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Non-invasive prenatal testing (NIPT) to detect fetal aneuploidy by sequencing the cell-free DNA (cfDNA) in maternal plasma is being broadly adopted. To detect fetal aneuploidies from maternal plasma, where fetal DNA is mixed with far-larger amounts of maternal DNA, NIPT requires a minimum fraction of the circulating cfDNA to be of placental origin, a level which is usually attained beginning at 10 weeks gestational age. We present an approach that leverages the arrangement of alleles along homologous chromosomes—also known as chromosomal phase—to make NIPT analyses more conclusive. We validate our approach with in silico simulations, then re-analyze data from a pregnant mother who, due to a fetal DNA fraction of 3.4%, received an inconclusive aneuploidy determination through NIPT. We find that the presence of a trisomy 18 fetus can be conclusively inferred from the patient’s same molecular data when chromosomal phase is incorporated into the analysis. Key to the effectiveness of our approach is the ability of homologous chromosomes to act as natural controls for each other and the ability of chromosomal phase to integrate subtle quantitative signals across very many sequence variants. These results show that chromosomal phase increases the sensitivity of a common laboratory test, an idea that could also advance cfDNA analyses for cancer detection.

Meiosis, although essential for reproduction, is also variable and error-prone: rates of chromosome crossover vary among gametes, between the sexes, and among humans of the same sex, and chromosome missegregation leads to abnormal chromosome numbers (aneuploidy)1–8. To study diverse meiotic outcomes and how they covary across chromosomes, gametes and humans, we developed Sperm-seq, a way of simultaneously analysing the genomes of thousands of individual sperm. Here we analyse the genomes of 31,228 human gametes from 20 sperm donors, identifying 813,122 crossovers and 787 aneuploid chromosomes. Sperm donors had aneuploidy rates ranging from 0.01 to 0.05 aneuploidies per gamete; crossovers partially protected chromosomes from nondisjunction at the meiosis I cell division. Some chromosomes and donors underwent more-frequent nondisjunction during meiosis I, and others showed more meiosis II segregation failures. Sperm genomes also manifested many genomic anomalies that could not be explained by simple nondisjunction. Diverse recombination phenotypes—from crossover rates to crossover location and separation, a measure of crossover interference—covaried strongly across individuals and cells. Our results can be incorporated with earlier observations into a unified model in which a core mechanism, the variable physical compaction of meiotic chromosomes, generates interindividual and cell-to-cell variation in diverse meiotic phenotypes. Thousands of sperm genomes have been analysed with a new method called Sperm-seq, revealing interconnected meiotic variation at the single-cell and person-to-person levels, and suggesting chromosome compaction as a way to explain the relationships between diverse recombination phenotypes.

SUMMARYHuman pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

SUMMARYVariation in the human genome contributes to abundant diversity in human traits and vulnerabilities, but the underlying molecular and cellular mechanisms are not yet known, and will need scalable approaches to accelerate their recognition. Here, we advanced and applied an experimental platform that analyzes genetic, molecular, and phenotypic heterogeneity across cells from very many human donors cultured in a single, shared in vitro environment, with algorithms (Dropulation and Census-seq) for assigning phenotypes to individual donors. We used natural genetic variation and synthetic (CRISPR-Cas9) genetic perturbations to analyze the vulnerability of neural progenitor cells to infection with Zika virus. These analyses identified a common variant in the antiviral IFITM3 gene that regulated IFITM3 expression and explained most inter-individual variation in NPCs’ susceptibility to Zika virus infectivity. These and other approaches could provide scalable ways to recognize the impact of genes and genetic variation on cellular phenotypes.HIGHLIGHTSMeasuring cellular phenotypes in iPSCs and hPSC-derived NPCs from many donorsEffects of donor sex, cell source, genetic and other variables on hPSC RNA expressionNatural genetic variation and synthetic perturbation screens both identify IFITM3 in NPC susceptibility to Zika virusA common genetic variant in IFITM3 explains most inter-individual variation in NPC susceptibility to Zika virus

