Analysis and benchmarking of small and large genomic variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits and are linked to over 60 disease phenotypes. However, they are often excluded from at-scale studies because of challenges with variant calling and representation, as well as a lack of a genome-wide standard. Here, to promote the development of TR methods, we created a catalog of TR regions and explored TR properties across 86 haplotype-resolved long-read human assemblies. We curated variants from the Genome in a Bottle (GIAB) HG002 individual to create a TR dataset to benchmark existing and future TR analysis methods. We also present an improved variant comparison method that handles variants greater than 4 bp in length and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ~24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 'truth-set' TR benchmark. We demonstrate the utility of this pipeline across short-read and long-read technologies.

Benchmarking of small and large variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits, and are linked to over 60 disease phenotypes. However, their complexity often excludes them from at-scale studies due to challenges with variant calling, representation, and lack of a genome-wide standard. To promote TR methods development, we create a comprehensive catalog of TR regions and explore its properties across 86 samples. We then curate variants from the GIAB HG002 individual to create a tandem repeat benchmark. We also present a variant comparison method that handles small and large alleles and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ∼24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 TR benchmark. We work with the GIAB community to demonstrate the utility of this benchmark across short and long read technologies.

A retrospective analysis of clinical outcomes between hospitalized patients with COVID-19 who received famotidine or pantoprazole.

Background and Aim: There is limited research on the use of histamine-H2 receptor antagonists and proton pump inhibitors for treating COVID-19. We compare clinical outcomes between patients hospitalized with COVID-19 receiving famotidine or pantoprazole. Methods: This retrospective study included 2184 patients (famotidine: n = 638, pantoprazole: n = 727, nonuse: n = 819) aged 18 years or older treated for COVID-19 from March 2020 to March 2021. Patients who received both famotidine and pantoprazole treatments were excluded. Multivariate logistic regression was used for the primary outcome, namely all-cause mortality, and the secondary outcomes, namely mechanical ventilation, vasopressor use, acute kidney injury, and gastrointestinal bleeding. The main predictor variable was the use of famotidine or pantoprazole. Covariates were demographics, chronic diseases, and symptoms. Results: As compared to nonuse, famotidine (OR: 0.30, 95% CI: 0.20-0.44, P < 0.001) and pantoprazole (OR: 0.47, 95% CI: 0.33-0.66, P < 0.001) were significantly associated with lower odds for all-cause mortality. Comparison of famotidine and pantoprazole showed that the former had lower odds for all-cause mortality (OR: 0.65, 95% CI:0.45-0.95, P < 0.05), mechanical ventilation (OR: 0.38, 95% CI: 0.25-0.58, P < 0.001), vasopressor use (OR: 0.33, 95% CI: 0.22-0.48, P < 0.001), acute kidney injury (OR: 0.40, 95% CI: 0.30-0.54, P < 0.001), and gastrointestinal bleeding (OR: 0.15, 95% CI: 0.08, 0.29, P < 0.001). Conclusions: Famotidine is associated with lower odds for all-cause mortality, mechanical ventilation, vasopressor use, acute kidney injury, and gastrointestinal bleeding as compared to pantoprazole in patients hospitalized with COVID-19. We recommend that clinicians consider the use of famotidine over pantoprazole for hospitalized COVID-19 patients. Future research with a clinical trial would be beneficial to further support such use of famotidine.

Multiscale analysis of pangenomes enables improved representation of genomic diversity for repetitive and clinically relevant genes.

Advancements in sequencing technologies and assembly methods enable the regular production of high-quality genome assemblies characterizing complex regions. However, challenges remain in efficiently interpreting variation at various scales, from smaller tandem repeats to megabase rearrangements, across many human genomes. We present a PanGenome Research Tool Kit (PGR-TK) enabling analyses of complex pangenome structural and haplotype variation at multiple scales. We apply the graph decomposition methods in PGR-TK to the class II major histocompatibility complex demonstrating the importance of the human pangenome for analyzing complicated regions. Moreover, we investigate the Y-chromosome genes, DAZ1/DAZ2/DAZ3/DAZ4, of which structural variants have been linked to male infertility, and X-chromosome genes OPN1LW and OPN1MW linked to eye disorders. We further showcase PGR-TK across 395 complex repetitive medically important genes. This highlights the power of PGR-TK to resolve complex variation in regions of the genome that were previously too complex to analyze.

