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bioRxiv2018

Sketching Algorithms for Genomic Data Analysis and Querying in a Secure Enclave

Current practices in collaborative genomic data analysis (e.g. PCAWG [1]) necessitate all involved parties to exchange individual patient data and perform all analysis locally, or use a trusted server for maintaining all data to perform analysis in a single site (e.g. the Cancer Genome Collaboratory). Since both approaches involve sharing genomic sequence data - which is typically not feasible due to privacy issues, collaborative data analysis remains to be a rarity in genomic medicine.\n\nIn order to facilitate efficient and effective collaborative or remote genomic computation we introduce SkSES (Sketching algorithms for Secure Enclave based genomic data analysiS), a computational framework for performing data analysis and querying on multiple, individually encrypted genomes from several institutions in an untrusted cloud environment. Unlike other techniques for secure/privacy preserving genomic data analysis, which typically rely on sophisticated cryptographic techniques with prohibitively large computational overheads, SkSES utilizes the secure enclaves supported by current generation microprocessor architectures such as Intels SGX. The key conceptual contribution of SkSES is its use of sketching data structures that can fit in the limited memory available in a secure enclave.\n\nWhile streaming/sketching algorithms have been developed for many applications in computer science, their feasibility in genomics has remained largely unexplored. On the other hand, even though privacy and security issues are becoming critical in genomic medicine, available cryptographic techniques based on, e.g. homomorphic encryption or garbled circuits, fail to address the performance demands of this rapidly growing field. The alternative offered by Intels SGX, a combination of hardware and software solutions for secure data analysis, is severely limited by the relatively small size of a secure enclave, a private region of the memory protected from other processes. SkSES addresses this limitation through the use of sketching data structures to support efficient secure and privacy preserving SNP analysis across individually encrypted VCF files from multiple institutions. In particular SkSES provides the users the ability to query for the \"k most significant SNPs\" among any set of user specified SNPs and any value of k - even when the total number of SNPs to be maintained is far beyond the memory capacity of the secure enclave.\n\nResultsWe tested SkSES on the complete iDASH-2017 competition data set comprised of 1000 case and 1000 control samples related to an unknown phenotype. SkSES was able to identify the top SNPs with respect to the{chi} 2 statistic, among any user specified subset of SNPs across this data set of 2000 individually encrypted complete human genomes quickly and accurately - demonstrating the feasibility of secure and privacy preserving computation for genomic medicine via Intels SGX.\n\nAvailabilityhttps://github.com/ndokmai/sgx-genome-variants-search

bioinformatics↗

bioRxiv2018

Closing Target Trimming: a Perl Package for Discovering Hidden Superfamily Loci in Genomes

The contemporary capacity of genome sequence analysis significantly lags behind the rapidly evolving sequencing technologies. Retrieving biological meaningful information from an ever-increasing amount of genome data would be significantly beneficial for functional genomic studies. For example, the duplication, organization, evolution, and function of superfamily genes are arguably important in many aspects of life. However, the incompleteness of annotations in many sequenced genomes often results in biased conclusions in comparative genomic studies of superfamilies. Here, we present a Perl software, called Closing Target Trimming, for automatically identifying most, if not all, members of a gene family in any sequenced genomes. Our test data on the F-box gene superfamily showed 78.2 and 79% gene finding accuracies in two well annotated plant genomes, Arabidopsis thaliana and rice, respectively. This annotation performance is clearly higher than the best ab initio methods that are currently available. To further demonstrate the effectiveness of this program, we ran it through 18 plant genomes and five non-plant genomes to compare the expansion of the F-box and the BTB superfamilies. The program discovered that on average 12.7 and 9.3% of the total F-box and BTB members, respectively, are new loci in plant genomes while it only found a small number of new members in vertebrate genomes. Therefore, different evolutionary and regulatory mechanisms of cullin-RING ubiquitin ligases may be present in the plant and the animal kingdoms. Further studies may shed light on new discoveries in the ubiquitin-26S proteasome system-mediated regulatory pathways in eukaryotic organisms. With a detailed compiling instruction and a simple running operation, we expect that this software will assist many biological scientists with little programming experience to smoothly obtain a comprehensive dataset of a gene superfamily in any sequenced eukaryotic genomes.

