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Multiple summer events, including large indoor gatherings, in Provincetown, Massachusetts (MA), in July 2021 contributed to an outbreak of over one thousand COVID-19 cases among residents and visitors. Most cases were fully vaccinated, many of whom were also symptomatic, prompting a comprehensive public health response, motivating changes to national masking recommendations, and raising questions about infection and transmission among vaccinated individuals. To characterize the outbreak and the viral population underlying it, we combined genomic and epidemiological data from 467 individuals, including 40% of known outbreak-associated cases. The Delta variant accounted for 99% of sequenced outbreak-associated cases. Phylogenetic analysis suggests over 40 sources of Delta in the dataset, with one responsible for a single cluster containing 83% of outbreak-associated genomes. This cluster was likely not the result of extensive spread at a single site, but rather transmission from a common source across multiple settings over a short time. Genomic and epidemiological data combined provide strong support for 25 transmission events from, including many between, fully vaccinated individuals; genomic data alone provides evidence for an additional 64. Together, genomic epidemiology provides a high-resolution picture of the Provincetown outbreak, revealing multiple cases of transmission of Delta from fully vaccinated individuals. However, despite its magnitude, the outbreak was restricted in its onward impact in MA and the US, likely due to high vaccination rates and a robust public health response.

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.

The advent of gene panel testing is challenging the previous practice of using clinically defined cancer family syndromes to inform single-gene genetic screening. Individual and family cancer histories that would have previously indicated testing of a single gene or a small number of related genes are now, increasingly, leading to screening across gene panels that contain larger numbers of genes. We have applied a gene panel test that included four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) to an Australian population-based case-control-family study of breast cancer. Altogether, eight pathogenic variants in MMR genes were identified: six in 1421 case-families (0.4%, 4 MSH6 and 2 PMS2) and two in 833 control-families (0.2%, one each of MLH1 and MSH2). This testing highlights the current and future challenges for clinical genetics in the context of anticipated gene panel-based population-based screening that includes the MMR genes. This testing is likely to provide additional opportunities for cancer prevention via cascade testing for Lynch syndrome and precision medicine for breast cancer treatment.

Metagenomic sequencing is revolutionizing the detection and characterization of microbial species, and a wide variety of software tools are available to perform taxonomic classification of this data. The fast pace of development of these tools, alongside the complexity of metagenomic data, make it important that researchers are able to benchmark their performance. Here, we review current approaches for metagenomic analysis and evaluate the performance of 20 metagenomic classifiers using simulated and experimental datasets. We describe the key metrics used to assess performance, offer a framework for the comparison of additional classifiers, and discuss the future of metagenomic data analysis.

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific tests, sometimes over days to weeks, whereas metagenomic next-generation sequencing (mNGS) profiles all nucleic acid in a sample. In this single-center, prospective study, 68 hospitalized patients with known (n = 44) or suspected (n = 24) CNS infections underwent mNGS from RNA and DNA to identify potential pathogens and also targeted sequencing of viruses using hybrid capture. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects, 3 of whom had no clinical diagnosis by routine workup. Among subjects diagnosed with infection by serology and/or peripheral samples, we demonstrated the utility of mNGS to detect pathogen nucleic acid in CSF, importantly for the Ixodes scapularis tick-borne pathogens Powassan virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. We also evaluated two methods to enhance the detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed the detection of varicella-zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with efficacy similar to that of pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. IMPORTANCE Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40 to 60% of people with meningitis or encephalitis have no clear cause of CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 prospectively enrolled patients with known (n = 44) or suspected (n = 24) CNS viral infection from a single center in New England and evaluated enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become integrated into the diagnostic toolkit for CNS infections.

Purpose: Brain imaging exams typically take 10-20 min and involve multiple sequential acquisitions. A low-distortion whole-brain echo planar imaging (EPI)-based approach was developed to efficiently encode multiple contrasts in one acquisition, allowing for calculation of quantitative parameter maps and synthetic contrast-weighted images.Methods: inversion prepared spin- and gradient-echo EPI was developed with sliceorder shuffling across measurements for efficient acquisition with T-1, T-2, and T-2* weighting. A dictionary-matching approach was used to fit the images to quantitative parameter maps, which in turn were used to create synthetic weighted images with typical clinical contrasts. Dynamic slice-optimized multi-coil shimming with a B-0 shim array was used to reduce B-0 inhomogeneity and, therefore, image distortion by >50%. Multi-shot EPI was also implemented to minimize distortion and blurring while enabling high in-plane resolution. A low-rank reconstruction approach was used to mitigate errors from shot-to-shot phase variation.Results: The slice-optimized shimming approach was combined with in-plane parallel-imaging acceleration of 4x to enable single-shot EPI with more than eightfold distortion reduction. The proposed sequence efficiently obtained 40 contrasts across the whole-brain in just over 1 min at 1.2 x 1.2 x 3 mm resolution. The multishot variant of the sequence achieved higher in-plane resolution of 1 x 1 x 4 mm with good image quality in 4 min. Derived quantitative maps showed comparable values to conventional mapping methods.Conclusion: The approach allows fast whole-brain imaging with quantitative parameter maps and synthetic weighted contrasts. The slice-optimized multi-coil shimming and multi-shot reconstruction approaches result in minimal EPI distortion, giving the sequence the potential to be used in rapid screening applications.