Variant calling and benchmarking in an era of complete human genome sequences.

Genetic variant calling from DNA sequencing has enabled understanding of germline variation in hundreds of thousands of humans. Sequencing technologies and variant-calling methods have advanced rapidly, routinely providing reliable variant calls in most of the human genome. We describe how advances in long reads, deep learning, de novo assembly and pangenomes have expanded access to variant calls in increasingly challenging, repetitive genomic regions, including medically relevant regions, and how new benchmark sets and benchmarking methods illuminate their strengths and limitations. Finally, we explore the possible future of more complete characterization of human genome variation in light of the recent completion of a telomere-to-telomere human genome reference assembly and human pangenomes, and we consider the innovations needed to benchmark their newly accessible repetitive regions and complex variants.

Quantum-cascade-laser-absorption-spectroscopy diagnostic for temperature, pressure, and NO X 2 Π 1/2 at 500 kHz in shock-heated air at elevated pressures.

The design, validation, and application of a quantum-cascade-laser-absorption-spectroscopy diagnostic for measuring gas temperature, pressure, and nitric oxide (NO) in high-temperature air are presented. A distributed-feedback quantum-cascade laser (QCL) centered near 1976c m -1 was used to scan across two transitions of NO in its ground electronic state (X 2 Π 1/2). A measurement rate of 500 kHz was achieved using a single QCL by: (1) performing current modulation through a bias-tee, and (2) targeting closely spaced transitions with a large difference in lower-state energy. The diagnostic was validated in a mixture of 95% argon and 5% NO, which was shock-heated to ≈2000 to 3700 K. The average mean percent differences between laser-absorption-spectroscopy (LAS) measurements and predictions from shock-jump relations for temperature, pressure, and NO mole fraction were 3.1%, 4.1%, and 6.5%, respectively. The diagnostic was then applied to characterize shock-heated air at high temperatures (up to ≈5500K) and high pressures (up to 12 atm) behind either incident or reflected shocks. The LAS measurements were compared to theoretical predictions from shock-jump relations, pressure sensors mounted in the wall of the shock tube, and equilibrium values of the NO mole fraction. The average mean percent differences between LAS measurements and their aforementioned reference values were 3.2%, 10.8%, and 10.4% for temperature, pressure, and NO mole fraction, respectively. Last, a comparison between a measured NO mole fraction time history and a time-stepped homogeneous reactor simulation performed using two different chemical kinetics mechanisms is presented.

Long-term safety of intraluminal spring-mediated bowel lengthening.

PURPOSE: The purpose of the study is to examine the long-term safety of an endoluminal bowel lengthening device prior to its use in the first human trial. In addition, device performance and natural passage will be evaluated. METHODS: Endoluminal lengthening springs were surgically placed into the jejunum of Yucatan minipigs using the Eclipse XL1 device. A matching internal control segment of jejunum was marked at the time of operation. Weekly weights and fluoroscopic studies were obtained to evaluate spring deployment and position until devices passed. Animals were euthanized at 28, 60, 90, and 180 days. At necropsy, length measurements were recorded, and histopathologic analysis was performed. RESULTS: There were no bowel obstructions or overt perforations attributable to the device. All surviving animals gained weight and were clinically thriving. All devices passed out of the rectum by 180 days. Bowel lengthening was seen in all experimental segments, and minimal fibrosis was observed by 180 days. CONCLUSION: Jejunal lengthening persisted after device had passed through the intestinal tract after 180 days. Early histopathologic changes of the jejunum during distraction enterogenesis normalized over time.

Hypertension and Gastrointestinal Bleed in COVID-19 Patients: Associations with Mortality, Acute Kidney Injury, Vasopressor Use, and Mechanical Ventilation Use.