bioinformatics↗

bioRxiv2019

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable isotope-informed genome-resolved metagenomics

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after feeding 13C-labeled butyrate. Differential abundance analysis on recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in the heavy 13C DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas, and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (< 95%) with any cultured representative species, indicating it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for aceticlastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to elucidate the nature of metabolic cooperation in slow-growing uncultured microbial populations, such as syntrophic bacteria and methanogens, that are important to waste treatment as well as global carbon cycling. ImportancePredicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in-situ metabolic activities of uncultured species could enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in-situ function of mixed microbiomes by combining DNA-stable isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve who is doing what by obtaining confirmatory assimilation of labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

microbiology↗

bioRxiv2019

Population genomics of Vibrionaceae isolated from an endangered oasis reveals local adaptation after an environmental perturbation.

BackgroundIn bacteria, pan-genomes are the result of the evolutionary \"tug of war\" between selection and horizontal gene transfer (HGT). High rates of HGT increase the genetic pool and the effective population size, resulting in open pan-genomes. In contrast, selective pressures can lead to local adaptation by purging the variation introduced by HGT, resulting in closed pan-genomes and clonal lineages. In this study, we explored both hypotheses elucidating the pan-genome of Vibrionaceae isolates after a perturbation event in the endangered oasis of Cuatro Cienegas Basin (CCB), Mexico, and looking for signals of adaptation to the environments in their genomes.\n\nResultsWe obtained 42 genomes of Vibrionaceae distributed in six lineages, two of them did not showed any close reference strain in databases. Five of the lineages showed closed pan-genomes and were associated to either water or sediment environment; their high effective population size (Ne) estimates suggest that these lineages are not from a recent origin. The only clade with an open pan-genome was found in both environments and was formed by ten genetic groups with low Ne, suggesting a recent origin. The recombination and mutation estimators (r/m) ranged from 0.0052 to 2.7249, which are similar to oceanic Vibrionaceae estimations; however, we identified 367 gene families with signals of positive selection, most of them found in the core genome; suggesting that despite recombination, natural selection moves the Vibrionaceae CCB lineages to local adaptation purging the genomes and keeping closed pan-genome patterns. Moreover, we identify 598 SNPs associated with an unstructured environment; some of the genes under this SNPs were related to sodium transport.\n\nConclusionsDifferent lines of evidence suggest that the sampled Vibrionaceae, are part of the rare biosphere usually living under famine conditions. Two of these lineages were reported by the first time. Most Vibrionaceae lineages of CCB are adapted to their microhabitats rather than to the sampled environments. This pattern of adaptation agrees with the association of closed pan-genomes and local adaptation.

evolutionary biology↗

bioRxiv2019

The RGG Domain of the Yeast Nucleolin Nsr1 Is Required for the Genome Instability Associated with Co-transcriptionally Formed G4 DNA

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin NCL is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that G4 DNA-binding is a conserved function in the yeast nucleolin Nsr1. Furthermore, we demonstrate that the Nsr1-G4 DNA complex formation results in replication obstruction and is a major factor in inducing the genome instability associated with the co-transcriptionally formed G4 DNA in the yeast genome. The G4-associated genome instability and the G4 DNA-binding in vivo requires the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vitro and in vivo and restores unhindered replication, overall resulting in a sharp reduction in the G4-associated genome instability. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and obstructing replication. AUTHOR SUMMARYGenome instability is uniquely elevated at sequences containing multiple runs of guanines, which can fold into the unusual, four-stranded G-quadruplex (G4) DNA. In this study, we report a novel finding that a highly conserved G4 DNA binding protein Nsr1 can elevate the rate of recombination and chromosomal rearrangement occurring at a G4 DNA-forming sequence in the genome of Saccharomyces cerevisiae. The elevated genome instability requires the C-terminally located RGG domain of Nsr1, which supports the high-affinity interaction between the protein and G4 DNA. The connection between G4-specific genome instability and the function of Nsr1 to form stable complex with G4 DNA led to the hypothesis that the high-affinity Nsr1-G4 DNA complexes can become a barrier to replication. We demonstrate here that the presence of Nsr1 in fact slows the replication past a G4 DNA-containing genomic site and that the RGG domain is required to facilitate such replication block.