PURPOSE: This pilot study sought to (1) validate the use of a novel technology for single-sperm-cell genome sequencing (Sperm-seq) in infertile men who may not have optimal quantity or quality of sperm for genomic analysis and (2) compare these results to fertile donors. METHODS: Infertile men undergoing IVF with female partners with a previous history of failed fertilization with ICSI (FF) or poor blastulation of embryos (PB) were recruited from a large IVF center. Sperm-seq was used to analyze thousands of individual sperm and was carried out at an affiliated university research institute. Global aneuploidy rate, crossover locations, and crossover frequencies were assessed in the infertile population, and compared with a control group of 20 fertile donors, which were analyzed previously at the same laboratory. RESULTS: Eight patients were initially included, but 3 samples did not yield high-quality genomic data for analysis. A total of 10,042 sperm were analyzed from 5 patients, 2 in the FF group, and 3 in the PB group. The global aneuploidy rate among the samples was 2–4%, similar to the control group. Likewise, crossover locations and frequencies were similar. CONCLUSION: Sperm-seq provides a robust analysis but may not be applicable to all male infertility cases due to technical limitations. This group of male infertility patients did not have higher rates of aneuploidy or abnormal crossover patterns compared to a fertile donor population. Our data may suggest that FF and PB phenotypes may not be related to sperm aneuploidy or meiotic errors but rather to other intrinsic nuclear anomalies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02300-3.

SummaryTens of thousands of genetic variants shape human phenotypes, mostly by unknown cellular mechanisms. Here we describe Census-seq, a way to measure cellular phenotypes in cells from many people simultaneously. Analogous to pooled CRISPR screens but for natural variation, Census-seq associates cellular phenotypes to donors’ genotypes by quantifying the presence of each donor’s DNA in cell “villages” before and after sorting or selection for cellular traits of interest. Census-seq enables population-scale cell-biological phenotyping with low cost and high internal control. We demonstrate Census-seq through investigation of genetic effects on the SMN protein whose deficiency underlies spinal muscular atrophy (SMA). Census-seq quantified and mapped effects of many common alleles on SMN protein levels and response to SMN-targeted therapeutics, including a common, cryptic non-responder allele. We provide tools enabling population-scale cell experiments and explain how Census-seq can be used to map genetic effects on diverse cell phenotypes.Abstract FigureHighlightsCensus-seq reveals how inherited genetic variation affects cell phenotypesGenetic analysis of cellular traits in cell villages of >100 donorsCharacterizing human alleles that shape SMN protein expression and drug responsesDevelopment of protocols and software to enable cellular population genetics

Quantifying SARS-CoV-2 infectivity, formulating well-calibrated public-health policy, and managing the safety of workplaces would all be facilitated by precise measurement of the extent to which SARS-CoV-2 RNA is present in an intact form in biological specimens and human environments. We describe assays that use digital PCR in nanoliter droplets (droplet digital PCR) to measure these properties. Such assays could be broadly deployed to inform COVID-19 epidemiology, measure symptomatic and asymptomatic infectivity, and help manage the safety of environments in which people live, move, and work.

Sperm-seq is a high-throughput, droplet-based single-sperm sequencing technology capable of generating thousands of cell-barcoded single-sperm sequencing libraries at one time. This protocol describes Sperm-seq library generation, featuring a full protocol for sperm preparation and suggestions for droplet-based sequencing methods from 10X Genomics to employ. Sperm preparation can be completed in half a day and the full protocol can be completed in 2-3 days, with several wait times and break points.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

