylation sites (t-test p < 6.28 × 10
−8
). Immune cell hypomethylated sites are mapped to genes enriched for immune pathways (p < 3.2 × 10
−5
). Epithelial cell hypomethylated sites were enriched for cornification (p = 5.2 × 10
−4
), a key process for hard palette formation. Saliva immune and epithelial cells have distinct DNA methylation profiles which can drive whole-saliva DNA methylation measures. A primary saliva DNA methylation reference panel, easily implemented with an R package, will allow estimates of cell proportions from whole saliva samples and improve epigenetic epidemiology studies by accounting for measurement heterogeneity by cell-type proportions.
The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae ("Oophila amblystomatis" Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show ...here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA amplification reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26-44 through chlorophyll autofluorescence and algal 18S rDNA amplification. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae through FISH, suggesting that the decline in autofluorescent cells is primarily due to algal cell death within the host. However, early embryonic egg capsules also contained encysted algal cells on the inner capsule wall, and algal 18S rDNA was amplified from adult reproductive tracts, consistent with oviductal transmission of algae from one salamander generation to the next. The invasion of algae into salamander host tissues and cells represents a unique association between a vertebrate and a eukaryotic alga, with implications for research into cell-cell recognition, possible exchange of metabolites or DNA, and potential congruence between host and symbiont population structures.
DNA methylation microarrays have been the platform of choice for epigenome-wide association studies in epidemiology, but declining costs have rendered targeted bisulphite sequencing a feasible ...alternative. Nonetheless, the literature for researchers seeking guidance on which platform to choose is sparse. To fill this gap, we conducted a comparison study in which we processed cord blood samples from four newborns in duplicates using both the Illumina HumanMethylationEPIC BeadChip and the Illumina TruSeq Methyl Capture EPIC Kit, and evaluated both platforms in regard to coverage, reproducibility, and identification of differential methylation. We conclude that with current analytic goals microarrays still outperform bisulphite sequencing for precise quantification of DNA methylation.
Abstract
Mantamonads were long considered to represent an “orphan” lineage in the tree of eukaryotes, likely branching near the most frequently assumed position for the root of eukaryotes. Recent ...phylogenomic analyses have placed them as part of the “CRuMs” supergroup, along with collodictyonids and rigifilids. This supergroup appears to branch at the base of Amorphea, making it of special importance for understanding the deep evolutionary history of eukaryotes. However, the lack of representative species and complete genomic data associated with them has hampered the investigation of their biology and evolution. Here, we isolated and described two new species of mantamonads,
Mantamonas vickermani
sp. nov. and
Mantamonas sphyraenae
sp. nov., for each of which we generated transcriptomic sequence data, as well as a high-quality genome for the latter. The estimated size of the
M. sphyraenae
genome is 25 Mb; our de novo assembly appears to be highly contiguous and complete with 9,416 predicted protein-coding genes. This near-chromosome-scale genome assembly is the first described for the CRuMs supergroup.
Diseases of the gastrointestinal tract due to changes in the bacterial flora have been described with increasing incidence in the European brown hare. Despite extensive demographic and ...phylogeographic research, little is known about the composition of its gut microbiota and how it might vary based on potential environmental or host factors. We analysed the intestinal and faecal microbiota of 3 hare populations by Illumina MiSeq 16S rRNA gene amplicon sequencing. The phyla and OTU abundance composition differed significantly between intestinal and faecal samples (PERMANOVA: P = 0.002 and P = 0.031, respectively), but in both sample types Firmicutes and Bacteroidetes dominated the microbial community composition (45.51% and 19.30% relative abundance). Intestinal samples contained an enrichment of Proteobacteria compared with faecal samples (15.71-fold change, P < 0.001). At OTU level, a significant enrichment with best BLAST hits to the Escherichia-Shigella group, Eubacterium limosum, Sphingomonas kyeonggiensis, Flintibacter butyricus and Blautia faecis were detected in intestinal samples (P < 0.05). In our statistical model, geographic location and possibly associated environmental factors had a greater impact on the microbiota composition than host factors. Population had a significant effect on the composition of abundant intestinal and faecal OTUs, and on the abundance of potential pathogenic bacteria of the family Enterobacteriaceae, regularly associated with intestinal dysbiosis in hares, in faecal samples. Our study is the first to describe the microbiota in brown hares and provides a foundation to generate hypothesis aiming to test the role of gut health in population fluctuations of the species.
Modern syntheses of eukaryote diversity assign almost all taxa to one of three groups: Amorphea, Diaphoretickes and Excavata (comprising Discoba and Metamonada). The most glaring exception is ...Malawimonadidae, a group of small heterotrophic flagellates that resemble Excavata by morphology, but branch with Amorphea in most phylogenomic analyses. However, just one malawimonad, Malawimonas jakobiformis, has been studied with both morphological and molecular-phylogenetic approaches, raising the spectre of interpretation errors and phylogenetic artefacts from low taxon sampling. We report a morphological and phylogenomic study of a new deep-branching malawimonad, Gefionella okellyi n. gen. n. sp. Electron microscopy revealed all canonical features of ‘typical excavates’, including flagellar vanes (as an opposed pair, unlike M. jakobiformis but like many metamonads) and a composite fibre. Initial phylogenomic analyses grouped malawimonads with the Amorphea-related orphan lineage Collodictyon, separate from a Metamonada+Discoba clade. However, support for this topology weakened when more sophisticated evolutionary models were used, and/or fast-evolving sites and long-branching taxa (FS/LB) were excluded. Analyses of ‘–FS/LB’ datasets instead suggested a relationship between malawimonads and metamonads. The ‘malawimonad+metamonad signal’ in morphological and molecular data argues against a strict Metamonada+Discoba clade (i.e. the predominant concept of Excavata). A Metamonad+Discoba clade should therefore not be assumed when inferring deep-level evolutionary history in eukaryotes.
The Illumina Infinium HumanMethylation450 BeadChip is frequently used in epigenetic research. Besides quantile normalization there is currently no standard method to normalize the data between ...arrays. We describe some properties of the data generated by this platform and present a normalization method based on local regression. We compare the performance of this method with other commonly used approaches in three benchmarks (correlation between 21 pairs of technical replicates, detection of differential methylation and correlation of methylation levels for smoking-associated CpG sites with smoking behavior of 655 participants of an epidemiological study). Results indicate that the proposed method improves reproducibility, whereas some commonly used methods can have adverse effects.
Abstract
This paper presents a method for organising the movement of unmanned aerial vehicles in formation. In the presented method of UAV collisions are prevented by a modification of the potential ...field method. The movement of the group is accomplished by incrementing the origin coordinates of the formation at each iteration of data exchange between UAVs. This paper presents a graph showing the negligible positioning error of drones in formation over time as the heterogeneous swarm of quadcopters moves towards the target position. Simulation results show that the flight time of a group of UAVs to the target swarm position using the proposed method is longer than using the master-slave method. The model of a Wi-Fi network as an information exchange network between UAVs was used in the developed simulation environment. This approach facilitates the implementation of the presented method on real aircrafts.
PDF
