The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Prebiotic Treatment in People With Schizophrenia.

BACKGROUND: Preliminary evidence suggests that people with schizophrenia have decreased relative abundance of butyrate-producing bacteria in the gut microbiota. Butyrate plays a critical role in maintaining the integrity of the gut-blood barrier and has a number of anti-inflammatory effects. This proof-of-concept study was designed to assess whether the addition of the oligofructose-enriched inulin (OEI) prebiotic: Prebiotin could increase the production of butyrate. METHODS: Twenty-seven people who met the criteria for either Diagnostic and Statistical Manual of Mental Disorders, 5th Edition, schizophrenia or schizoaffective disorder were entered into a 10-day, double-blind, placebo-controlled, randomized clinical trial. The study was conducted on an inpatient unit to standardize the participant diet and environment. Participants were randomized to either OEI (4 g, 3 times a day) or a placebo (4 g of maltodextrin, 3 times a day). In order to assess the effect of OEI treatment on butyrate levels, participants underwent pretreatment and posttreatment OEI challenges. The primary outcome measure was relative change in postchallenge plasma butyrate levels after 10 days of OEI treatment. RESULTS: In both the intent-to-treat and completer analyses, OEI treatment was associated with a greater number of participants who met the OEI challenge responder criteria than those treated with placebo. OEI treatment was also associated with an increase in baseline butyrate levels (effect size for the group difference in the change of baseline butyrate levels was 0.58). CONCLUSIONS: We were able to demonstrate that treatment with the prebiotic OEI selectively increased the level of plasma butyrate in people with schizophrenia.Trial registration:ClinicalTrials.gov identifier NCT03617783.

Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.

LgDel mice, which model the heterozygous deletion of genes at human chromosome 22q11.2 associated with DiGeorge/22q11.2 deletion syndrome (22q11DS), have cranial nerve and craniofacial dysfunction as well as disrupted suckling, feeding and swallowing, similar to key 22q11DS phenotypes. Divergent trigeminal nerve (CN V) differentiation and altered trigeminal ganglion (CNgV) cellular composition prefigure these disruptions in LgDel embryos. We therefore asked whether a distinct transcriptional state in a specific population of early differentiating LgDel cranial sensory neurons, those in CNgV, a major source of innervation for appropriate oropharyngeal function, underlies this departure from typical development. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior-posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17 000 genes expressed in common in LgDel versus WT CNgV. Thus, quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice.

Changes in the Gut Microbiota Following Bariatric Surgery Are Associated with Increased Alcohol Intake in a Female Rat Model.

AIMS: We aimed to investigate if differences in gut microbiota diversity and composition are associated with post-operative alcohol intake following bariatric surgery in a rat model. METHODS: Twenty-four female rats were randomized to three treatment groups: sham surgery, vertical sleeve gastrectomy (VSG) or Roux-en-Y gastric bypass (RYGB). Stool was collected pre- and post-operatively and 16S rRNA gene amplification and sequencing was performed. Analysis focused on correlating microbial diversity, type of surgery and alcohol (EtOH) intake. RESULTS: Pre-operative stools samples on regular diet showed similar taxonomic composition and Shannon diversity among the three treatment groups. There was a significant decrease in Shannon diversity and a change in taxonomic composition of the gut microbiota after rats was fed high fat diet. Post-operatively, the RYGB group showed significantly lower taxonomic diversity than the VSG and sham groups, while the VSG and sham groups diversity were not significantly different. Taxonomic composition and function prediction based on PICRUSt analysis showed the RYGB group to be distinct from the VSG and sham groups. Shannon diversity was found to be negatively associated with EtOH intake. CONCLUSIONS: Changes in the taxonomic profile of the gut microbiota following bariatric surgery, particularly RYGB, are associated with increased EtOH intake and may contribute to increased alcohol use disorder risk through the gut-brain-microbiome axis.

Establishing What Constitutes a Healthy Human Gut Microbiome: State of the Science, Regulatory Considerations, and Future Directions.