There are mixed findings on the association of hypertension or gastrointestinal bleed (GIB) with mortality in COVID-19 patients but no research on the combination of both hypertension and GIB with mortality in COVID-19 patients. We study in COVID-19 patients the association of hypertension and GIB with mortality, acute kidney injury (AKI), vasopressor use, and/or mechanical ventilation. This is a retrospective study of COVID-19 patients who were categorized into groups of no GIB/no hypertension (n = 653), yes hypertension/no GIB (n = 1,620), yes GIB/no hypertension (n = 104), or yes GIB/yes hypertension (n = 334). Covariates included demographics and medical history variables. In the multi-variate logistic regression analysis for the composite outcome of mortality, AKI, vasopressor use, and/or mechanical ventilation use, yes hypertension/no GIB (OR: 1.47, 95% CI: 1.13, 1.89, p <0.001) and yes GIB/no hypertension (OR: 1.68, 95% CI: 1.02, 2.78, p <0.001) were each significantly positively associated with the composite outcome. The yes GIB/yes hypertension group was not significantly associated with the composite outcome. In conclusion, we found that hypertension or GIB alone were each significantly associated with increased odds for the composite outcome while having both hypertension and GIB was protective and not significantly associated with the composite outcome. We recommend that clinicians be aware of such findings when treating patients with COVID-19, as those with both hypertension and GIB may not need as aggressive treatment as compared to those with either hypertension or GIB.

A Comprehensive Evaluation of Generalizability of Deep Learning-Based Hi-C Resolution Improvement Methods.

Hi-C is a widely used technique to study the 3D organization of the genome. Due to its high sequencing cost, most of the generated datasets are of a coarse resolution, which makes it impractical to study finer chromatin features such as Topologically Associating Domains (TADs) and chromatin loops. Multiple deep learning-based methods have recently been proposed to increase the resolution of these datasets by imputing Hi-C reads (typically called upscaling). However, the existing works evaluate these methods on either synthetically downsampled datasets, or a small subset of experimentally generated sparse Hi-C datasets, making it hard to establish their generalizability in the real-world use case. We present our framework-Hi-CY-that compares existing Hi-C resolution upscaling methods on seven experimentally generated low-resolution Hi-C datasets belonging to various levels of read sparsities originating from three cell lines on a comprehensive set of evaluation metrics. Hi-CY also includes four downstream analysis tasks, such as TAD and chromatin loops recall, to provide a thorough report on the generalizability of these methods. We observe that existing deep learning methods fail to generalize to experimentally generated sparse Hi-C datasets, showing a performance reduction of up to 57%. As a potential solution, we find that retraining deep learning-based methods with experimentally generated Hi-C datasets improves performance by up to 31%. More importantly, Hi-CY shows that even with retraining, the existing deep learning-based methods struggle to recover biological features such as chromatin loops and TADs when provided with sparse Hi-C datasets. Our study, through the Hi-CY framework, highlights the need for rigorous evaluation in the future. We identify specific avenues for improvements in the current deep learning-based Hi-C upscaling methods, including but not limited to using experimentally generated datasets for training.

Benchmarking challenging small variants with linked and long reads.

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine.

The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts' function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPMSW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10-3 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Hospital Variation in Surgical Technique for Repair of Uncomplicated Gastroschisis.

Practices in surgical repair of uncomplicated gastroschisis are varied. Data regarding hospital volume, surgical technique, clinical outcomes, and costs remain limited. Neonatal patients with uncomplicated gastroschisis were identified using the 2015-2019 National Readmissions Database. Hospital volume tertiles were determined, and sutureless or fascial repair techniques were enumerated. High volume centers (HVC) comprised the top tertile. Hospital-level variability in surgical technique was determined. Adjusted multivariable analysis was performed to compare clinical outcomes and costs among HVC and lower-volume centers and among repair techniques. Of an estimated 2903 hospitalizations meeting inclusion criteria, 23.5% occurred at HVC. There was 42.4% variation among sutureless and fascial repair techniques across all hospitals. Among HVC and lower-volume centers, there were no significant differences in rates of 30-day readmission or complication; however, HVC were associated with greater cost and length of stay. Those with codes for fascial repair technique experienced greater lengths of stay, costs, and rates of complication. Codes for surgical repair technique for uncomplicated gastroschisis vary widely, while outcomes are equivalent across strata of hospital volume. Those with codes for sutureless technique were associated with favorable clinical outcomes, irrespective of hospital volume. Guidelines for management of uncomplicated gastroschisis should account for hospital volume, variation in technique, outcomes, and resource utilization.