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium’s amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

Most voxels in white matter contain multiple fiber populations with different orientations and levels of myelination. Conventional TWe perform simulations to evaluate the accuracy and precision of our crossing fiber IR-DWI signal model for various fiber parameters. The proposed sequence and signal model are tested in a phantom consisting of crossing asparagus pieces doped with gadolinium to vary TOur simulations show that tract-specific TWhole-brain tract-specific T

Phylogenetics of superspreading One important characteristic of coronavirus epidemiology is the occurrence of superspreading events. These are marked by a disproportionate number of cases originating from often-times asymptomatic individuals. Using a rich sequence dataset from the early stages of the Boston outbreak, Lemieux et al. identified superspreading events in specific settings and analyzed them phylogenetically (see the Perspective by Alizon). Using ancestral trait inference, the authors identified several importation events, further investigated the context and contribution of particular superspreading events to the establishment of local and wider SARS-CoV-2 transmission, and used viral phylogenies to describe sustained transmission. Science , this issue p. eabe3261 ; see also p. 574

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Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific diagnostic tests, sometimes over days to weeks. Metagenomic next-generation sequencing (mNGS) is a high-throughput platform that profiles all nucleic acid in a sample. We prospectively enrolled 68 patients from New England with known or suspected CNS infection and performed mNGS from both RNA and DNA to identify potential pathogens. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects. This included some pathogens traditionally diagnosed by serology or not typically identified in CSF, including three transmitted by Ixodes scapularis ticks (Powassan virus, Borrelia burgdorferi, Anaplasma phagocytophilum). Among 24 subjects with no clinical diagnosis, we detected enterovirus in two subjects and Epstein Barr virus in one subject. We also evaluated two methods to enhance detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed detection of varicella zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with similar efficacy to pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis.ImportanceMeningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40-60% of people with meningitis or encephalitis have no clear cause of their CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 patients with suspected CNS infection, and define enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become a mainstay in the diagnostic toolkit for CNS infections.

Dengue virus is a major and rapidly growing public health concern in tropic and subtropic regions across the globe. In late 2018, Senegal experienced its largest dengue virus outbreak to date, covering several regions. However, little is known about the genetic diversity of dengue virus (DENV) in Senegal. Here we used molecular tools including metagenomic sequencing to identify 19 previously undetected dengue virus cases from the city of Thiès and assemble 17 complete virus genomes. DENV3 was the most frequent serotype; 11 sequences (65%) were DENV3, 4 sequences were DENV2 and 2 were DENV1. Sequences were closest to recent sequences from West Africa, suggesting ongoing local circulation of viral populations; however, detailed inference is limited by the scarcity of available genomic data. We did not find clear associations with reported clinical signs or symptoms, highlighting the importance of testing for diagnosing febrile diseases. Overall, these findings expand the known range of DENV in Senegal, and underscore the need for better genomic characterization of DENV in West Africa.

Three-dimensional plasmonic superlattice microcavities, made from programmable atom equivalents comprising gold nanoparticles functionalized with DNA, are used as a testbed to study directional light emission. DNA-guided nanoparticle colloidal crystallization allows for the formation of micrometer-scale single-crystal body-centered cubic gold nanoparticle superlattices, with dye molecules coupled to the DNA strands that link the particles together, in the form of a rhombic dodecahedron. Encapsulation in silica allows one to create robust architectures with the plasmonically active particles and dye molecules fixed in space. At the micrometer scale, the anisotropic rhombic dodecahedron crystal habit couples with photonic modes to give directional light emission. At the nanoscale, the interaction between the dye dipoles and surface plasmons can be finely tuned by coupling the dye molecules to specific sites of the DNA particle-linker strands, thereby modulating dye-nanoparticle distance (three different positions are studied). The ability to control dye position with subnanometer precision allows one to systematically tune plasmon-excition interaction strength and decay lifetime, the results of which have been supported by electrodynamics calculations that span length scales from nanometers to micrometers. The unique ability to control surface plasmon/exciton interactions within such superlattice microcavities will catalyze studies involving quantum optics, plasmon laser physics, strong coupling, and nonlinear phenomena.