On December 17, 2018, the North American branch of the International Life Sciences Institute (ILSI North America) convened a workshop "Can We Begin to Define a Healthy Gut Microbiome Through Quantifiable Characteristics?" with >40 invited academic, government, and industry experts in Washington, DC. The workshop objectives were to 1) develop a collective expert assessment of the state of the evidence on the human gut microbiome and associated human health benefits, 2) see if there was sufficient evidence to establish measurable gut microbiome characteristics that could serve as indicators of "health," 3) identify short- and long-term research needs to fully characterize healthy gut microbiome-host relationships, and 4) publish the findings. Conclusions were as follows: 1) mechanistic links of specific changes in gut microbiome structure with function or markers of human health are not yet established; 2) it is not established if dysbiosis is a cause, consequence, or both of changes in human gut epithelial function and disease; 3) microbiome communities are highly individualized, show a high degree of interindividual variation to perturbation, and tend to be stable over years; 4) the complexity of microbiome-host interactions requires a comprehensive, multidisciplinary research agenda to elucidate relationships between gut microbiome and host health; 5) biomarkers and/or surrogate indicators of host function and pathogenic processes based on the microbiome need to be determined and validated, along with normal ranges, using approaches similar to those used to establish biomarkers and/or surrogate indicators based on host metabolic phenotypes; 6) future studies measuring responses to an exposure or intervention need to combine validated microbiome-related biomarkers and/or surrogate indicators with multiomics characterization of the microbiome; and 7) because static genetic sampling misses important short- and long-term microbiome-related dynamic changes to host health, future studies must be powered to account for inter- and intraindividual variation and should use repeated measures within individuals.

A Distinct Gut Microbiota Exists Within Crohn's Disease-Related Perianal Fistulae.

BACKGROUND: Gut bacteria are strongly suspected to play a key role in the pathogenesis of Crohn's disease (CD). Studies have demonstrated alterations in the gut microbiota in this patient population. The purpose of this study was to characterize the gut microbiota of fistulizing perianal CD. MATERIALS AND METHODS: Stool and fistula samples were obtained from patients undergoing surgery for CD-related anorectal fistulae. Microbial compositions of matched stool and fistula samples were characterized using 16S rRNA gene profiling. The effect of sample type, patient gender, disease classification (Montreal A/B), disease activity (Harvey Bradshaw Index), antibiotic use, and presence of active proctitis on microbial composition was assessed. RESULTS: Samples were obtained from 18 patients. Bacteroides was the most abundant genera across all samples collected, followed by Streptococcus and Bifidobacterium. Bifidobacterium was present at significantly higher levels in fecal samples than fistula samples, whereas Achromobacter and Corynebacterium were present at significantly higher levels in fistula samples. Antibiotic, but not thiopurine or antitumor necrosis factor medication, exposure affected the gut microbial composition. Patient gender, disease classification, disease activity, and presence of active proctitis did not alter stool or fistula microbiota. CONCLUSIONS: Our data show that the gut microbiota within CD-related anorectal fistulae is distinct from that in stool samples obtained from the same patients. We also observe a dysbiosis in patients treated with antibiotics compared with those not treated with antibiotics.

Primordial origin and diversification of plasmids in Lyme disease agent bacteria.

BACKGROUND: With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species. RESULTS: We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types. CONCLUSIONS: The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

New signaling pathways govern the host response to C. albicans infection in various niches.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.

Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

BACKGROUND: Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. RESULTS: We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). CONCLUSIONS: Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

CloVR-Comparative: automated, cloud-enabled comparative microbial genome sequence analysis pipeline.

BACKGROUND: The benefit of increasing genomic sequence data to the scientific community depends on easy-to-use, scalable bioinformatics support. CloVR-Comparative combines commonly used bioinformatics tools into an intuitive, automated, and cloud-enabled analysis pipeline for comparative microbial genomics. RESULTS: CloVR-Comparative runs on annotated complete or draft genome sequences that are uploaded by the user or selected via a taxonomic tree-based user interface and downloaded from NCBI. CloVR-Comparative runs reference-free multiple whole-genome alignments to determine unique, shared and core coding sequences (CDSs) and single nucleotide polymorphisms (SNPs). Output includes short summary reports and detailed text-based results files, graphical visualizations (phylogenetic trees, circular figures), and a database file linked to the Sybil comparative genome browser. Data up- and download, pipeline configuration and monitoring, and access to Sybil are managed through CloVR-Comparative web interface. CloVR-Comparative and Sybil are distributed as part of the CloVR virtual appliance, which runs on local computers or the Amazon EC2 cloud. Representative datasets (e.g. 40 draft and complete Escherichia coli genomes) are processed in <36 h on a local desktop or at a cost of <$20 on EC2. CONCLUSIONS: CloVR-Comparative allows anybody with Internet access to run comparative genomics projects, while eliminating the need for on-site computational resources and expertise.

Pulmonary Nontuberculous Mycobacterial Infection. A Multisystem, Multigenic Disease.

