Metagenome sequencing enables the genetic characterization of complex microbial communities. However, determining the activity of isolates within a community presents several challenges, including the wide range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many sites. To address these limitations, we developed "targeted expression analysis sequencing" or TEAL-seq, enabling sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools. For proof of concept, we targeted about 1,700 core and accessory genes of Staphylococcus aureus and S. epidermidis, two key species of the skin microbiome. Two targeting methods were applied to laboratory cultures and human nasal swab specimens. Both methods showed a high degree of specificity, with >90% reads on target, even in the presence of complex microbial or human background DNA/RNA. Targeting using molecular inversion probes demonstrated excellent correlation in inferred expression levels with bulk RNA-seq. Furthermore, we show that a linear pre-amplification step to increase the number of nucleic acids for analysis yielded consistent and predictable results when applied to complex samples and enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling, enables detection across a greater dynamic range, and uses a strategy that is readily configurable for determining the transcriptional status of organisms in any microbial community.IMPORTANCEThe gene expression patterns of bacteria in microbial communities reflect their activity and interactions with other community members. Measuring gene expression in complex microbiome contexts is challenging, however, due to the large dynamic range of microbial abundances and transcript levels. Here we describe an approach to assessing gene expression for specific species of interest using highly multiplexed pools of targeting probes. We show that an isothermal amplification step enables the profiling of low biomass samples. TEAL-seq should be widely adaptable to the study of microbial activity in natural environments.

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes observed in this individual were global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, failure to thrive, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical phenotypic spectrum.

SUMMARYP21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelicPAK2variants cause Knobloch syndrome type 2 (KNO2)—a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novelde novoheterozygous missense variant inPAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging byin silicoanalysis. Previous clinical genetic testing did not report this variant due to unknown relevance ofPAK2variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy ofPAK2variants in KNO2 and expand the KNO2 clinical spectrum.

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.

Abstract Background Truvian has developed a fully automated blood testing analyzer (TruAnalyzer) capable of running a panel of clinical chemistry, immunoassay and hematology assays from a low-volume blood sample. Truvian's compact instrument was designed to deliver a convenient and affordable alternative to off-site traditional labs, providing lab-accurate rapid insights from a single, small sample of blood with no upfront pre-processing required to inform health decisions at the point of care. Here, we evaluate the precision and accuracy of a comprehensive wellness panel tested on the TruAnalyzer and compare its performance to FDA cleared devices. Methods TruAnalyzer performance was assessed for both precision and accuracy. For the precision studies, two commercially available controls (one for plasma-based assays and one for blood-based assays) were added to the Truvian consumables and loaded into the TruAnalyzer. Six replicates were run on each of the ten instruments (N = 60). Coefficient of variance (CV) analysis was performed and compared against pre-established requirements. For method comparison studies, 50 unique donors were consented and enrolled in an IRB approved study and 68 donor samples were collected over a 3-day period. For each donor, three 4-mL tubes of blood were drawn: 1) a Lithium Heparin (LiHep) sample for the TruAnalyzer clinical chemistry and hematology panel, 2) a LiHep gel tube for the Roche Cobas c311 clinical chemistry assays, and 3) an EDTA sample for the Sysmex PocH-100i complete blood count with 3-part differential. All samples were processed within 1 hour of collection. Using only 300 µL sample volume, the TruAnalyzer reported results for 23 analytes including CMP (no electrolyes), Lipid, HbA1c, and CBC with 3-part differential. Concordance to comparator devices was determined by Bland-Altman analysis. Results For the precision study, 13 out of 14 (93%) chemistry analytes had a between-system CV of ≤5.8%. For hematology, 5 out of 5 (100%) analytes had a between-system CV of ≤6%. For the method comparison study, RBC counts showed 98% of samples tested being within 6% of comparator values. WBC and platelet counts had 97% of samples tested being within 10% and 15% of comparator values respectively. 95% of hematocrit samples were within 6% of comparator values and 98% of hemoglobin samples were within 7% of comparator values. All hematology analytes showed equivalency to Sysmex PocH-100i as determined by Bland-Altman equivalency analysis. For clinical chemistry analytes, 14 out of 15 (93%) analytes showed equivalency to Roche Cobas c311 as determined by Bland-Altman equivalency analysis when CLIA ATE guidelines were applied. 7 out of 15 clinical chemistry analytes had ≥90% of results within ATE. 5 out of 15 clinical chemistry analytes had ≥80% of results within ATE. Conclusion The Truvian blood testing analyzer is the first multi-modality system capable of running hematology, clinical chemistry and immunoassays from a single, low-volume blood sample. Results from this study demonstrate good precision for the majority of panel analytes and excellent concordance to FDA cleared comparators for assays evaluated in this study.

