Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis

Sequencing targeted DNA regions in large samples is necessary to discover the full spectrum of rare variants. We report an effective Illumina sequencing strategy utilizing pooled samples with novel quality (Srfim) and filtering (SERVIC4E) algorithms. We sequenced 24 exons in two cohorts of 480 samples each, identifying 47 coding variants, including 30 present once per cohort. Validation by Sanger sequencing revealed an excellent combination of sensitivity and specificity for variant detection in pooled samples of both cohorts as compared to publicly available algorithms

Centroacinar cells are progenitors that contribute to endocrine pancreas regeneration

Diabetes is associated with a paucity of insulin-producing β-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in β-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after β-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both β-cell neogenesis and β-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis

Cellular Resolution Maps of X Chromosome Inactivation: Implications for Neural Development, Function, and Disease

SummaryFemale eutherian mammals use X chromosome inactivation (XCI) to epigenetically regulate gene expression from ∼4% of the genome. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters—GFP on one X chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers, we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left versus right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals

Establishing the baseline level of repetitive element expression in the human cortex

Background: Although nearly half of the human genome is comprised of repetitive sequences, the expression profile of these elements remains largely uncharacterized. Recently developed high throughput sequencing technologies provide us with a powerful new set of tools to study repeat elements. Hence, we performed whole transcriptome sequencing to investigate the expression of repetitive elements in human frontal cortex using postmortem tissue obtained from the Stanley Medical Research Institute. Results: We found a significant amount of reads from the human frontal cortex originate from repeat elements. We also noticed that Alu elements were expressed at levels higher than expected by random or background transcription. In contrast, L1 elements were expressed at lower than expected amounts. Conclusions: Repetitive elements are expressed abundantly in the human brain. This expression pattern appears to be element specific and can not be explained by random or background transcription. These results demonstrate that our knowledge about repetitive elements is far from complete. Further characterization is required to determine the mechanism, the control, and the effects of repeat element expression

Extensive somatic L1 retrotransposition in colorectal tumors

L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5′ and 3′ junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5′ truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer

Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-Poly(A) Termination Pathway: Nrd1, Nab3, and Sen1

RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination

Short-term inhibition of 11β-hydroxysteroid dehydrogenase type 1 reversibly improves spatial memory but persistently impairs contextual fear memory in aged mice

AbstractHigh glucocorticoid levels induced by stress enhance the memory of fearful events and may contribute to the development of anxiety and posttraumatic stress disorder. In contrast, elevated glucocorticoids associated with ageing impair spatial memory. We have previously shown that pharmacological inhibition of the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) improves spatial memory in aged mice. However, it is not known whether inhibition of 11β-HSD1 will have any beneficial effects on contextual fear memories in aged mice. Here, we examined the effects of UE2316, a selective 11β-HSD1 inhibitor which accesses the brain, on both spatial and contextual fear memories in aged mice using a vehicle-controlled crossover study design.Short-term UE2316 treatment improved spatial memory in aged mice, an effect which was reversed when UE2316 was substituted with vehicle. In contrast, contextual fear memory induced by foot-shock conditioning was significantly reduced by UE2316 in a non-reversible manner. When the order of treatment was reversed following extinction of the original fear memory, and a second foot-shock conditioning was given in a novel context, UE2316 treated aged mice (previously on vehicle) now showed increased fear memory compared to vehicle-treated aged mice (previously on UE2316). Renewal of the original extinguished fear memory triggered by exposure to a new environmental context may explain these effects. Thus 11β-HSD1 inhibition reverses spatial memory impairments with ageing while reducing the strength and persistence of new contextual fear memories. Potentially this could help prevent anxiety-related disorders in vulnerable elderly individuals

Inverting the model of genomics data sharing with the NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space

The NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL; https://anvilproject.org) was developed to address a widespread community need for a unified computing environment for genomics data storage, management, and analysis. In this perspective, we present AnVIL, describe its ecosystem and interoperability with other platforms, and highlight how this platform and associated initiatives contribute to improved genomic data sharing efforts. The AnVIL is a federated cloud platform designed to manage and store genomics and related data, enable population-scale analysis, and facilitate collaboration through the sharing of data, code, and analysis results. By inverting the traditional model of data sharing, the AnVIL eliminates the need for data movement while also adding security measures for active threat detection and monitoring and provides scalable, shared computing resources for any researcher. We describe the core data management and analysis components of the AnVIL, which currently consists of Terra, Gen3, Galaxy, RStudio/Bioconductor, Dockstore, and Jupyter, and describe several flagship genomics datasets available within the AnVIL. We continue to extend and innovate the AnVIL ecosystem by implementing new capabilities, including mechanisms for interoperability and responsible data sharing, while streamlining access management. The AnVIL opens many new opportunities for analysis, collaboration, and data sharing that are needed to drive research and to make discoveries through the joint analysis of hundreds of thousands to millions of genomes along with associated clinical and molecular data types

Spidey: A Tool for mRNA-to-Genomic Alignments

We have developed a computer program that aligns spliced sequences to genomic sequences, using local alignment algorithms and heuristics to put together a global spliced alignment. Spidey can produce reliable alignments quickly, even when confronted with noise from alternative splicing, polymorphisms, sequencing errors, or evolutionary divergence. We show how Spidey was used to align reference sequences to known genomic sequences and then to the draft human genome, to align mRNAs to gene clusters, and to align mouse mRNAs to human genomic sequence. We compared Spidey to two other spliced alignment programs; Spidey generally performed quite well in a very reasonable amount of time

GeneDesign: Rapid, automated design of multikilobase synthetic genes

Modern molecular biology has brought many new tools to the geneticist as well as an exponentially expanding database of genomes and new genes for study. Of particular use in the analysis of these genes is the synthetic gene, a nucleotide sequence designed to the specifications of the investigator. Typically, synthetic genes encode the same product as the gene of interest, but the synthetic nucleotide sequence for that protein may contain modifications affecting expression or base composition. Other desirable changes typically involve the revision of restriction sites. Designing synthetic genes by hand is a time-consuming and error-prone process that may involve several computer programs. We have developed a tools environment that combines many modules to provide a platform for rapid synthetic gene design for multikilobase sequences. We have used GeneDesign to successfully design a synthetic Ty1 element and a large variety of other synthetic sequences. GeneDesign has been implemented as a publicly accessible Web-based resource and can be found at http://slam.bs.jhmi.edu/gd