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We report recent progress on the ongoing NuSTAR observational campaign of 8 TeV-detected pulsar wind nebulae (PWNe). This campaign constitutes a major part of our NuSTAR study of some of the most energetic TeV sources in our galaxy detected by VERITAS and HAWC. NuSTAR is the first focusing X-ray telescope operating above 10 keV in space with sub-arcminute angular resolution. Broad-band X-ray imaging and spectroscopy data, obtained by NuSTAR, allow us to probe sub-PeV electron populations through detecting synchrotron X-ray radiation. Our targets include PeVatron candidates detected by HAWC, the Boomerang nebula, PWNe crushed by supernova remnant shocks (or relic PWNe), and G0.9+0.1 in the Galactic Center. Using Fermi-LAT data and available TeV data, we aim to provide a complete, multi-wavelength view of a diverse class of middle-aged (~10-100 kyrs old) PWNe. Our NuSTAR analysis detected hard X-ray emission from the Eel and Boomerang PWNe and characterized their broad-band X-ray spectra most accurately. We plan to apply both time-evolution and multi-zone PWN models to multi-wavelength spectral energy distribution (SED) data over the radio, X-ray, GeV, and TeV bands. In this proceeding, we will review our observational campaign and discuss the preliminary results for some PWNe.

Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Background Orang-utans comprise three critically endangered species endemic to the islands of Borneo and Sumatra. Though whole-genome sequencing has recently accelerated our understanding of their evolutionary history, the costs of implementing routine genome screening and diagnostics remain prohibitive. Capitalizing on a tri-fold locus discovery approach, combining data from published whole-genome sequences, novel whole-exome sequencing, and microarray-derived genotype data, we aimed to develop a highly informative gene-focused panel of targets that can be used to address a broad range of research questions. Results We identified and present genomic co-ordinates for 175,186 SNPs and 2315 Y-chromosomal targets, plus 185 genes either known or presumed to be pathogenic in cardiovascular (N = 109) or respiratory (N = 43) diseases in humans – the primary and secondary causes of captive orang-utan mortality – or a majority of other human diseases (N = 33). As proof of concept, we designed and synthesized ‘SeqCap’ hybrid capture probes for these targets, demonstrating cost-effective target enrichment and reduced-representation sequencing. Conclusions Our targets are of broad utility in studies of orang-utan ancestry, admixture and disease susceptibility and aetiology, and thus are of value in addressing questions key to the survival of these species. To facilitate comparative analyses, these targets could now be standardized for future orang-utan population genomic studies. The targets are broadly compatible with commercial target enrichment platforms and can be utilized as published here to synthesize applicable probes.

International audience; Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias isinherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mmbiopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB butlow clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

While hundreds of thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g. 6mm punch) from a single spatial location, which becomes grossly under-powered as tumor volume scales. Indeed our analysis of current clinical and research practice shows that existing protocols sample from only 0.0005% to 2.0% of the total tumor mass, raising the prospect of considerable sampling bias. Failure to address this bias risks undermining the clinical utility of genomic medicine in cancer; through lack of sensitivity to detect actionable mutations, miss-assignment of subclonal variants as clonal, and unreliable estimate of tumor mutational burden (TMB). Here we demonstrate Representative Sequencing (Rep-Seq), as a novel method to achieve unbiased sampling of solid tumor tissue. The Rep-Seq protocol comprises homogenization of all residual tumor material not taken for pathology into a well-mixed solution, coupled with next generation sequencing. Rep-Seq was implemented on a proof of concept basis, and benchmarked against current methods across multiple solid tumor types including renal cell carcinoma, lung cancer, melanoma, breast and colorectal cancer. Analysis of intra-tumor TMB variability showed a high level of misclassification with current single biopsy methods, with 20% of lung tumors, and 52% of bladder tumors, having ≥1 biopsy with high-TMB, but low clonal TMB overall (based on 10.0 mutations/Mb threshold for immune checkpoint inhibitor therapy (CPI)). Misclassification rates by contrast were reduced to 2% (lung) and 4% (bladder) when a more representative sampling frame was used. Clonal clustering analysis revealed rapid convergence of cancer cell fraction estimates in Rep-Seq towards true values, as validated in >60 biopsies, and >5ctDNA samples, taken from a single tumor. As a consequence 100% of variants were correctly classified as clonal by Rep-Seq, compared to ~85% in single biopsy sequencing. In terms of subclonal mutation detection, Rep-Seq achieved a greater sensitivity to detect variants, as compared to single-biopsy sequencing at equivalent depth (p=6.6x10-11). Finally, in a rapid autopsy setting Rep-Seq was able to accurately reconstruct the clonal phylogeny of advanced stage disease, correctly identifying polyclonal metastases, whereas single biopsy sequencing predicted monoclonal disease. Clinically this case showed lack of response to three lines of immune CPI therapy, which was observed in the context of a heterogeneous subclonal neoantigen repertoire. In conclusion, Rep-Seq effectively implements an unbiased tumor sampling approach, drawing DNA molecules from a well-mixed solution of the entire tumor mass, hence removing spatial bias inherent in current approaches. As a result Rep-Seq detects more mutations, achieves greater accuracy in determining clonal from subclonal variants. Rep-Seq offers an improved sampling protocol for tumor profling, with the same equivalent sequencing costs as current methods (single biopsy sequencing), but with significant potential for improved clinical utility.