Unintended Perioperative Hypothermia and Surgical Site Infection in Children.

INTRODUCTION: Unintended perioperative hypothermia is associated with surgical site infection (SSI) in adults, prompting exhaustive efforts to maintain perioperative normothermia. Although these efforts are also made for pediatric patients, the association between hypothermia and SSI has not been demonstrated in children. We sought to determine whether perioperative hypothermia and other risk factors and clinical outcomes are associated with SSI in the pediatric population. MATERIALS AND METHODS: This case-control study took place from January 2014 through December 2016 and included patients at a National Surgical Quality Improvement Program-participant academic children's hospital. All surgical patients were included in this retrospective analysis. SSI rates were determined. A univariate analysis was performed to determine clinical factors associated with SSI. A multivariate regression analysis was then performed to determine the predictive effect of minimum perioperative temperature for SSI. RESULTS: This study included 3541 patients, of which 92 (2.6%) developed SSI. A univariate analysis showed associations among SSI and higher perioperative temperatures, surgical specialty of otolaryngology and general surgery, and wound classification (American Society of Anesthesiologists [ASA] classification III and IV). A multivariate analysis determined the odds of SSI increased by a factor of 1.6 for every 1°C increase in minimum perioperative temperature. CONCLUSIONS: Unintended perioperative hypothermia in our pediatric patients was inversely associated with SSI. This finding suggests that pediatric SSI prevention may not require the efforts made for adult patients to maintain normothermia.

A complete reference genome improves analysis of human genetic variation.

Compared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 million base pairs of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome for clinical and functional study. We show how this reference universally improves read mapping and variant calling for 3202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of variants per sample in previously unresolved regions, showcasing the promise of the T2T-CHM13 reference for evolutionary and biomedical discovery. Simultaneously, this reference eliminates tens of thousands of spurious variants per sample, including reduction of false positives in 269 medically relevant genes by up to a factor of 12. Because of these improvements in variant discovery coupled with population and functional genomic resources, T2T-CHM13 is positioned to replace GRCh38 as the prevailing reference for human genetics.

Association of research timing with surgery resident perceptions of operative autonomy and satisfaction: A multi-institutional study.

BACKGROUND: General surgery residents commonly engage in research years after the second (Post-postgraduate year 2 [PostPGY2]) or third (PostPGY3) clinical training year. The impact of dedicated research training timing on training experience is unknown. Our aim was to examine the progression of residents' perceived meaningful operative autonomy and evaluate career satisfaction, in relation to research timing. METHODS: Categorical surgery residents with 2-year research requirements were surveyed regarding perceived autonomy for laparoscopic appendectomy, laparoscopic cholecystectomy, and right hemicolectomy and satisfaction with the impact of dedicated research training on professional development. Meaningful operative autonomy was defined as Zwisch scores ≥3 (passive help or supervision only). RESULTS: Residents from 17 programs participated (n = 233, 30.6%); 48% were PostPGY2. PostPGY3 residents were more likely to perceive meaningful operative autonomy when starting dedicated research training (laparoscopic appendectomy: 98% vs 74%, P < .001; laparoscopic cholecystectomy: 87% vs 48%, P < .001; right hemicolectomy: 27% vs 3%, P < .001). Meaningful operative autonomy declined during dedicated research training but was still higher for PostPGY3 residents for laparoscopic appendectomy (84% vs 42%, P < .001) and laparoscopic cholecystectomy (68% vs 30%, P < .001). By PGY4, PostPGY2 residents reported rates of meaningful operative autonomy comparable to PostPGY3 through training completion. A higher proportion of PostPGY3 residents reported dedicated research training satisfaction (90% vs 78%, P = .01). Training at PostPGY3 programs (odds ratio, 3.06, 95% confidence interval, 1.38-6.80) and postresearch training stage (compared with preresearch residents, odds ratio, 3.25, 95% confidence interval, 1.06-10.0) were independently associated with satisfaction. CONCLUSION: Significant differences existed in the progression of perceived operative autonomy and dedicated research training satisfaction between PostPGY2 and PostPGY3 residents. These results could help surgical educators make individualized decisions regarding research timing to promote surgical skill acquisition and resident well-being.