SARS-CoV-2 has caused a severe, ongoing outbreak of COVID-19 in Massachusetts with 111,070 confirmed cases and 8,433 deaths as of August 1, 2020. To investigate the introduction, spread, and epidemiology of COVID-19 in the Boston area, we sequenced and analyzed 772 complete SARS-CoV-2 genomes from the region, including nearly all confirmed cases within the first week of the epidemic and hundreds of cases from major outbreaks at a conference, a nursing facility, and among homeless shelter guests and staff. The data reveal over 80 introductions into the Boston area, predominantly from elsewhere in the United States and Europe. We studied two superspreading events covered by the data, events that led to very different outcomes because of the timing and populations involved. One produced rapid spread in a vulnerable population but little onward transmission, while the other was a major contributor to sustained community transmission, including outbreaks in homeless populations, and was exported to several other domestic and international sites. The same two events differed significantly in the number of new mutations seen, raising the possibility that SARS-CoV-2 superspreading might encompass disparate transmission dynamics. Our results highlight the failure of measures to prevent importation into MA early in the outbreak, underscore the role of superspreading in amplifying an outbreak in a major urban area, and lay a foundation for contact tracing informed by genetic data.

Purpose To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. Methods Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. Results We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). Conclusions These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks., Outbreaks of viral hemorrhagic fevers highlight the need for sensitive, field-deployable diagnostics. Here the authors present a CRISPR-based SHERLOCK platform with field protocol and mobile app for Ebola and Lassa fever outbreaks.

BackgroundMolecular epidemiology can provide important information regarding the genetic diversity and transmission ofPlasmodium falciparum, which can assist in designing and monitoring elimination efforts. However, malaria molecular epidemiology including understanding the genetic diversity of the parasite and performing molecular surveillance of transmission has been poorly documented in Senegal. Next Generation Sequencing (NGS) offers a practical, fast and high-throughput approach to understand malaria population genetics. This study aims to unravel the population structure ofP. falciparumand to estimate the allelic diversity, multiplicity of infection (MOI), and evolutionary patterns of the malaria parasite using the NGS platform.MethodsMultiplex amplicon deep sequencing of merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) in fifty-threeP. falciparumisolates from two epidemiologically different areas in the South and North of Senegal, was carried out.ResultsA total of 76Pfmsp1and 116Pfmsp2clones were identified and 135 different alleles were found, 56 and 79 belonged to thepfmsp1andpfmsp2genes, respectively. K1 and IC3D7 allelic families were most predominant in both sites. The local haplotype diversity (Hd) and nucleotide diversity (π) were higher in the South than in the North for both genes. Forpfmsp1, a high positive Tajima’s D (TD) value was observed in the South (D = 2.0453) while negative TD value was recorded in the North (D = − 1.46045) and F-Statistic (Fst) was 0.19505. Forpfmsp2, non-directional selection was found with a highly positive TD test in both areas and Fst was 0.02111. The mean MOI for both genes was 3.07 and 1.76 for the South and the North, respectively, with a statistically significant difference between areas (p = 0.001).ConclusionThis study revealed a high genetic diversity ofpfmsp1andpfmsp2genes and low genetic differentiation inP. falciparumpopulation in Senegal. The MOI means were significantly different between the Southern and Northern areas. Findings also showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology ofP. falciparuminfections.

Purpose To alleviate the spatial encoding limitations of single-shot EPI by developing multi-shot segmented EPI for ultra-high-resolution fMRI with reduced ghosting artifacts from subject motion and respiration. Methods Segmented EPI can reduce readout duration and reduce acceleration factors, however, the time elapsed between segment acquisitions (on the order of seconds) can result in intermittent ghosting, limiting its use for fMRI. Here, "FLEET" segment ordering--where segments are looped over before slices--was combined with a variable flip angle progression (VFA-FLEET) to improve inter-segment fidelity and maximize signal for fMRI. Scaling a sinc pulse's flip angle for each segment (VFA-FLEET-Sinc) produced inconsistent slice profiles and ghosting, therefore, a recursive Shinnar-Le Roux (SLR) RF pulse design was developed (VFA-FLEET-SLR) to generate unique pulses for every segment that together produce consistent slice profiles and signals. Results The temporal stability of VFA-FLEET-SLR was compared against conventional-segmented EPI and VFA-FLEET-Sinc at 3 T and 7 T. VFA-FLEET-SLR showed reductions in both intermittent and stable ghosting compared to conventional-segmented and VFA-FLEET-Sinc, resulting in improved image quality with a minor trade-off in temporal SNR. Combining VFA-FLEET-SLR with acceleration, we achieved a 0.6-mm isotropic acquisition at 7 T--without zoomed imaging or partial Fourier--demonstrating reliable detection of BOLD responses to a visual stimulus. To counteract the increased repetition time from segmentation, simultaneous multi-slice VFA-FLEET-SLR was demonstrated using RF-encoded controlled aliasing. Conclusions VFA-FLEET with a recursive RF pulse design supports acquisitions with low levels of artifact and spatial blur, enabling fMRI at previously inaccessible spatial resolutions with a "full-brain" field of view., 51 pages (including supplement), 8 main figures, 6 supporting figures. For supporting videos (8), please visit https://github.com/aveberman/vfa-fleet. Note: this work has been accepted for publication at Magnetic Resonance in Medicine