Study objectives Workers in the nylon flockingindustry recently have been found to be at increased risk of chronicnongranulomatous interstitial lung disease. Although a spectrum ofcytologic and histopathologic abnormalities has been observed, nonspecific interstitial pneumonia, lymphoid nodules, and lymphocyticbronchiolitis predominated in the 19 previously reported cases of flockworker's lung. Here we describe five additional patients who appear toexpand the histopathologic spectrum and add to the evidence suggestinga causative role for respirable-sized nylon fragments. Methods We studied all North American patients(n = 5) found in 1998 to satisfy our previous case definition offlock worker's lung. Two pulmonary pathologists independently reviewedeach biopsy specimen. Results All five patientsreported cough and dyspnea. Only one patient had crackles on chestauscultation. High-resolution CT scan, interpreted with attention tosubtle ground-glass attenuation, remained a highly sensitive diagnostictest. Pulmonary function tests and plain chest radiograph were lesssensitive. One patient's wedge biopsy showed previously describedprototypical findings. Two others had transbronchial biopsies showingsome of the same features. The fourth patient's wedge biopsy showeddesquamative interstitial pneumonia. The fifth patient had bilateralsynchronous adenocarcinoma but with radiographic evidence of diffuseinterstitial fibrosis. These 5 patients and the 19 patients studiedpreviously were exposed to nylon flock manufactured by a rarely usedcutting technology. Conclusion Findings in these fivepatients appear to broaden the spectrum of the clinicopathology offlock worker's lung and add to the evidence incriminatingrespirable-sized nylon particulates produced during the manufacture anduse of rotary-cut nylon flock.

The diagnosis of papillary carcinoma of the thyroid metastatic to the lung frequently requires a battery of noninvasive tests. Occasionally, invasive procedures such as open lung biopsy, transthoracic needle biopsy, and transbronchial lung biopsy are employed to confirm the diagnosis. A 31-yr-old woman with papillary thyroid carcinoma treated previously by a near-total thyroidectomy and 131I ablation presented to our clinic with shortness of breath and a clear chest roentgenogram. A post-131I treatment whole body scan revealed widespread 131I pulmonary uptake, and the presence of papillary thyroid cancer was confirmed by bronchoalveolar lavage. We conclude that bronchoalveolar lavage should be considered when tissue confirmation of metastatic papillary carcinoma to the lung is needed. During the evaluation and follow-up of this patient, we were able to determine that metastatic papillary carcinoma to the lung may cause a methacholine bronchoprovocation test to be falsely positive for asthma.

Background: It is not clear whether careful history taking with detailed questioning of the characteristics of cough is diagnostically useful. Objective: To determine if the character, timing, or complications of chronic cough were helpful in determining its cause. Methods: A prospective, descriptive study of consecutive, unselected, immunocompetent patients referred to our university outpatient clinic because of chronic cough. All patients were evaluated by a previously published and validated systematic diagnostic protocol, a self-administered questionnaire, and by observing the character of involuntary and voluntary coughs. The final diagnosis of the cause of cough required fulfillment of pre pretreatment criteria plus having cough disappear or substantially improve as a complaint with specific therapy. Results: Eighty-eight patients met inclusion criteria and were fully evaluated. The mean±SD age was 53.1 ± 16 years (range, 15-83 years) and 24 were males and 64 were females with a mean±SD history of cough for 6.6±9.8 years (range, 1 month-44 years). The cause of chronic cough was established in 86 (98%) of 88 patients. Eighty-one (92%) of 88 had cough disappear as a complaint. Cough was as a result of a single cause in 39% and multiple causes in 59%. Gastroesophageal reflux disease, postnasal drip syndrome, and asthma were the 3 most common causes of chronic cough and accounted for 90% % of diagnoses. Gastroesophageal reflux disease, postnasal drip syndrome, and asthma were again found to be the 3 most common causes of chronic cough irrespective of patient estimated quantity of daily sputum production. These 3 conditions caused chronic cough in 99.4% of patients with the following characteristics: (1) nonsmoker; (2) not receiving an angiotensin-converting enzyme inhibitor drug; and (3) normal or nearly normal and stable chest radiograph. With respect to the spectrum and frequency of diagnoses and their interrelationships with the character, timing, and complications of cough, multiple stepwise linear regression analysis showed that none of the variability of the character, timing, or complications of cough could be explained by any specific diagnosis. Conclusions: A carefully taken history with detailed questioning of the character, timing, and complications of chronic cough is not likely to be useful in diagnosing the cause of cough. The cause can be determined and successfully treated with specific therapy in the greatest majority of cases. Chronic cough is often caused by multiple, simultaneously contributing causes. Postnasal drip syndrome, asthma, and gastroesophageal reflux disease remain the 3 most common causes of chronic cough and there is a clinical profile that nearly always predicts their presence in immunocompetent patients. (Arch Intern Med. 1996;156:997-1003)