genetics↗

bioRxiv2019

Addressing dereplication crisis: Taxonomy-free reduction of massive genome collections using embeddings of protein content

Many recent microbial genome collections curate hundreds of thousands of genomes. This volume complicates many genomic analyses such as taxon assignment because the associated computational burden is substantial. However, the number of representatives of each species is highly skewed towards human pathogens and model organisms. Thus many genomes contain little additional information and could be removed. We created a frugal dereplication method that can reduce massive genome collections based on genome sequence alone, without the need for manual curation nor taxonomic information. We recently created a genome representation for bacteria and archaea called "nanotext". This method embeds each genome in a low-dimensional vector of numbers. Extending nanotext, our proposed algorithm called "thinspace" uses these vectors to group and dereplicate similar genomes. We dereplicated the Genome Taxonomy Database (GTDB) from about 150 thousand genomes to less than 22 thousand. The resulting collection increases the percent of classified reads in a metagenomic dataset by a factor of 5 compared to NCBI RefSeq and performs equal to both a larger as well as a manually curated GTDB subset. With thinspace, massive genome collections can be dereplicated on regular hardware, without affecting downstream results. It is released under a BSD-3 license (github.com/phiweger/thinspace).

bioinformatics↗

bioRxiv2019

Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive

Multiply antibiotic resistant Acinetobacter baumannii infections are a global public health concern and accurate tracking of the spread of specific lineages is needed. Variation in the composition and structure of capsular polysaccharide (CPS), a critical determinant of virulence and phage susceptibility, makes it an attractive epidemiological marker. The outer core (OC) of lipooligosaccharide also exhibits variation. To take better advantage of the untapped information available in whole genome sequences, we have created a curated reference database of the 92 publicly available gene clusters at the locus encoding proteins responsible for biosynthesis and export of CPS (K locus), and a second database for the 12 gene clusters at the locus for outer core biosynthesis (OC locus). Each entry has been assigned a unique KL or OCL number, and is fully annotated using a simple, transparent and standardised nomenclature. These databases are compatible with Kaptive, a tool for in silico typing of bacterial surface polysaccharide loci, and their utility was validated using a) >630 assembled A. baumannii draft genomes for which the KL and OCL regions had been previously typed manually, and b) 3386 A. baumannii genome assemblies downloaded from NCBI. Among the previously typed genomes, Kaptive was able to confidently assign KL and OCL types with 100% accuracy. Among the genomes retrieved from NCBI, Kaptive detected known KL and OCL in 87% and 90% of genomes, respectively indicating that the majority of common KL and OCL types are captured within the databases; 13 KL were not detected in any public genome assembly. The failure to assign a KL or OCL type may indicate incomplete or poor-quality genomes. However, further novel variants may remain to be documented. Combining outputs with multi-locus sequence typing (Institut Pasteur scheme) revealed multiple KL and OCL types in collections of a single sequence type (ST) representing each of the two predominant globally-distributed clones, ST1 of GC1 and ST2 of GC2, and in collections of other clones comprising >20 isolates each (ST10, ST25, and ST140), indicating extensive within-clone replacement of these loci. The databases are available at https://github.com/katholt/Kaptive and will be updated as further locus types become available. Data Summary1. Databases including fully annotated gene cluster sequences for A. baumannii K loci and OC loci are available for download at https://github.com/katholt/Kaptive 2. The Kaptive software, which can be used to screen new genomes against the K and O locus database is available at https://github.com/katholt/Kaptive (command-line code) and http://kaptive.holtlab.net/ (interactive web service). 3. Details of the Kaptive search results validating in silico serotyping of K and O loci using our approach are provided as supplementary files, Dataset 1 (92 KL reference sequences and 12 OCL reference sequences), Dataset 2 (642 genomes assembled from reads available in NCBI SRA) and Dataset 3 (3415 genome assemblies downloaded from NCBI GenBank). Impact statementThe ability to identify and track closely related isolates is key to understanding, and ultimately controlling, the spread of multiply antibiotic resistant A. baumannii causing difficult to treat infections, which are an urgent public health threat. Extensive variation in the KL and OCL gene clusters responsible for biosynthesis of capsule and the outer core of lipooligosaccharide, respectively, are potentially highly informative epidemiological markers. However, clear, well-documented identification of each variant and simple-to-use tools and procedures are needed to reliably identify them in genome sequence data. Here, we present curated databases compatible with the available web-based and command-line Kaptive tool to make KL and OCL typing readily accessible to assist epidemiological surveillance of this species. As many bacteriophage recognise specific properties of the capsule and attach to it, capsule typing is also important in assessing the potential of specific phage for therapy on a case by case basis.