Structural variation (SV) is typically defined as variation within the human genome that exceeds 50 base pairs (bp). SV may be copy number neutral or it may involve duplications, deletions, and complex rearrangements. Recent studies have shown SV to be associated with many human diseases. However, studies of SV have been challenging due to technological constraints. With the advent of third generation (long-read) sequencing technology, exploration of longer stretches of DNA not easily examined previously has been made possible. In the present study, we utilized third generation (long-read) sequencing techniques to examine SV in the EGFR landscape of four haplotypes derived from two human samples. We analyzed the EGFR gene and its landscape (+/- 500,000 base pairs) using this approach and were able to identify a region of non-coding DNA with over 90% similarity to the most common activating EGFR mutation in non-small cell lung cancer. Based on previously published Alu-element genome instability algorithms, we propose a molecular mechanism to explain how this non-coding region of DNA may be interacting with and impacting the stability of the EGFR gene and potentially generating this cancer-driver gene. By these techniques, we were also able to identify previously hidden structural variation in the four haplotypes and in the human reference genome (hg38). We applied previously published algorithms to compare the relative stabilities of these five different EGFR gene landscape haplotypes to estimate their relative potentials to generate the EGFR exon 19, 15 bp canonical deletion. To our knowledge, the present study is the first to use the differences in genomic architecture between targeted cancer-linked phased haplotypes to estimate their relative potentials to form a common cancer-linked driver mutation.

Cytosine methylation has been shown to have a role in a host of biological processes. In mammalian biology these include stem cell differentiation, embryonic development, genomic imprinting, inflammation, and silencing of transposable elements. Given the central importance of these processes, it is not surprising to find aberrant cytosine methylation patterns associated with many disorders in humans, including cancer, cardiovascular disease, and neurological disease. While whole genome shotgun bisulfite sequencing (WGBS) has recently become feasible, generating high sequence coverage data for the entire genome is expensive, both in terms of money and analysis time, when generally only a small subset of the genome is of interest to most researchers. This report details a procedure for the targeted enrichment of bisulfite treated DNA via SeqCap Epi, allowing high resolution focus of next generation sequencing onto a subset of the genome for high resolution cytosine methylation analysis. Regions ranging in size from only a few kb up to over 200 Mb may be targeted, including the use of the SeqCap Epi CpGiant design which is designed to target 5.5 million CpGs in the human genome. Finally, multiple samples may be multiplexed and sequenced together to provide an inexpensive method of generating methylation data for a large number of samples in a high throughput fashion.

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.

Sequencing-based whole-transcriptome analysis (i.e., RNA-Seq) can be a powerful tool when used to measure gene expression, detect novel transcripts, characterize transcript isoforms, and identify sequence polymorphisms. However, this method can be inefficient when the goal is to study only one component of the transcriptome, such as long noncoding RNAs (lncRNAs), which constitute only a small fraction of transcripts in a total RNA sample. Here, we describe a target enrichment method where a total RNA sample is converted to a sequencing-ready cDNA library and hybridized to a complex pool of lncRNA-specific biotinylated long oligonucleotide capture probes prior to sequencing. The resulting sequence data are highly enriched for the targets of interest, dramatically increasing the efficiency of next-generation sequencing approaches for the analysis of lncRNAs.

Sequencing-based whole-transcriptome analysis (i.e., RNA-Seq) can be a powerful tool when used to measure gene expression, detect novel transcripts, characterize transcript isoforms, and identify sequence polymorphisms. However, this method can be inefficient when the goal is to study only one component of the transcriptome, such as long noncoding RNAs (lncRNAs), which constitute only a small fraction of transcripts in a total RNA sample. Here, we describe a target enrichment method where a total RNA sample is converted to a sequencing-ready cDNA library and hybridized to a complex pool of lncRNA-specific biotinylated long oligonucleotide capture probes prior to sequencing. The resulting sequence data are highly enriched for the targets of interest, dramatically increasing the efficiency of next-generation sequencing approaches for the analysis of lncRNAs.