RATIONALE: The clinical features of patients infected with pulmonary nontuberculous mycobacteria (PNTM) are well described, but the genetic components of infection susceptibility are not. OBJECTIVES: To examine genetic variants in patients with PNTM, their unaffected family members, and a control group. METHODS: Whole-exome sequencing was done on 69 white patients with PNTM and 18 of their white unaffected family members. We performed a candidate gene analysis using immune, cystic fibrosis transmembrance conductance regulator (CFTR), cilia, and connective tissue gene sets. The numbers of patients, family members, and control subjects with variants in each category were compared, as was the average number of variants per person. MEASUREMENTS AND MAIN RESULTS: A significantly higher number of patients with PNTM than the other subjects had low-frequency, protein-affecting variants in immune, CFTR, cilia, and connective tissue categories (35, 26, 90, and 90%, respectively). Patients with PNTM also had significantly more cilia and connective tissue variants per person than did control subjects (2.47 and 2.55 compared with 1.38 and 1.40, respectively; P = 1.4 × 10(-6) and P = 2.7 × 10(-8), respectively). Patients with PNTM had an average of 5.26 variants across all categories (1.98 in control subjects; P = 2.8 × 10(-17)), and they were more likely than control subjects to have variants in multiple categories. We observed similar results for family members without PNTM infection, with the exception of the immune category. CONCLUSIONS: Patients with PNTM have more low-frequency, protein-affecting variants in immune, CFTR, cilia, and connective tissue genes than their unaffected family members and control subjects. We propose that PNTM infection is a multigenic disease in which combinations of variants across gene categories, plus environmental exposures, increase susceptibility to the infection.

Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli.

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin (stx)-encoding phage, which are labeled Shiga toxin-producing E. coli; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus (bfp) genes. All other LEE+/bfp-/stx- isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+/stx+/bfp- genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE+/bfp+/stx- AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE+ isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.

Defining the phylogenomics of Shigella species: a pathway to diagnostics.

Shigellae cause significant diarrheal disease and mortality in humans, as there are approximately 163 million episodes of shigellosis and 1.1 million deaths annually. While significant strides have been made in the understanding of the pathogenesis, few studies on the genomic content of the Shigella species have been completed. The goal of this study was to characterize the genomic diversity of Shigella species through sequencing of 55 isolates representing members of each of the four Shigella species: S. flexneri, S. sonnei, S. boydii, and S. dysenteriae. Phylogeny inferred from 336 available Shigella and Escherichia coli genomes defined exclusive clades of Shigella; conserved genomic markers that can identify each clade were then identified. PCR assays were developed for each clade-specific marker, which was combined with an amplicon for the conserved Shigella invasion antigen, IpaH3, into a multiplex PCR assay. This assay demonstrated high specificity, correctly identifying 218 of 221 presumptive Shigella isolates, and sensitivity, by not identifying any of 151 diverse E. coli isolates incorrectly as Shigella. This new phylogenomics-based PCR assay represents a valuable tool for rapid typing of uncharacterized Shigella isolates and provides a framework that can be utilized for the identification of novel genomic markers from genomic data.

Genetic variation in vitro and in vivo of an attenuated Lassa vaccine candidate.

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

BorreliaBase: a phylogeny-centered browser of Borrelia genomes.

BACKGROUND: The bacterial genus Borrelia (phylum Spirochaetes) consists of two groups of pathogens represented respectively by B. burgdorferi, the agent of Lyme borreliosis, and B. hermsii, the agent of tick-borne relapsing fever. The number of publicly available Borrelia genomic sequences is growing rapidly with the discovery and sequencing of Borrelia strains worldwide. There is however a lack of dedicated online databases to facilitate comparative analyses of Borrelia genomes. DESCRIPTION: We have developed BorreliaBase, an online database for comparative browsing of Borrelia genomes. The database is currently populated with sequences from 35 genomes of eight Lyme-borreliosis (LB) group Borrelia species and 7 Relapsing-fever (RF) group Borrelia species. Distinct from genome repositories and aggregator databases, BorreliaBase serves manually curated comparative-genomic data including genome-based phylogeny, genome synteny, and sequence alignments of orthologous genes and intergenic spacers. CONCLUSIONS: With a genome phylogeny at its center, BorreliaBase allows online identification of hypervariable lipoprotein genes, potential regulatory elements, and recombination footprints by providing evolution-based expectations of sequence variability at each genomic locus. The phylo-centric design of BorreliaBase (http://borreliabase.org) is a novel model for interactive browsing and comparative analysis of bacterial genomes online.

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis.

BACKGROUND: More than 20% of the world's population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries. RESULTS: Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts. CONCLUSIONS: The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

High-level relatedness among Mycobacterium abscessus subsp. massiliense strains from widely separated outbreaks.

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.