microbiology↗

bioRxiv2025

Rapid and Consistent Genome Clustering for Navigating Bacterial Diversity with Millions of MAGs and Isolates

Bacterial genome and metagenome databases collectively contain over 5 million high-quality assemblies. However, the redundancy of these databases and the limited scalability of existing tools create bottlenecks for fully comprehensive, tree-of-life-scale genomic analyses. A fundamental task is to first break this data into smaller chunks, guided by their genome similarity. However, alignment-based comparative methods struggle to handle more than a few tens of thousands of genomes at a time, making the global organisation computationally complex and expensive. Here, we present gemsparcl (https://github.com/johannahelene/gemsparcl), a tool that clusters bacterial genomes into genomic cohesive units (GCUs), at approximately species-level resolution, over 500 times faster than existing methods. As part of developing gemsparcl, we developed sketchlib.rust, a one-permutation MinHash approach that implements an auxiliary inverted index to further accelerate all-versus-all comparisons. We added a statistical correction for incomplete metagenome-assembled genomes (MAGs) to enable accurate distance estimation and network-based edge quality filtering. After genome completeness quality control, we clustered 5.6 million high-quality bacterial genomes (2.88 million isolates and 2.77 million MAGs) into 92,954 GCUs in [~]14 hours using 48 CPU threads and less than 16.5 GB of memory. Using taxonomic validation of the GCUs, the method achieves very high (99.76%) cluster purity (meaning only one species label occurs per GCU). We demonstrate that the clustering also highlights cases where taxonomic naming can be potentially harmonised or improved. Furthermore, we identify the most frequently reconstructed MAGs that lack a corresponding isolate genome and are thus priorities for culturing. The enhanced speed of gemsparcl enables routine database updates to incorporate the latest genomes. It also makes reference-free microbiome analysis across millions of genomes computationally tractable for the first time.

bioinformatics↗

bioRxiv2026

Genome Assembly of the Iconic Samba Mahsuri Delineates Locus-specific Population Structure within Indica Rice