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing.

Viral hemorrhagic fevers (VHFs) remain some of the most devastating human diseases, and recent outbreaks of Ebola virus disease (EVD) 1,2 and Lassa fever (LF) 3,4 highlight the urgent need for sensitive, field-deployable tests to diagnose them 5,6. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. Our EBOV diagnostic detects both the L and NP genes, thereby eliminating the potential for false positive results caused by the rVSVΔG-ZEBOV-GP live attenuated vaccine. Our two LASV diagnostics together capture 90% of known viral diversity and demonstrate that CRISPR-RNAs (crRNAs) can be effectively multiplexed to provide greater coverage of known viral diversity. We performed safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating and chemically treating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We developed a user-friendly field protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deployed our tests in Sierra Leone and Nigeria in response to recent outbreaks.

Summary During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017–2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer’s University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.

An outbreak of over one thousand COVID-19 cases in Provincetown, Massachusetts, in July 2021—the first large outbreak mostly in vaccinated individuals in the US—prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined genomic and epidemiological data from 467 individuals, including 40% of known outbreak-associated cases. The Delta variant accounted for 99% of outbreak-associated cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US, likely due to high vaccination rates and a robust public health response., Rapid integration of epidemiological and genomic data helped to support public health measures during a recent large outbreak of the SARS-CoV-2 Delta variant. This outbreak stemmed primarily from a single source that spread the virus at multiple locations and frequent transmission between vaccinated individuals was observed and contained with robust intervention and community engagement.

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA-Exchange-Imaging (DEI), for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DNA-Exchange-Imaging allows us to apply it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ~300 nm down to sub-20-nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Loss-of-function germline mutations in the PALB2 gene are associated with an increase of breast cancer risk. The purpose of this study was to characterise the spectrum of PALB2 mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of PALB2 in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. Targeted-sequencing identified two frameshift deletions: PALB2:c.509_510del; p.R170Ifs in three women affected with breast cancer and PALB2:c.172_175del;p.Q60Rfs in one woman affected with ovarian cancer. A number of other previously described missense (some predicted to be damaging by PolyPhen-2 and CADD) and synonymous mutations were also identified in this population. This study is consistent with previous reports that PALB2:c.509_510del and PALB2:c.172_175del are recurrent mutations associated with breast cancer predisposition in Polish women with a family history of the disease. Our study contributes to the accumulating evidence indicating that PALB2 should be included in genetic testing for breast cancer susceptibility in these populations to enhance risk assessment and management of women at high-risk of developing breast cancer. This data could also contribute to ongoing work that is assessing the possible association between ovarian cancer risk and PALB2 mutations for which there is currently no evidence.

Purpose To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. Methods Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. Results We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). Conclusions These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

Few genetic risk factors have been demonstrated to be specifically associated with aggressive prostate cancer (PrCa). Here, we report a case-case study of PrCa comparing the prevalence of germline pathogenic/likely pathogenic (P/LP) genetic variants in 787 men with aggressive disease and 769 with nonaggressive disease. Overall, we observed P/LP variants in 11.4% of men with aggressive PrCa and 9.8% of men with nonaggressive PrCa (two-tailed Fisher's exact tests, P = .28). The proportion of BRCA2 and ATM P/LP variant carriers in men with aggressive PrCa exceeded that observed in men with nonaggressive PrCa; 18/787 carriers (2.3%) and 4/769 carriers (0.5%), P = .004, and 14/787 carriers (0.02%) and 5/769 carriers (0.01%), P = .06, respectively. Our findings contribute to the extensive international effort to interpret the genetic variation identified in genes included on gene-panel tests, for which there is currently an insufficient evidence-base for clinical translation in the context of PrCa risk.

