An essential step in the discovery of molecular mechanisms contributing to disease phenotypes and efficient experimental planning is the development of weighted hypotheses that estimate the functional effects of sequence variants discovered by high-throughput genomics. With the increasing specialization of the bioinformatics resources, creating analytical workflows that seamlessly integrate data and bioinformatics tools developed by multiple groups becomes inevitable. Here we present a case study of a use of the distributed analytical environment integrating four complementary specialized resources, namely the Lynx platform, VISTA RViewer, the Developmental Brain Disorders Database (DBDB), and the RaptorX server, for the identification of high-confidence candidate genes contributing to pathogenesis of spina bifida. The analysis resulted in prediction and validation of deleterious mutations in the SLC19A placental transporter in mothers of the affected children that causes narrowing of the outlet channel and therefore leads to the reduced folate permeation rate. The described approach also enabled correct identification of several genes, previously shown to contribute to pathogenesis of spina bifida, and suggestion of additional genes for experimental validations. The study demonstrates that the seamless integration of bioinformatics resources enables fast and efficient prioritization and characterization of genomic factors and molecular networks contributing to the phenotypes of interest.

High-throughput RNA sequencing (RNA-seq) enables comprehensive scans of entire transcriptomes, but best practices for analyzing RNA-seq data have not been fully defined, particularly for data collected with multiple sequencing platforms or at multiple sites. Here we used standardized RNA samples with built-in controls to examine sources of error in large-scale RNA-seq studies and their impact on the detection of differentially expressed genes (DEGs). Analysis of variations in guanine-cytosine content, gene coverage, sequencing error rate and insert size allowed identification of decreased reproducibility across sites. Moreover, commonly used methods for normalization (cqn, EDASeq, RUV2, sva, PEER) varied in their ability to remove these systematic biases, depending on sample complexity and initial data quality. Normalization methods that combine data from genes across sites are strongly recommended to identify and remove site-specific effects and can substantially improve RNA-seq studies.

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ~3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ~2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ~10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage-a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests.

RNA-based next-generation sequencing (RNA-Seq) provides a tremendous amount of new information regarding gene and transcript structure, expression and regulation. This is particularly true for non-coding RNAs where whole transcriptome analyses have revealed that the much of the genome is transcribed and that many non-coding transcripts have widespread functionality. However, uniform resources for raw, cleaned and processed RNA-Seq data are sparse for most organisms and this is especially true for non-human primates (NHPs). Here, we describe a large-scale RNA-Seq data and analysis infrastructure, the NHP reference transcriptome resource (http://nhprtr.org); it presently hosts data from12 species of primates, to be expanded to 15 species/subspecies spanning great apes, old world monkeys, new world monkeys and prosimians. Data are collected for each species using pools of RNA from comparable tissues. We provide data access in advance of its deposition at NCBI, as well as browsable tracks of alignments against the human genome using the UCSC genome browser. This resource will continue to host additional RNA-Seq data, alignments and assemblies as they are generated over the coming years and provide a key resource for the annotation of NHP genomes as well as informing primate studies on evolution, reproduction, infection, immunity and pharmacology.

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

Although B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children and while highly curable, it remains a leading cause of cancer-related mortality. The outgrowth of tumor subclones carrying mutations in genes responsible for resistance to therapy has led to a Darwinian model of clonal selection. Previous work has indicated that alterations in the epigenome might contribute to clonal selection, yet the extent to which the chromatin state is altered under the selective pressures of therapy is unknown. To address this, we performed chromatin immunoprecipitation, gene expression analysis, and enhanced reduced representation bisulfite sequencing on a cohort of paired diagnosis and relapse samples from individual patients who all but one relapsed within 36 months of initial diagnosis. The chromatin state at diagnosis varied widely among patients, while the majority of peaks remained stable between diagnosis and relapse. Yet a significant fraction was either lost or newly gained, with some patients showing few differences and others showing massive changes of the epigenetic state. Evolution of the epigenome was associated with pathways previously linked to therapy resistance as well as novel candidate pathways through alterations in pyrimidine biosynthesis and downregulation of polycomb repressive complex 2 targets. Three novel, relapse-specific superenhancers were shared by a majority of patients including one associated with S100A8, the top upregulated gene seen at relapse in childhood B-ALL. Overall, our results support a role of the epigenome in clonal evolution and uncover new candidate pathways associated with relapse. SIGNIFICANCE: This study suggests a major role for epigenetic mechanisms in driving clonal evolution in B-ALL and identifies novel pathways associated with drug resistance.