High-quality reference genomes enable detailed analysis of structural variation and its consequences for genome organization in crops. Here, we present a chromosome-scale genome assembly of Oryza sativa cv. Samba Mahsuri (SM), an elite Indian mega rice variety cultivated for its grain and cooking quality. Using PacBio HiFi sequencing in combination with Illumina reads and Bionano optical mapping, we generated a [~]395 Mb assembly (SMv1.0) with 97.7% BUSCO completeness. A robust annotation framework identified 31,138 evidence-guided protein-coding gene models alongside 59,152 ab initio predictions. Comparative genomic analyses revealed extensive macrosynteny with established rice reference genomes, while uncovering pronounced locus-specific sequence and structural polymorphisms. Notably, a complex inversion-match-inversion (IMI) configuration on chromosome 6 differentiates SM from the japonica reference Nipponbare, but not from the indica reference R498. Population-scale analyses of 533 cultivated and 4 wild rice accessions demonstrate that genetic variation within the IMI region produces a markedly sharper and more coherent population structure than is observed in flanking regions or genome-wide, including tight subpopulation-based clustering and segregation of alternative IMI configurations within indica rice. Together, these results establish SMv1.0 as a robust chromosome-scale reference genome sequence for rice and demonstrate how large structural polymorphisms can shape locus-specific patterns of relatedness that diverge from genome-wide ancestry. Significance StatementWe present a high-quality chromosome-scale genome sequence of the elite Indian rice variety Samba Mahsuri (SM), which, to the best of our knowledge, represents the first chromosome-scale reference genome from Indian rice germplasm assembled using a map-based method. Using this reference to analyze population-scale genotyping data from 533 cultivated and four wild rice accessions reveals markedly tighter population clustering within a megabase-scale inversion (IMI) region, than at the whole-genome scale, along with a pronounced split within indica rice that is independent of genome-wide ancestry.

plant biology↗

bioRxiv2026

ArchaeaHQ: A Curated Reference Database of Archaeal Genomes

Archaea have proven to be major players in biogeochemical cycles across diverse ecosystems, yet we still see an underrepresentation of archaeal genomes in the datasets used by popular computational biology tools. Here we present ArchaeaHQ, a quality-controlled, systematically curated reference database of 21,644 archaeal genomes compiled initially from 35,993 assemblies from all four archaeal kingdoms retrieved from NCBI: Methanobacteriati (Euryarchaeota), Thermoproteati (TACK), Nanobdellati (DPANN), and Promethearchaeati (Asgard). All genomes in the database passed standardized quality control, requiring [≥]70% completeness and [≤]10% contamination. A total of 44.2% of genomes in ArchaeaHQ achieved [≥]90% completeness, while 93.1% exhibited [≤]5% contamination. ArchaeaHQ comprises 16,199 metagenome-assembled genomes (MAGs; 74.8%) and 5,445 isolate genomes (25.2%). Approximately 75% of MAGs are assigned to 17 ecologically meaningful categories based on sampling origin, and around 65% of genomes include geographic metadata. ArchaeaHQ is available at https://doi.org/10.6084/m9.figshare.32266599 and provides an analysis-ready reference set for metagenomic classification, biogeochemical and ecological studies, comparative genomics, and development of archaeal-specific bioinformatic tools. Impact StatementArchaea are key drivers of the global carbon, nitrogen and methane cycles, yet their genomes remain underrepresented and inconsistently curated in the public databases that power modern computational biology tools. We present ArchaeaHQ, a quality-controlled, systematically curated reference set of 21,644 archaeal genomes spanning all four archaeal kingdoms, each passing standardized completeness and contamination thresholds and enriched with environmental and geographic metadata. By providing an analysis-ready, downloadable resource compatible with standard pipelines, ArchaeaHQ fits the gap between taxonomy-focused frameworks and unfiltered genome archives supporting metagenomic classification, biogeochemical and ecological studies, comparative genomics, and the development of archaeal-specific bioinformatic tools.

microbiology↗

bioRxiv2025

Monitoring of vaccine targets and interventions using global genome data: vaccines.watch