Trigeminal neuralgia (TN) is characterized by recurrent attacks of lancinating facial pain in the dermatomal distribution of the trigeminal nerve. TN is rare, affecting 4 to 13 people per 100,000. Although there remains a debate surrounding the pathogenesis of TN, neurovascular compromise is the most currently accepted theory. Minimal stimulation caused by light touch, talking, or chewing can lead to debilitating pain and incapacitation of the patient. Pain may occur sporadically, though is primarily unilateral in onset. The diagnosis is typically determined clinically. Treatment options include medications, surgery, and complementary approaches. Anti-epileptic and tricyclic antidepressant medications are first-line treatments. Surgical management of patients with TN may be indicated in those who have either failed medical treatment with at least three medications, suffer from intolerable side-effects, or have non-remitting symptoms. Surgical treatment is categorized as either destructive or non-destructive. Deep brain and motor cortex neuro-modulatory stimulation are off label emerging techniques which may offer relief to TN that is otherwise refractory to pharmacological management and surgery. Still, sufficient data has yet to be obtained and more studies are needed.

Unusually large outbreaks of mumps across the United States in 2016 and 2017 raised questions about the extent of mumps circulation and the relationship between these and prior outbreaks. We paired epidemiological data from public health investigations with analysis of mumps virus whole genome sequences from 201 infected individuals, focusing on Massachusetts university communities. Our analysis suggests continuous, undetected circulation of mumps locally and nationally, including multiple independent introductions into Massachusetts and into individual communities. Despite the presence of these multiple mumps virus lineages, the genomic data show that one lineage has dominated in the US since at least 2006. Widespread transmission was surprising given high vaccination rates, but we found no genetic evidence that variants arising during this outbreak contributed to vaccine escape. Viral genomic data allowed us to reconstruct mumps transmission links not evident from epidemiological data or standard single-gene surveillance efforts and also revealed connections between apparently unrelated mumps outbreaks., A study of recent mumps outbreaks in the United States reveals transmission patterns within and between Massachusetts communities and highlights the importance of combining detailed genomic and epidemiological data, both locally and nationwide.

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

Background: Global DNA methylation has been reported to be associated with urothelial cell carcinoma (UCC) by studies using blood samples collected at diagnosis. Using the Illumina HumanMethylation450 assay, we derived genome-wide measures of blood DNA methylation and assessed them for their prospective association with UCC risk. Methods: We used 439 case–control pairs from the Melbourne Collaborative Cohort Study matched on age, sex, country of birth, DNA sample type, and collection period. Conditional logistic regression was used to compute odds ratios (OR) of UCC risk per s.d. of each genome-wide measure of DNA methylation and 95% confidence intervals (CIs), adjusted for potential confounders. We also investigated associations by disease subtype, sex, smoking, and time since blood collection. Results: The risk of superficial UCC was decreased for individuals with higher levels of our genome-wide DNA methylation measure (OR=0.71, 95% CI: 0.54–0.94; P=0.02). This association was particularly strong for current smokers at sample collection (OR=0.47, 95% CI: 0.27–0.83). Intermediate levels of our genome-wide measure were associated with decreased risk of invasive UCC. Some variation was observed between UCC subtypes and the location and regulatory function of the CpGs included in the genome-wide measures of methylation. Conclusions: Higher levels of our genome-wide DNA methylation measure were associated with decreased risk of superficial UCC and intermediate levels were associated with reduced risk of invasive disease. These findings require replication by other prospective studies.

Background.Kenema Government Hospital (KGH) has developed an advanced clinical and laboratory research capacity to manage the threat of Lassa fever, a viral hemorrhagic fever (VHF). The 2013–2016 Ebola virus (EBOV) disease (EVD) outbreak is the first to have occurred in an area close to a facility with established clinical and laboratory capacity for study of VHFs. Methods.Because of its proximity to the epicenter of the EVD outbreak, which began in Guinea in March 2014, the KGH Lassa fever Team mobilized to establish EBOV surveillance and diagnostic capabilities. Results.Augustine Goba, director of the KGH Lassa laboratory, diagnosed the first documented case of EVD in Sierra Leone, on 25 May 2014. Thereafter, KGH received and cared for numbers of patients with EVD that quickly overwhelmed the capacity for safe management. Numerous healthcare workers contracted and lost their lives to EVD. The vast majority of subsequent EVD cases in West Africa can be traced back to a single transmission chain that includes this first diagnosed case. Conclusions.Responding to the challenges of confronting 2 hemorrhagic fever viruses will require continued investments in the development of countermeasures (vaccines, therapeutic agents, and diagnostic assays), infrastructure, and human resources.

