Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Mycobacterium tuberculosis (Mtb) has generated a global health catastrophe that has been compounded by the emergence of drug resistant Mtb strains. We used whole genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent and reactivated disease. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. Our data suggest that Mtb acquires a similar number of chromosomal mutations during latency as occurs during active disease or in a logarithmically growing culture over the same period of time despite reduced bacterial replication during latent infection. The pattern of polymorphisms suggests that the mutational burden in vivo is due to oxidative DNA damage. Thus, we demonstrate that Mtb continues to acquire mutations during latency and provide a novel explanation for the observation that isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of INH resistance1,2.

Two common variants within CFH, the Y402H1–4 and the rs1410996 SNPs5,6, explain 17% of age-related macular degeneration (AMD) liability. However, proof for the involvement of CFH, as opposed to a neighboring transcript, and the potential mechanism of susceptibility alleles are lacking. Assuming that rare functional variants might provide mechanistic insights, we used genotype data and high throughput sequencing to discover a rare high-risk CFH haplotype containing an R1210C mutation. This allele has been implicated previously in atypical hemolytic uremic syndrome, and abrogates C-terminal ligand binding7,8. Genotyping R1210C in 2,423 AMD cases and 1,122 controls demonstrated high penetrance (present in 40 cases versus 1 control, p=7.0×10−6) and six year earlier onset of disease (p=2.3×10−6). This result suggests that loss of function alleles at CFH likely drive AMD risk. This finding represents one of the first instances where a common complex disease variant has led to discovery of a rare penetrant mutation.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of ∼90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

Rationale: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs.Objectives: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance.Methods: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first-and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. Measurements and Main Results:The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci.Conclusions: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes.

To exploit contemporary sequencing technologies for targeted genetic analyses, we developed a hybridization enrichment strategy for DNA capture that uses PCR products as subgenomic traps. We applied this strategy to 115 kb of the human genome encompassing 47 genes implicated in cardiovascular disease. Parallel sequencing of captured subgenomic libraries interrogated 99.8% of targeted nucleotides ≥20 times (~40,000-fold enrichment), enabling sensitive and specific detection of sequence and copy number variation.