The determination of the complete nucleotide sequence of the Human Genome is a goal that has captivated both scientists and the lay public for nearly a decade. We are rapidly progressing toward the determination of that sequence and the world-wide effort to sequence the human genome has set 2005 as the target date for the completion of that goal. The promise of the Genome Project has been that, as the sequence unfolds, we will be able to obtain a catalog of all human genes, as well as to begin investigation of the large-scale structure and organization of the genome. Comparisons within and between species promise to provide substantial information about the function of many genes. Knowledge of the sequence of the human genome will greatly enhance our ability to detect sequence variation among individuals. This, in turn, will be invaluable for identifying genes underlying human diseases, as sequence variation provides the necessary link between genes and phenotypes. Knowledge of the gene sequences alone, however, provides only part of the puzzle. The challenge for the future is to identify those genes that are responsible for the development of a specific cellular or organismal phenotype. Scientists at The Institute for Genomic Research have been leaders in genomic research, including both Expressed Sequence Tag and genomic sequencing, and are building a program that we hope will define the manner in which future researchers will address questions of gene function using genomic techniques.

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (R t ) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the R t of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%???80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant???s respective emergence period, finding that Delta emerged 1.37???2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%???167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1???10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta???s enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability.

Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. Methods Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. Results The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. Conclusions The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P . aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. Methods Twenty-two sequential P . aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. Results The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. Conclusions The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P . aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta's infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta's enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Ceftazidime (CAZ)-avibactam (AVI) is a β-lactam/β-lactamase inhibitor combination with activity against type A and type C β-lactamases. Resistance emergence has been seen, with multiple mechanisms accounting for the resistance. We performed four experiments in the dynamic hollow-fiber infection model, delineating the linkage between drug exposure and both the rate of bacterial kill and resistance emergence by all mechanisms.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g., 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. This approach, termed MA-GenTA (microbial abundances from genome tagged analysis), enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria present in mouse stool specimens. Comparison of the MA-GenTA data with mWGS data demonstrated excellent correlation down to 0.01% relative abundance and a similar number of organisms detected per sample. Despite the incompleteness of the reference database, nonmetric multidimensional scaling (NMDS) clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS, and 16S rRNA data sets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes.IMPORTANCE New methods for profiling the microbial communities can create new approaches to understanding the composition and function of those communities. In this study, we combined bacterial genome-specific probe design with a highly multiplexed single primer extension reaction as a new method to profile microbial communities, using stool from various mouse strains as a test case. This method, termed MA-GenTA, was benchmarked against 16S rRNA gene sequencing and metagenome sequencing methods and delivered similar relative abundance and clustering data. Since the probes were generated from reference genomes, MA-GenTA was also able to provide functional pathway data for the stool microbiome in the assayed samples. The method is more informative than 16S rRNA analysis while being less costly than metagenome shotgun sequencing.

Animal models play a critical role in establishing causal relationships between gut microbiota and disease. The laboratory mouse is widely used to study the role of microbes in various disorders; however, differences between mouse vendors, genetic lineages and husbandry protocols have been shown to contribute to variation in phenotypes and to non-reproducibility of experimental results. We sought to understand how gut microbiome profiles of mice vary by vendor, vendor production facility and health status upon receipt into an academic facility and how they change over 12 weeks in the new environment. C57BL/6 mice were sourced from two different production sites for each of three different vendors. Mice were shipped to an academic research vivarium, and fresh-catch stool samples were collected from mice immediately from the shipping box upon receipt, and again after 2, 6 and 12 weeks in the new facility. Substantial variation in bacterial proportional abundance was observed among mice from each vendor at the time of receipt, but shared microbes accounted for most sequence reads. Vendor-specific microbes were generally of low abundance. Microbial profiles of mice from all vendors exhibited shifts over time, highlighting the importance of environmental conditions on microbial dynamics. Our results emphasize the need for continued efforts to account for sources of variation in animal models and understand how they contribute to experimental reproducibility.

An increasing number of untreatable infections are recorded every year. Many studies have focused their efforts on developing new β-lactamase inhibitors to treat multi-drug resistant (MDR) isolates. In the present study, sulbactam/avibactam and sulbactam/relebactam combination were tested against 187 multi-drug resistant (MDR) Acinetobacter clinical isolates; both sulbactam/avibactam and sulbactam/relebactam restored sulbactam activity. A decrease ≥2 dilutions in sulbactam MICs was observed in 89% of the isolates when tested in combination with avibactam. Sulbactam/relebactam was able to restore sulbactam susceptibility in 40% of the isolates. In addition, the susceptibility testing using twenty-three A. baumannii AB5075 knockout strains revealed potential sulbactam and/or sulbactam/avibactam target genes. We observed that diazabicyclooctanes (DBOs) β-lactamase inhibitors combined with sulbactam restore sulbactam susceptibility against carbapenem-resistant Acinetobacter clinical isolates. However, relebactam was not as effective as avibactam when combined with sulbactam. Exploring novel combinations may offer new options to treat Acinetobacter spp. infections, especially for widespread oxacillinases and metallo-β-lactamases (MBLs) producers.

Objectives: To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. Methods: Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. Results: Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8kb, bounded by IS26 at both ends, and embedded in a new target location between an alpha/beta-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. Conclusions: A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying bla(NDM-1). Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. bla(NDM-1) was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying bla(PER-7) and other resistance determinants was found on a plasmid in some A. baumannii strains.