Purpose In oral cavity cancer surgery there are many factors that contribute to the surgical margin; thus, the factors determining patient outcomes are still not completely understood. The aim of this study was to determine which variable or variables had the greatest influence on increasing the size of the surgical margin. Materials and Methods A retrospective cohort study was conducted at the Royal Brisbane and Women's Hospital of patients who underwent resective surgery for a primary oral cavity cancer from January 1, 2008 through December 31, 2012. The primary outcome variable was the surgical margin, defined as the closest distance between the surgical edge and invasive cancer. A heterogeneous set of predictor variables was identified as potentially affecting the primary outcome variable: demographic, 5 surgical, and 7 histologic variables. The data then underwent statistical analysis using univariable linear regression, and variables that were found to have a statistical association were retained in a non-interaction multivariable model. Results This study included 250 patients. The results showed that high-volume surgeons delivered larger surgical margins than low-volume surgeons. The single most important variable associated with larger surgical margins was who performed the resective operation. The following variables also were associated with smaller surgical margins: retromolar trigone location, non–squamous cell carcinomas, perineural invasion, and a lip-split mandibulectomy surgical approach. Conclusion There was a strong association between high-volume surgeons and larger surgical margins, supporting the rationalization of oral cavity cancer management in high-volume centers and by high-volume surgeons.

BRA-STRAP is an Australia-wide study of breast cancer predisposition that brings together gene-panel data from 30,000 adult Australian women of all ages, across the breast cancer risk spectrum, with and without a diagnosis of breast cancer. The “BRA-STRAP panel” includes 24 genes* that are involved in, or putatively associated with, predisposition to breast and/or ovarian cancer. Despite insufficient evidence for clinical translation for some of these genes, all 24 are commonly included on panel tests for breast cancer predisposition. We present findings from the population-based case-control sub-study of BRA-STRAP, which involved 1451 women diagnosed with breast cancer and 857 age-matched controls participating in the Australian Breast Cancer Family Registry (ABCFR), and 6101 healthy, elderly Australian women enrolled in the ASPREE study. These analyses focus on rare genetic variants predicted to lead to loss of function and/or classified as pathogenic/likely pathogenic (P/LP) in ClinVar. Odds ratios (ORs) for their associations with breast cancer were estimated by aggregating genetic variants for each gene. For the women diagnosed with breast cancer, the median age at diagnosis (inter-quartile range, IQR) was 40.0 (14.0) years and the overall frequency of P/LP variant carriers across all genes was 156/1451 (10.8%). The median age (IQR) of the ABCFR and ASPREE controls were 39.4 (14.9) and 73.9 (5.8) years, respectively. The frequencies of P/LP variant carriers were 33/857 (3.9%) and 268/6101 (4.4%) in the ABCFR and ASPREE controls, respectively. We combined both control datasets and, after adjusting for age and other potential confounders, the ORs associated with P/LP variants in BRCA1 and BRCA2 were 4.1 [95% confidence interval (CI): 1.8-10.2] and 2.9 [95% CI: 1.5-6], respectively. We also found that the OR for P/LP variants in ATM was 4.0 [95% CI: 1.5-10.4] and the OR for P/LP variants in PALB2 was 2.2 [95% CI: 0.75-5.7] although this did not reach statistical significance. These results contribute to international efforts to refine the breast cancer risk estimates for genetic variants identified from population-based screening of unselected women using genes that are included on panel tests and thought to be potentially breast cancer predisposition genes.The case-control-family design of the ABCFR will also allow us to estimate the age specific cumulative risk (penetrance) of these genetic variants, which is important for genetic counselling and the clinical management of carrier families. *ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, FANCM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, RECQL, STK11 and TP53 Citation Format: Tu Nguyen-Dumont, James Dowty, Katherine Tucker, Judy Kirk, Paul James, Alison Trainer, Ingrid Winship, Nicholas Pachter, Nicola Poplawski, Scott Grist, Daniel J Park, Anne-Laure Renault, Fleur Hammet, Maryam Mahmoodi, Helen Tsimiklis, Jason A Steen, Derrick Theys, Amanda Rewse, Amanda Willis, April Morrow, Catherine Speechly, Rebecca Harris, Moeen Riaz, Robert Sebra, Eric Schadt, Paul Lacaze, John McNeil, John L Hopper, Melissa C Southey. Population-based estimates of breast cancer risk for germline pathogenic variants identified by gene-panel testing: An Australian perspective [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-04.