Curated variation benchmarks for challenging medically relevant autosomal genes.

The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

Effect of teleconferencing variables on faculty impression of mock residency applicants.

Purpose: The objective of this study was to assess how teleconferencing variables influence faculty impressions of mock residency applicants. Methods: In October 2020, we conducted an online experiment studying five teleconferencing variables: background, lighting, eye contact, internet connectivity, and audio quality. We created interview videos of three mock residency applicants and systematically modified variables in control and intervention conditions. Faculty viewed the videos and rated their immediate impression on a 1-10 scale. The effect of each variable was measured as the mean difference between the intervention and control impression ratings. One-way analysis of variance (ANOVA) was performed to assess whether ratings varied across applicants. Paired-samples Wilcoxon signed-rank tests were conducted to assess the significance of the effect of each variable. Results: Of 711 faculty members who were emailed a link to the experiment, 97 participated (13.6%). The mean ratings for control videos were 8.1, 7.2, and 7.6 (P < .01). Videos with backlighting, off-center eye contact, choppy internet connectivity, or muffled audio quality had lower ratings when compared with control videos (P < .01). There was no rating difference between home and conference room backgrounds (P = .77). Many faculty participants reported that their immediate impressions were very much or extremely influenced by audio quality (60%), eye contact (57%), and internet connectivity (49%). Conclusions: Teleconferencing variables may serve as a source of assessment bias during residency interviews. Mock residency applicants received significantly lower ratings when they had off-center eye contact, muffled audio, or choppy internet connectivity, compared to optimal teleconferencing conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s44186-022-00053-w.

Persistence of Gender Bias Over Four Decades of Surgical Training.

OBJECTIVE: Female surgeons face gender-specific obstacles during residency training, yet longitudinal data on gender bias experienced by female surgery residents are lacking. We aimed to investigate the evolution of gender bias, identify obstacles experienced by female general surgery residents, and discuss approaches to supporting female surgeons during residency training. METHODS: Between August 2019 and January 2021, we conducted a retrospective cohort study using structured telephone interviews of female graduates of the UCLA General Surgery Residency training program. Responses of early graduates (1981-2009) were compared with those of recent graduates (2010-2020). Quantitative data were compared with Fisher's exact tests and Chi-squared tests. Interview responses were reviewed to catalog gender bias, obstacles experienced by female surgeons, and advice offered to training programs to address women's concerns. RESULTS: Of 61 female surgery residency graduates, 37 (61%) participated. Compared to early graduates (N = 20), recent graduates (N = 17) were significantly more likely to pursue fellowship training (100% vs. 65%, p < 0.01) and have children before or during residency (65% vs. 25%, p = 0.02). A substantial proportion in each cohort experienced some form of gender bias (71% vs. 85%, p = 0.43). Compared to early graduates, recent graduates were significantly less likely to report experiencing explicit gender bias (12% vs. 50%, p = 0.02) but equally likely to report implicit gender bias (71% vs. 55%, p = 0.50). Female graduates across the decades advocated for specific measures to champion work-life balance in residency (51%), strengthen female mentorship (49%), increase childcare support (41%), and promote women into leadership positions (32%). CONCLUSIONS: While having children during residency has become more common and accepted over the decades, female surgery residents continue to experience implicit gender bias in the workplace. Female surgeons advocate for targeted interventions to establish systems for parental leave, address gender bias, and strengthen female mentorship.