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases. S.T. and H.X. are funded by Cancer Research UK (CRUK) (C50947/A18176). S.T., T.C., J.L., and M.G. are funded by the NIH Research (NIHR) Biomedical Research Centre (BRC) at the Royal Marsden Hospital and Institute of Cancer Research (A109). J.I.L. is funded by the Ministerio de Economia y Competitividad (MINECO, SAF2016-79847-R). M.S., A.S., J.L., R.F., L.A., and L.S. are funded by the Royal Marsden Cancer Charity. K.L. is funded by UK Medical Research Council (MR/P014712/1). N.M. receives funding from CRUK, Rosetrees, and the NIHR BRC at University College London Hospitals. C.S is Royal Society Napier Research Professor. C.S. is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees and Stoneygate Trusts, NovoNordisk Foundation (ID 16584), the Breast Cancer Research Foundation (BCRF), the European Research Council (THESEUS), Marie Curie Network PloidyNet, the NIHR BRC at University College London Hospitals, and the CRUK University College London Experimental Cancer Medicine Centre. The work presented in this manuscript was funded by Cancer Research UK (grant reference number C50947/A18176), Ventana Medical Systems (grant reference numbers 10467 and 10530), the Kidney Cancer Fund of The Royal Marsden Cancer Charity, NIHR BRC at the Royal Marsden Hospital and Institute of Cancer Research (grant reference number A109), and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001202), the UK Medical Research Council (FC001202), and the Wellcome Trust (FC001202). In particular, we acknowledge the support of the Advanced Sequencing Facility and the High-Performance Computing at the Francis Crick Institute. This project was enabled through access to the MRC eMedLab Medical Bioinformatics infrastructure, supported by the Medical Research Council (grant number MR/L016311/1).

Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.

Characterization of the somatic sequence variations that accrue in cells is critical for understanding the pronounced cellular and clinical heterogeneity observed in cancer. The ability to efficiently detect variations in tumors can help to identify biomarkers which may be relevant to clinical trials, support more accurate prognosis, and help guide more effective choices of therapy. Next-generation sequencing (NGS) has become a valuable tool for discovering somatic mutations in cancers. Here we present an alternative approach to current methodologies for addressing these needs. HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) is a targeted NGS method based on optimized, multiplexed molecular inversion probes (MIPs). This is a convenient, sensitive and cost-effective target enrichment technology for SNP discovery and SNP validation in cancer-related genes. HEAT-Seq probes target both DNA strands and were designed to facilitate bioinformatic error correction. Molecule identifiers (UIDs) incorporated into the probes tag PCR duplicates and support ultra-sensitive detection of low frequency variants, reduction of false positives, and accurate assessment of molecular complexity free of amplification bias. Sensitivities for allele detection have been measured to below 1%. The HEAT-Seq workflow from input DNA to sequence-ready sample can be completed in ≤8 hours without any requirement for a separate library preparation step, which saves both time and cost. Additionally, the capture, amplification and sample clean up steps are performed in a single reaction tube. This eliminates the need to transfer samples to subsequent reaction tubes, preserves sample identity, prevents cross contamination, limits sample loss, and makes the HEAT-Seq protocol easy to automate. A comparison of current PCR-based targeted sequencing with the new HEAT-Seq technology indicates significant advantages for HEAT-Seq in the accurate quantification of low frequency cancer variants. In summary, HEAT-Seq technology offers a rapid, convenient, automatable option to identify genomic DNA variants and enable advances in cancer research. Citation Format: Keynttisha Jefferson, Heather Halvensleben, Dawn Green, Ryan Bannen, Michael Brockman, Todd Richmond, Daniel Burgess. A novel molecular inversion probe (MIP) system for the streamlined identification of germline and somatic sequence variants in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5223.

Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking. Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common “best practice” NGS analysis pipelines. Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability. The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2×76bp reads for each of 8 replicates, we observe an on-target read rate of >95%. After duplicate removal, the% of target bases with at least 20x coverage is >90%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼90%. The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2×76bp reads for each of 24 replicates, we observe an on-target read rate of ∼80%. After duplicate removal, the% of target bases with at least 20x coverage is >95%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼95%. We also observed detection of validated low frequency variants down to 1%. In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants. Citation Format: Ryan Bannen, Michael Brockman, Mark D’Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5217.

Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility., Author Summary Severe short stature in humans has many causes including defects in skeletal and hormonal growth regulation. Primordial dwarfisms are congenital growth defects that involve mutations in genes for DNA repair, DNA replication, splicing of U12 introns, and centrosome dynamics. We discovered that the spontaneous, dwarf mouse mutant chagun is caused by loss-of-function of the gene Poc1a, which encodes protein of the centriole 1A. Mutants exhibit disproportionate dwarfism, and bones formed by endochondral and intramembranous ossification processes are affected. The epiphyseal growth plates of their long bones are disorganized. The chagun males are infertile due to Sertoli cell dysfunction and the failure of germ cells to complete meiosis. The chagun mouse is a model for human dwarfism and provides insight into the mechanism whereby this centriolar protein affects bone growth and spermatogenesis.