DNA methylation influences predisposition, development and prognosis for many diseases, including cancer. However, it is not uncommon to encounter samples with incorrect sex labelling or atypical sex chromosome arrangement. Sex is one of the strongest influencers of the genomic distribution of DNA methylation and, therefore, correct assignment of sex and filtering of abnormal samples are essential for the quality control of study data. Differences in sex chromosome copy numbers between sexes and X-chromosome inactivation in females result in distinctive sex-specific patterns in the distribution of DNA methylation levels. In this study, we present a software tool, sEst, which incorporates clustering analysis to infer sex and to detect sex-chromosome abnormalities from DNA methylation microarray data. Testing with two publicly available datasets demonstrated that sEst not only correctly inferred the sex of the test samples, but also identified mislabelled samples and samples with potential sex-chromosome abnormalities, such as Klinefelter syndrome and Turner syndrome, the latter being a feature not offered by existing methods. Considering that sex and the sex-chromosome abnormalities can have large effects on many phenotypes, including diseases, our method can make a significant contribution to DNA methylation studies that are based on microarray platforms.

Despite widespread vaccination, eleven thousand mumps cases were reported in the United States (US) in 2016–17, including hundreds in Massachusetts, primarily in college settings. We generated 203 whole genome mumps virus (MuV) sequences from Massachusetts and 15 other states to understand the dynamics of mumps spread locally and nationally, as well as to search for variants potentially related to vaccination. We observed multiple MuV lineages circulating within Massachusetts during 2016–17, evidence for multiple introductions of the virus to the state, and extensive geographic movement of MuV within the US on short time scales. We found no evidence that variants arising during this outbreak contributed to vaccine escape. Combining epidemiological and genomic data, we observed multiple co-circulating clades within individual universities as well as spillover into the local community. Detailed data from one well-sampled university allowed us to estimate an effective reproductive number within that university significantly greater than one. We also used publicly available small hydrophobic (SH) gene sequences to estimate migration between world regions and to place this outbreak in a global context, but demonstrate that these short sequences, historically used for MuV genotyping, are inadequate for tracing detailed transmission. Our findings suggest continuous, often undetected, circulation of mumps both locally and nationally, and highlight the value of combining genomic and epidemiological data to track viral disease transmission at high resolution.

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. We developed CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs compact probe sets that achieve full coverage of known sequence diversity and that scale well with this diversity. To illustrate applications of CATCH, we focused on capturing viral genomes. We designed, synthesized, and validated multiple probe sets, including one that targets whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriched unique viral content on average 18× and allowed us to assemble genomes that we could not otherwise recover, while accurately preserving within-sample diversity. We used this approach to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of viral infections in samples with unknown content. Together, this work demonstrates a path toward more sensitive, cost-effective metagenomic sequencing.

Aim: To examine whether peripheral blood methylation is associated with risk of developing mature B-cell neoplasms (MBCNs). Materials & methods: We conducted a case–control study nested within a large prospective cohort. Peripheral blood was collected from healthy participants. Cases of MBCN were identified by linkage to cancer registries. Methylation was measured using the Infinium® HumanMethylation450. Results: During a median of 10.6-year follow-up, 438 MBCN cases were evaluated. Global hypomethylation was associated with increased risk of MBCN (odds ratio: 2.27, [95% CI: 1.59–3.25]). Within high CpG promoter regions, hypermethylation was associated with increased risk (odds ratio: 1.76 [95% CI: 1.25–2.48]). Promoter hypermethylation was observed in HOXA9 and CDH1 genes. Conclusion: Aberrant global DNA methylation is detectable in peripheral blood collected years before diagnosis and is associated with increased risk of MBCN, suggesting changes to DNA methylation are an early event in MBCN development.

The stability and longevity of recordings obtained from intracortical microelectrodes continues to remain an area of concern for neural interfacing applications. The limited longevity of microelectrode performance has been associated with the integrity of the blood brain barrier (BBB) and the neuroinflammatory response to the microelectrode. Here, we report the investigation of an additive approach that targets both mechanical and chemical factors believed to contribute to chronic BBB instability and the neuroinflammatory response associated with implanted intracortical microelectrodes. The implants investigated were based on a mechanically adaptive, compliant nanocomposite (NC), which reduces the tissue response and tissue strain. This material was doped with various concentrations of the antioxidant resveratrol with the objective of local and rapid delivery. In vitro analysis of resveratrol release, antioxidant activity, and cytotoxicity suggested that a resveratrol content of 0.01% was optimal for in vivo assessment. Thus, probes made from the neat NC reference and probes containing resveratrol (NC Res) were implanted into the cortical tissue of rats for up to sixteen weeks. Histochemical analysis suggested that at three days post-implantation, neither materials nor therapeutic approaches (independently or in combination) could alter the initial wound healing response. However, at two weeks post-implantation, the NC Res implant showed a reduction in activated microglia/macrophages and improvement in neuron density at the tissue-implant interface when compared to the neat NC reference. However, sixteen weeks post-implantation, when the antioxidant was exhausted, NC Res and the neat NC reference exhibited similar tissue responses. The data show that NC Res provides short-term, short-lived benefits due to the antioxidant release, and a long-term reduction in neuroinflammation on account of is mechanical adaptive, compliant nature. Together, these results demonstrate that local delivery of resveratrol can provide an additive advantage by providing a consistent reduction in the tissue response.