The expansion of pathogen genome sequencing into routine disease surveillance programmes is set to bring rapidly-growing volumes of increasingly structured data on a global scale. This has the potential to deliver exciting opportunities for accelerating vaccine development and monitoring. Here we present an interactive platform, vaccines.watch (https://vaccines.watch), which aims to support decision-making around vaccine formulations and roll-out by enabling interrogation of vaccine target diversity from global genome data. We have initially focused on targets included in existing or prospective multivalent polysaccharide- based vaccines for Streptococcus pneumoniae, Klebsiella pneumoniae (and related species) and Acinetobacter baumannii. The platform currently displays data for >100k high-quality genomes with geotemporal sampling information (post-2010), with new genomes assembled, analysed and incorporated on an ongoing basis (every 4 hours) as public data are newly deposited. Crucially, users can view vaccine target information in the broader context of genotypic variants (e.g. sequence types) and antimicrobial resistance markers. The platform also enables users to review the composite serotypes of pneumococcal vaccine formulations among the available genomes. For example, using data in vaccines.watch from 3 June 2025, we observed that serotypes included in the PCV13 and PCV21 formulations accounted for 36.2% (11,907/32,918) and 87.4% (28,764/32,918) of global public genomes, respectively. The platform also enables continuous review of the global genomic landscape of the included pathogens, enabling identification of gaps (e.g. in geographic coverage) that should be targeted with increased genomic surveillance. Indeed we demonstrate that substantial geographic gaps remain in the coverage of available genomes, with over half of countries contributing no genomes for each of the three pathogens. However, while caution in interpretation is important, as global representativeness of genome data grows, vaccines.watch is positioned to support different stages of the vaccine pipeline, from selection of target antigens to post-rollout monitoring of population changes.

genomics↗

bioRxiv2016

The haplotype-resolved genome sequence of hexaploid Ipomoea batatas reveals its evolutionary history

Although the sweet potato, Ipomoea batatas, is the seventh most important crop in the world and the fourth most significant in China, its genome has not yet been sequenced. The reason, at least in part, is that the genome has proven very difficult to assemble, being hexaploid and highly polymorphic; it has a presumptive composition of two B1 and four B2 component genomes (B1B1B2B2B2B2). By using a novel haplotyping method based on de novo genome assembly, however, we have produced a half haplotype-resolved genome from [~]267Gb of paired-end sequence reads amounting to roughly 60-fold coverage. By phylogenetic tree analysis of homologous chromosomes, it was possible to estimate the time of two whole genome duplication events as occurring about 525,000 and 341,000 years ago. Our analysis also identified many clusters of genes for specialized compounds biosynthesis in this genome. This half haplotype-resolved hexaploid genome represents the first successful attempt to investigate the complexity of chromosome sequence composition directly in a polyploid genome, using direct sequencing of the polyploid organism itself rather than of any of its simplified proxy relatives. Adaptation and application of our approach should provide higher resolution in future genomic structure investigations, especially for similarly complex genomes.

Genomics↗

bioRxiv2016

A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens

Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. The 2001 anthrax letter attacks involved less than gram quantities of Bacillus anthracis spores while the earlier Soviet weapons program produced tons. A Soviet industrial production facility in Sverdlovsk proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreak. In order to understand this outbreak and others, we have generated a B. anthracis population genetic database based upon whole genome analysis to identify all SNPs across a reference genome. Only ~12,000 SNPs were identified in this low diversity species and represents the breadth of its known global diversity. Phylogenetic analysis has defined three major clades (A, B and C) with B and C being relatively rare compared to A. The A clade has numerous subclades including a major polytomy named the Trans-Eurasian (TEA) group. The TEA radiation is a dominant evolutionary feature of B. anthracis, many contemporary populations, and must have resulted from large-scale dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deeply sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. Only 13 SNPs differentiated the virulent Sverdlovsk strain from its common ancestor with two vaccine strains. The Soviet Sverdlovsk strain genome is consistent with a wild type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world's largest biological weapons program and provides an extensive B. anthracis phylogenetic reference valuable for future anthrax investigations.\n\nImportanceThe 1979 Russian anthrax outbreak resulted from an industrial accident at the Soviet anthrax spore production facility in the city of Sverdlovsk. Deep genomic sequencing of two autopsy specimens generated a draft genome and phylogenetic placement of the Soviet Sverdlovsk anthrax strain. While it is known that Soviet scientists had genetically manipulated Bacillus anthracis, with the potential to evade vaccine prophylaxis and antibiotic therapeutics, there was no genomic evidence of this from the Sverdlovsk production strain genome. The whole genome SNP genotype of the Sverdlovsk strain was used to precisely identify it and its close relatives in the context of an extensive global B. anthracis strain collection. This genomic identity can now be used for forensic tracking of this weapons material on a global scale and for future anthrax investigations.