We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.

The vertebrate lens is composed of proliferating epithelial cells in the anterior portion and postmitotic, terminally differentiated fiber cells in the posterior part. This polarity is maintained throughout life by precise coordination of proliferation, cell cycle exit, and differentiation. An inductive signal from the retina is thought to induce lens epithelial cells at the equatorial region to withdraw from the cell cycle and initiate the fiber differentiation program.1 In vitro and in vivo studies show that FGF stimulation of lens epithelial cells is sufficient to induce premature fiber differentiation.2–5 Expression of secreted dominant negative FGF receptor 3 (FGFR3) or deletion of all six alleles of FGFR1, -2, and -3 in the lens in transgenic or knockout mice, respectively, is sufficient to inhibit initiation of lens fiber differentiation.6,7 These results suggest that FGFR-meditated signaling is necessary for normal fiber differentiation. Sproutys play an essential role in regulation of Ras-Raf-Erk signaling downstream of FGFR stimulation. Four vertebrate Sprys, Spry1-4, have been identified and have been shown to antagonize Ras-Raf-ERK signaling downstream of FGFR.8–11 During murine embryogenesis, the sites of Spry expression often coincide with centers of FGF signaling.11–15 Spry-knockout mice have revealed interesting roles for these proteins in regulation of organogenesis. Spry1-null mice show kidney and urinary tract defects with ectopic branching of the Wolffian duct and increased ERK activation.16,17 Spry2-null mice show alterations in inner ear development that are rescued by reduction in FGF8 dosage.18 Spry3 is not expressed in the mouse embryo but is expressed in the adult brain and testes.11 Effects of Spry3 deletion in the murine germline have not been reported. Spry4-null mice show growth retardation and polysyndactyly.19 In addition, Spry2 and -4 have been shown to be critical for repression of ERK activation and diastema tooth formation.20 In the brain, Spry1 and -2 regulate cortical patterning, proliferation, and differentiation by repression of FGF-ERK signaling.9 These results establish Sprys as critical modulators of FGF-ERK signaling during organogenesis. Expression of Spry1 and -2 at different stages of lens maturation has been described previously.21 Nonetheless, the question of whether Sprys are necessary for lens development has not been addressed. By conditional deletion of Spry1 and -2 in ocular tissues, we show that these genes play critical roles in not only lens but also corneal development.

Background Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood. Results We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS. Conclusions We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.

SummarySerum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed−/− serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

We report recent progress on the on-going NuSTAR observational campaign of 8 TeV-detected pulsar wind nebulae (PWNe). This campaign constitutes a major part of our NuSTAR study of some of the most energetic TeV sources in our Galaxy detected by VERITAS and HAWC. NuSTAR is the first focusing X-ray telescope operating above 10 keV in space with sub-arcminute angular resolution. Broad-band X-ray imaging and spectroscopy data, obtained by NuSTAR, allow us to probe sub-PeV electron populations through detecting synchrotron X-ray radiation. Our targets include PeVatron candidates detected by HAWC, the Boomerang nebula, PWNe crushed by supernova remnant shocks (or relic PWNe) and G0.9+0.1 in the Galactic Center. Using Fermi-LAT data and available TeV data, we aim to provide a complete, multi-wavelength view of a diverse class of middle-aged (~10-100 kyrs old) PWNe. Our NuSTAR analysis detected hard X-ray emission from the Eel and Boomerang PWNe and characterized their broad-band X-ray spectra most accurately. We plan to apply both time-evolution and multi-zone PWN models to multi-wavelength spectral energy distribution (SED) data over the radio, X-ray, GeV and TeV bands. In this proceeding, we will review our observational campaign and discuss the preliminary results for some PWNe., Comment: Presented at the 37th International Cosmic Ray Conference (ICRC 2021)

Page 4 of cover has a verse pointing out the moral of this book., Illustrations are hand-colored., Electronic reproduction. [Florida] : Florida Board of Education, Division of Colleges and Universities, PALMM Project, 2001. (Preservation and Access for American and British Children's Literature, 1850-1869 (NEH PA-23536-00) ) Mode of access: World Wide Web. System requirements: Internet connectivity; Web browser software; Adobe Acrobat Reader to view and print PDF files. Electronically reproduced from collection in the George A. Smathers Libraries of the University of Florida.

Background The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1 -/- mice using a Cre/loxP system. Results Both male and female Ehd1 -/- mice survived at sub-Mendelian ratios. A proportion of Ehd1 -/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1 -/- adult males were infertile and displayed decreased testis size, whereas Ehd1 -/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1 -/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1 -/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1 -/- mice. Conclusions Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.