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Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Associative Pavlovian conditioning leads to an increase in spinophilin-immunoreactive dendritic spines in the lateral amygdala

Changes in dendritic spine number and shape are believed to reflect structural plasticity consequent to learning. Previous studies have strongly suggested that the dorsal subnucleus of the lateral amygdala is an important site of physiological plasticity in Pavlovian fear conditioning. In the present study, we examined the effect of auditory fear conditioning on dendritic spine numbers in the dorsal subnucleus of the lateral amygdala using an immunolabelling procedure to visualize the spine-associated protein spinophilin. Associatively conditioned rats that received paired tone and shock presentations had 35% more total spinophilin-immunoreactive spines than animals that had unpaired stimulation, consistent with the idea that changes in the number of dendritic spines occur during learning and account in part for memory

Clean room microbiome complexity impacts planetary protection bioburden.

BackgroundThe Spacecraft Assembly Facility (SAF) at the NASA's Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom.ResultsCleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter.ConclusionThis study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays. Video abstract

Visualizing 'omic feature rankings and log-ratios using Qurro.

Many tools for dealing with compositional ' 'omics' data produce feature-wise values that can be ranked in order to describe features' associations with some sort of variation. These values include differentials (which describe features' associations with specified covariates) and feature loadings (which describe features' associations with variation along a given axis in a biplot). Although prior work has discussed the use of these 'rankings' as a starting point for exploring the log-ratios of particularly high- or low-ranked features, such exploratory analyses have previously been done using custom code to visualize feature rankings and the log-ratios of interest. This approach is laborious, prone to errors and raises questions about reproducibility. To address these problems we introduce Qurro, a tool that interactively visualizes a plot of feature rankings (a 'rank plot') alongside a plot of selected features' log-ratios within samples (a 'sample plot'). Qurro's interface includes various controls that allow users to select features from along the rank plot to compute a log-ratio; this action updates both the rank plot (through highlighting selected features) and the sample plot (through displaying the current log-ratios of samples). Here, we demonstrate how this unique interface helps users explore feature rankings and log-ratios simply and effectively

A comparison of six DNA extraction protocols for 16S, ITS and shotgun metagenomic sequencing of microbial communities

Microbial communities contain a broad phylogenetic diversity of organisms; however, the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include data from the fungal internal transcribed spacer (ITS) region, five candidate DNA extraction kits were compared against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. The results are presented considering a diverse panel of host-associated and environmental sample types and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection and microbial community composition among protocols. Across all criteria, the MagMAX Microbiome kit was found to perform best. The PowerSoil Pro kit performed comparably but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food and Norgen Stool kits were included

Gilles de la Tourette Syndrome

Gilles de la Tourette syndrome (GTS) is a childhood-onset neurodevelopmental disorder that is characterized by several motor and phonic tics. Tics usually develop before 10 years of age, exhibit a waxing and waning course and typically improve with increasing age. A prevalence of approximately 1% is estimated in children and adolescents. The condition can result in considerable social stigma and poor quality of life, especially when tics are severe (for example, with coprolalia (swearing tics) and self-injurious behaviours) or when GTS is accompanied by attention-deficit/hyperactivity disorder, obsessive–compulsive disorder or another neuropsychiatric disorder. The aetiology is complex and multifactorial. GTS is considered to be polygenic, involving multiple common risk variants combined with rare, inherited or de novo mutations. These as well as non-genetic factors (such as perinatal events and immunological factors) are likely to contribute to the heterogeneity of the clinical phenotype, the structural and functional brain anomalies and the neural circuitry involvement. Management usually includes psychoeducation and reassurance, behavioural methods, pharmacotherapy and, rarely, functional neurosurgery. Future research that integrates clinical and neurobiological data, including neuroimaging and genetics, is expected to reveal the pathogenesis of GTS at the neural circuit level, which may lead to targeted interventions