Genomics↗

bioRxiv2017

Comparative Genomics Of Two Sequential Candida glabrata Clinical Isolates

Candida glabrata is an important fungal pathogen which develops rapidly antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. We were interested in this study to compare genomes of two specific C. glabrata related isolates, one of which was azole-susceptible (DSY562) while the other was azole-resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time lapse of 50 days of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacbBio technology used here yielded 14 major contigs (sizes 0.18 Mb-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 contained each a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (66) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 non-synonymous SNPs (among which the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes were containing a DNA mismatch repair allele of MSH2 known to be involved in the so-called hypermutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points were exhibiting limited variations, even if submitted to the host pressure.

genomics↗

bioRxiv2017

Genomic architecture of codfishes featured by expansions of innate immune genes and short tandem repeats

BackgroundIncreased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates including teleosts. The sequencing and assembly of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss 100 million years ago of the major histocompatibility complex (MHC) II genes. The recent publication of an improved version of the Atlantic cod genome assembly reported an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous genome assemblies are needed to further investigate the unusual immune system of the Gadiformes, and the high density of tandem repeats in this group.\n\nResultsHere, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) - a relative of Atlantic cod - using a combination of PacBio and Illumina reads. Comparative analyses uncover that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. An in-depth characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For another gene family associated with the innate immune system, the NOD-like receptors (NLRs), we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.\n\nConclusionsThe generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod - but most likely a feature common to all codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization, i.e. the number of NLRs might be underestimates in the different teleost species.

genomics↗

bioRxiv2017

Novel metrics for quantifying bacterial genome composition skews

BackgroundBacterial genomes have characteristic compositional skews, which are differences in nucleotide frequency between the leading and lagging DNA strands across a segment of a genome. It is thought that these strand asymmetries arise as a result of mutational biases and selective constraints, particularly for energy efficiency. Analysis of compositional skews in a diverse set of bacteria provides a comparative context in which mutational and selective environmental constraints can be studied. These analyses typically require finished and well-annotated genomic sequences.\n\nResultsWe present three novel metrics for examining genome composition skews; all three metrics can be computed for unfinished or partially-annotated genomes. The first two metrics, (dot-skew and cross-skew) depend on sequence and gene annotation of a single genome, while the third metric (residual skew) highlights unusual genomes by subtracting a GC content-based model of a library of genome sequences. We applied these metrics to all 7738 available bacterial genomes, including partial drafts, and identified outlier species. A number of these outliers (i.e., Borrelia, Ehrlichia, Kinetoplastibacterium, and Phytoplasma) display similar skew patterns despite only distant phylogenetic relationship. While unrelated, some of the outlier bacterial species share lifestyle characteristics, in particular intracellularity and biosynthetic dependence on their hosts.\n\nConclusionsOur novel metrics appear to reflect the effects of biosynthetic constraints and adaptations to life within one or more hosts on genome composition. We provide results for each analyzed genome, software and interactive visualizations at http://db.systemsbiology.net/gestalt/skew_metrics.

genomics↗

bioRxiv2017

Comprehensive, Integrated, and Phased Whole-Genome Analysis of the Primary ENCODE Cell Line K562

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and Indels (both corrected for CN in aneuploid regions), loss of heterozygosity, mega-base-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs) including small and large-scale complex SVs and non-reference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT. Taking aneuploidy into account, we re-analyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.

genomics↗

bioRxiv2020

Reference genome for the highly transformable Setaria viridis cultivar ME034V

Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis cultivar ME034V is exceptionally transformable, but the lack of a sequenced genome for this cultivar has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50=41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis cultivars. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.

genomics↗