Constitutional biological processes involve the generation of DNA double-strand breaks (DSBs). The production of such breaks and their subsequent resolution are also highly relevant to neurodegenerative diseases and cancer, in which extensive DNA fragmentation has been described Stephens et al. (2011), Blondet et al. (2001). Tchurikov et al. Tchurikov et al. (2011, 2013) have reported previously that frequent sites of DSBs occur in chromosomal domains involved in the co-ordinated expression of genes. This group report that hot spots of DSBs in human HEK293T cells often coincide with H3K4me3 marks, associated with active transcription Kravatsky et al. (2015) and that frequent sites of DNA double-strand breakage are likely to be relevant to cancer genomics Tchurikov et al. (2013, 2016) . Recently, they applied a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended DSB sites and mapped these to the human genome within defined co-ordinate ‘windows’. In this paper, we re-analyse public RAFT data to derive sites of DSBs at the single-nucleotide level across the built genome for human HEK293T cells (https://figshare.com/s/35220b2b79eaaaf64ed8). This refined mapping, combined with accessory ENCODE data tracks and ribosomal DNA-related sequence annotations, will likely be of value for the design of clinically relevant targeted assays such as those for cancer susceptibility, diagnosis, treatment-matching and prognostication.

Loss-of-function mutations in PALB2 are associated with an increased risk of breast cancer, with recent data showing that female breast cancer risks for PALB2 mutation carriers are comparable in magnitude to those for BRCA2 mutation carriers. This study applied targeted massively parallel sequencing to characterize the mutation spectrum of PALB2 in probands attending breast cancer genetics clinics in the USA. The coding regions and proximal intron-exon junctions of PALB2 were screened in probands not known to carry a mutation in BRCA1 or BCRA2 from 1,250 families enrolled through familial cancer clinics by the Breast Cancer Family Registry. Mutation screening was performed using Hi-Plex, an amplicon-based targeted massively parallel sequencing platform. Screening of PALB2 was successful in 1,240/1,250 probands and identified nine women with protein-truncating mutations (three nonsense mutations and five frameshift mutations). Four of the 33 missense variants were predicted to be deleterious to protein function by in silico analysis using two different programs. Analysis of tumors from carriers of truncating mutations revealed that the majority were high histological grade, invasive ductal carcinomas. Young onset was apparent in most families, with 19 breast cancers under 50 years of age, including eight under the age of 40 years. Our data demonstrate the utility of Hi-Plex in the context of high-throughput testing for rare genetic mutations and provide additional timely information about the nature and prevalence of PALB2 mutations, to enhance risk assessment and risk management of women at high risk of cancer attending clinical genetic services.

In cardiac perfusion imaging, choice of flip angle is an important factor for steady state acquisition. This work focuses on presenting an analytical framework for understanding how non-ideal slice excitation profiles affect contrast in ungated 2D steady state cardiac perfusion studies, and to study a technique for estimating flip angle that maximizes enhanced/unenhanced myocardial contrast-to-noise ratio (CNR) in single slice and multi-slice acquisitions. A numerical simulation of ungated 2D golden ratio radial spoiled gradient echo (SPGR) was created that takes into consideration the actual (Bloch simulated) slice excitation profile. The effect of slice excitation profile on myocardial CNR as a function of flip angle was assessed in phantoms and in vivo. For fast RF pulses, the flip angle that yields maximum CNR (considering the actual slice excitation profile) was considerably higher than expected, assuming an ideal excitation. The simulation framework presented accurately predicts the flip angle yielding maximum CNR when the actual slice excitation profile is taken into consideration. The prescribed flip angle for optimal contrast in ungated 2D steady-state SPGR cardiac perfusion studies can vary significantly from that calculated when an ideal slice excitation profile is assumed. Consideration of the actual slice excitation can yield a more optimal flip angle estimate in both the single slice and multi-slice cases.

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.