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has emerged as the cause of a global pandemic. We used RNA sequencing to analyze 286 nasopharyngeal (NP) swab and 53 whole-blood (WB) samples from 333 patients with COVID-19 and controls. Overall, a muted immune response was observed in COVID-19 relative to other infections (influenza, other seasonal coronaviruses, and bacterial sepsis), with paradoxical down-regulation of several key differentially expressed genes. Hospitalized patients and outpatients exhibited up-regulation of interferon-associated pathways, although heightened and more robust inflammatory responses were observed in hospitalized patients with more clinically severe illness. Two-layer machine learning-based host classifiers consisting of complete (>1000 genes), medium (<100), and small (<20) gene biomarker panels identified COVID-19 disease with 85.1-86.5% accuracy when benchmarked using an independent test set. SARS-CoV-2 infection has a distinct biosignature that differs between NP swabs and WB and can be leveraged for COVID-19 diagnosis.

Telomere length dynamics and DNA damage responses were assessed before, during, and after one-year or shorter duration missions aboard the International Space Station (ISS) in a comparatively large cohort of astronauts (n = 11). Although generally healthy individuals, astronauts tended to have significantly shorter telomeres and lower telomerase activity than age- and sex-matched ground controls before and after spaceflight. Although telomeres were longer during spaceflight irrespective of mission duration, telomere length shortened rapidly upon return to Earth, and overall astronauts had shorter telomeres after spaceflight than they did before; inter-individual differences were identified. During spaceflight, all crewmembers experienced oxidative stress, which positively correlated with telomere length dynamics. Significantly increased frequencies of chromosomal inversions were observed during and after spaceflight; changes in cell populations were also detected. We propose a telomeric adaptive response to chronic oxidative damage in extreme environments, whereby the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway is transiently activated in normal somatic cells.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses 1-3 , there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK 4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa 6 and P.1 (also 501Y.V3) recently detected in Brazil 7 . We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern 8 . Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.

Clean rooms of the Space Assembly Facility (SAF) at the Jet Propulsion Laboratory (JPL) at NASA are the final step of spacecraft cleaning and assembly before launching into space. Clean rooms have stringent methods of air-filtration and cleaning to minimize microbial contamination for exoplanetary research and minimize the risk of human pathogens, but they are not sterile. Clean rooms make a selective environment for microorganisms that tolerate such cleaning methods. Previous studies have attempted to characterize the microbial cargo through sequencing and culture-dependent protocols. However, there is not a standardized metagenomic workflow nor analysis pipeline for spaceflight hardware cleanroom samples to identify microbial contamination. Additionally, current identification methods fail to characterize and profile the risk of low-abundance microorganisms.

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

Microbial ecosystems of the built environments have become key mediators of health as people worldwide tend to spend large amount of time indoors. Underexposure to microbes at an early age is linked to increased risks of allergic and autoimmune diseases. Transportation systems are of particular interest, as they are globally the largest space for interactions between city-dwellers. Here we performed the first pilot study of the Moscow subway microbiome by analyzing swabs collected from 5 types of surfaces at 4 stations using high-throughput 16S rRNA gene sequencing. The study was conducted as a part of The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) project. The most abundant microbial taxa comprising the subway microbiome originated from soil and human skin. Microbiome diversity was positively correlated with passenger traffic. No substantial evidence of major human pathogens presence was found. Co-occurrence analysis revealed clusters of microbial genera including combinations of microbes likely originating from different niches. The clusters as well as the most abundant microbes were similar to ones obtained for the published data on New-York City subway microbiome. Our results suggest that people are the main source and driving force of diversity in subway-associated microbiome. The data form a basis for a wider survey of Moscow subway microbiome to explore its longitudinal dynamics by analyzing an extended set of sample types and stations. Complementation of methods with viability testing, "shotgun" metagenomics, sequencing of bacterial isolates and culturomics will provide insights for public health, biosafety, microbial ecology and urban design.

The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 lineages that have spread throughout the world. In this study, we investigated 129 RNA-seq data sets and 6928 consensus genomes to contrast the intra-host and inter-host diversity of SARS-CoV-2. Our analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights intra-host single nucleotide variant (iSNV) and SNP similarity, albeit with differences in C > U changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of insertions and deletions contribute to the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.

Mutations affecting enhancer chromatin regulators CREBBP and KMT2D are highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other's stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8 + T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions.