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Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (approximately 100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.

Advances in modern sequencing technologies allow us to generate sufficient data to analyze hundreds of bacterial genomes from a single machine in a single day. This potential for sequencing massive numbers of genomes calls for fully automated methods to produce high-quality assemblies and variant calls. We introduce Pilon, a fully automated, all-in-one tool for correcting draft assemblies and calling sequence variants of multiple sizes, including very large insertions and deletions. Pilon works with many types of sequence data, but is particularly strong when supplied with paired end data from two Illumina libraries with small e.g., 180 bp and large e.g., 3-5 Kb inserts. Pilon significantly improves draft genome assemblies by correcting bases, fixing mis-assemblies and filling gaps. For both haploid and diploid genomes, Pilon produces more contiguous genomes with fewer errors, enabling identification of more biologically relevant genes. Furthermore, Pilon identifies small variants with high accuracy as compared to state-of-the-art tools and is unique in its ability to accurately identify large sequence variants including duplications and resolve large insertions. Pilon is being used to improve the assemblies of thousands of new genomes and to identify variants from thousands of clinically relevant bacterial strains. Pilon is freely available as open source software.

While emerging fungi threaten global biodiversity, the paucity of fungal genome assemblies impedes thoroughly characterizing epidemics and developing effective mitigation strategies. Here, we generate de novo genomic assemblies for six outbreaks of the emerging pathogen Batrachochytrium salamandrivorans ( Bsal ). We reveal the European epidemic currently damaging amphibian populations to comprise multiple, highly divergent lineages demonstrating isolate-specific adaptations and metabolic capacities. In particular, we show extensive gene family expansions and acquisitions, through a variety of evolutionary mechanisms, and an isolate-specific saprotrophic lifecycle. This finding both explains the chytrid’s ability to divorce transmission from host density, producing Bsal ’s enigmatic host population declines, and is a key consideration in developing successful mitigation measures. B. salamandrivorans (Bsal) is a fungus of amphibians that has been implicated in the collapse of salamander populations. Here, the authors sequence nine Bsal isolates from six epidemic sites and describe variation in genome size and composition.

Bacterial strains that make up as little as 0.00001% of a microbial community are detected in metagenomic sequencing data. Analyses of metagenomic datasets that are sequenced to a depth of billions or trillions of bases can uncover hundreds of microbial genomes, but naive assembly of these data is computationally intensive, requiring hundreds of gigabytes to terabytes of RAM. We present latent strain analysis (LSA), a scalable, de novo pre-assembly method that separates reads into biologically informed partitions and thereby enables assembly of individual genomes. LSA is implemented with a streaming calculation of unobserved variables that we call eigengenomes. Eigengenomes reflect covariance in the abundance of short, fixed-length sequences, or k -mers. As the abundance of each genome in a sample is reflected in the abundance of each k -mer in that genome, eigengenome analysis can be used to partition reads from different genomes. This partitioning can be done in fixed memory using tens of gigabytes of RAM, which makes assembly and downstream analyses of terabytes of data feasible on commodity hardware. Using LSA, we assemble partial and near-complete genomes of bacterial taxa present at relative abundances as low as 0.00001%. We also show that LSA is sensitive enough to separate reads from several strains of the same species.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae. There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by bla KPC2, bla KPC3, and bla KPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution.

The gut mycobiota is crucial for intestinal homeostasis and immune function 1 . Yet its variability and inconsistent fungal colonization of laboratory mice hinders the study of the evolutionary and immune processes that underpin commensalism 2 , 3 . Here, we show that Kazachstania pintolopesii is a fungal commensal in wild urban and rural mice, with an exceptional ability to colonize the mouse gastrointestinal tract and dominate the gut mycobiome. Kazachstania pintolopesii colonization occurs in a bacteria-independent manner, results in enhanced colonization resistance to other fungi and is shielded from host immune surveillance, allowing commensal presence. Following changes in the mucosal environment, K. pintolopesii colonization triggers a type 2 immune response in mice and induces gastrointestinal eosinophilia. Mechanistically, we determined that K. pintolopesii activates type 2 immunity via the induction of epithelial IL-33 and downstream IL-33–ST2 signalling during mucus fluctuations. Kazachstania pintolopesii -induced type 2 immunity enhanced resistance to helminth infections or aggravated gastrointestinal allergy in a context-dependent manner. Our findings indicate that K. pintolopesii is a mouse commensal and serves as a valuable model organism for studying gut fungal commensalism and immunity in its native host. Its unnoticed presence in mouse facilities highlights the need to evaluate its influence on experimental outcomes and phenotypes. Kazachstania pintolopesii is a highly prevalent fungal symbiont that can trigger type 2 immunity and influence the composition of the gut mycobiome, as well as immune and disease phenotypes.

Alexander Pym, Ashlee Earl and colleagues use the whole-genome sequences from 498 strains of Mycobacterium tuberculosis to identify new genotypes conferring resistance to antitubercular drugs. They find that loss-of-function mutations in ald (Rv2780), encoding L -alanine dehydrogenase, are associated with unexplained drug resistance and demonstrate that these mutations confer resistance to D -cycloserine. A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D -cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L -alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D -cycloserine, and susceptibility to the drug was partially restored by complementation of ald . Clinical strains with mutations in ald and alr exhibited increased resistance to D -cycloserine when cultured in vitro . Incorporation of D -cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.

Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe. Histoplasma capsulatum , a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent hosts. Histoplasma is endemic in the Ohio and Mississippi River Valleys in the United States and is also distributed worldwide. Previous studies have revealed at least eight clades, each specific to a geographic location: North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B), Eurasian, Netherlands, Australian and African, and an additional distinct lineage (H81) comprised of Panamanian isolates. Previously assembled Histoplasma genomes are highly fragmented, with the highly repetitive G217B (NAm 2) strain, which has been used for most whole-genome-scale transcriptome studies, assembled into over 250 contigs. In this study, we set out to fully assemble the repeat regions and characterize the large-scale genome architecture of Histoplasma species. We resequenced five Histoplasma strains (WU24 [NAm 1], G217B [NAm 2], H88 [African], G186AR [Panama], and G184AR [Panama]) using Oxford Nanopore Technologies long-read sequencing technology. Here, we report chromosomal-level assemblies for all five strains, which exhibit extensive synteny among the geographically distant Histoplasma isolates. The new assemblies revealed that RYP2 , a major regulator of morphology and virulence, is duplicated in G186AR. In addition, we mapped previously generated transcriptome data sets onto the newly assembled chromosomes. Our analyses revealed that the expression of transposons and transposon-embedded genes are upregulated in yeast phase compared to mycelial phase in the G217B and H88 strains. This study provides an important resource for fungal researchers and further highlights the importance of chromosomal-level assemblies in analyzing high-throughput data sets. IMPORTANCE Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe. Phylogenetically distinct species of Histoplasma have been isolated and sequenced. However, for the commonly used strains, genome assemblies have been fragmented, leading to underutilization of genome-scale data. This study provides chromosome-level assemblies of the commonly used Histoplasma strains using long-read sequencing technology. Comparative analysis of these genomes shows largely conserved gene order within the chromosomes. Mapping existing transcriptome data on these new assemblies reveals clustering of transcriptionally coregulated genes. The results of this study highlight the importance of obtaining chromosome-level assemblies in understanding the biology of human fungal pathogens.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella . Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion–fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus . Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.