Molecular pixelation: spatial proteomics of single cells by sequencing.

The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.

An uncertain uphill battle - experiences and consequences of living with lipedema.

PURPOSE: To describe and analyse experiences of living with lipedema. Methods: Individual, semi-structured interviews with a purposive sample of 12 women diagnosed with lipedema and analysed by qualitative content analysis utilizing an inductive approach. RESULTS: The overarching theme, "An uncertain uphill battle against a divergent body and societal ignorance", covers the experiences of living with lipedema and is based on five categories; "Captivated by a disintegrating body", "Face the impairments of a chronic condition", "Experience social exclusion", "Need emotional support to go on" and "Mull over an insecure future". The women felt entrapped within their bodies and experienced social exclusion due to the chronic symptoms and the progressive body shape alteration caused by their illness. Having experienced deficient information on the illness, varying support from other people, and a deteriorating economic situation, the women face an uncertain future. CONCLUSIONS: Symptoms and restrictions caused by lipedema affect women's livelihood and future, as there are no indications for disease improvement. Preventive work aimed at reducing health deterioration should be a priority. More research is needed to raise healthcare awareness regarding difficulties experienced by patients with lipedema.

Educational Differences in Cohort Fertility Across Sub-national Regions in Europe.

Educational differences in female cohort fertility vary strongly across high-income countries and over time, but knowledge about how educational fertility differentials play out at the sub-national regional level is limited. Examining these sub-national regional patterns might improve our understanding of national patterns, as regionally varying contextual conditions may affect fertility. This study provides for the first time for a large number of European countries a comprehensive account of educational differences in the cohort fertility rate (CFR) at the sub-national regional level. We harmonise data from population registers, censuses, and large-sample surveys for 15 countries to measure women's completed fertility by educational level and region of residence at the end of the reproductive lifespan. In order to explore associations between educational differences in CFRs and levels of economic development, we link our data to regional GDP per capita. Empirical Bayesian estimation is used to reduce uncertainty in the regional fertility estimates. We document an overall negative gradient between the CFR and level of education, and notable regional variation in the gradient. The steepness of the gradient is inversely related to the economic development level. It is steepest in the least developed regions and close to zero in the most developed regions. This tendency is observed within countries as well as across all regions of all countries. Our findings underline the variability of educational gradients in women's fertility, suggest that higher levels of development may be associated with less negative gradients, and call for more in-depth sub-national-level fertility analyses by education.

Death Is Not the End: A Register-Based Study of the Effect of Parental Death on Adult Children's Childbearing Behavior in Sweden.

Macro-level studies have shown that rapid increases in mortality can affect fertility rates. Parental death has also been linked to negative psychological and physical outcomes, reduced relationship quality, and making bereaved children attach more importance to their families. No prior study has examined whether parental death influences adult children's fertility at the microlevel. This study applies event history techniques to Swedish multigeneration registers listing 1.5 million individuals with micro data on mortality and fertility to investigate short-term (first birth risk) and long-term (childlessness at age 45) effects of parental death on adult children's fertility. The principal finding is that parental death during reproductive age affects children's fertility and this effect is mainly short term. The effects differ to some degree between men and women and depend on the stage of the life course in which the bereavement occurs. Younger individuals experiencing a parental death have a significantly higher first birth risk after the parental death compared with peers who did not experience a parental death. Individuals older than 23 who experience a parental death have no or lower first birth risk after the parental death compared with baseline. Men, compared with women, are more likely to end childless if they experience a parental death.

Arteria: An automation system for a sequencing core facility.

BACKGROUND: In recent years, nucleotide sequencing has become increasingly instrumental in both research and clinical settings. This has led to an explosive growth in sequencing data produced worldwide. As the amount of data increases, so does the need for automated solutions for data processing and analysis. The concept of workflows has gained favour in the bioinformatics community, but there is little in the scientific literature describing end-to-end automation systems. Arteria is an automation system that aims at providing a solution to the data-related operational challenges that face sequencing core facilities. FINDINGS: Arteria is built on existing open source technologies, with a modular design allowing for a community-driven effort to create plug-and-play micro-services. In this article we describe the system, elaborate on the underlying conceptual framework, and present an example implementation. Arteria can be reduced to 3 conceptual levels: orchestration (using an event-based model of automation), process (the steps involved in processing sequencing data, modelled as workflows), and execution (using a series of RESTful micro-services). This creates a system that is both flexible and scalable. Arteria-based systems have been successfully deployed at 3 sequencing core facilities. The Arteria Project code, written largely in Python, is available as open source software, and more information can be found at https://arteria-project.github.io/ . CONCLUSIONS: We describe the Arteria system and the underlying conceptual framework, demonstrating how this model can be used to automate data handling and analysis in the context of a sequencing core facility.

Education, Gender, and Cohort Fertility in the Nordic Countries.

Systematic comparisons of fertility developments based on education, gender and country context are rare. Using harmonized register data, we compare cohort total fertility and ultimate childlessness by gender and educational attainment for cohorts born beginning in 1940 in four Nordic countries. Cohort fertility (CTF) initially declined in all four countries, although for cohorts born in the 1950s and later, the CTF remained stable or declined only modestly. Childlessness, which had been increasing, has plateaued in Denmark, Norway and Sweden. Women's negative educational gradient in relation to total fertility has vanished, except in Finland, while men's positive gradient has persisted. The highest level of men's childlessness appears among the least educated. In the oldest female cohorts, childlessness was highest among the highly educated, but these patterns have changed over the cohorts as childlessness has increased among the low educated and remained relatively stable among higher educated women. In Denmark, Norway and Sweden, childlessness is now highest among the least educated women. We witness both a new gender similarity and persistent (among men) and new (among women) educational disparities in childbearing outcomes in the Nordic region. Overall, the number of low educated has decreased remarkably over time. These population segments face increasing social and economic disadvantages that are reflected as well in their patterns of family formation.

Fecundity and human birth seasonality in Sweden: a register-based study.

BACKGROUND: It is well-established that couples' fecundities vary widely. Each couple has a relatively constant monthly probability of conceiving, which can vary from zero to quite high. This underlying probability is usually expressed as the time (number of menstrual cycles) the couple requires to conceive. Couples with high fecundity will, on average, need fewer cycles than couples with low fecundity. It is also well-documented that almost all human populations exhibit seasonal variation in births. Most European countries show seasonal variation that usually peak in the spring and are the lowest during the last quarter of the year. The increasingly strong pattern of depressed birth rates in November and December is likely explained by the December-January cut-off threshold for Swedish pupils' school entry and their parents increasing awareness of the negative effects on school outcomes for children who are juniors in the school-entry cohort they belong to. To actively plan births for a specific time of the year, couples need to have some knowledge of the time required for them to conceive. METHODS: We use the duration between marriage of childless couples and first birth as a proxy measure of couples' fecundity. Based on this time-to-pregnancy measure we study to what extent couples' capacity to conceive affects the seasonality of their second births. We hypothesize that in a society with highly controlled fertility and a strong norm of having at least two children, sub-fertile couples will on average show less seasonal variation in second births. Sub-fertile couples, requiring more time to conceive the first time, will be less likely to try to target a desired birth month for their second child because doing so could jeopardize fulfilling their desired family size. We apply multinomial logistic regressions on 81,998 Swedish couples who married while being childless and subsequently gave birth to at least two children between 1990 and 2012, to investigate fecundity's role in seasonal variation in second births. RESULTS: We found that seasonal variation in second births was strongly associated with couples' observed fecundity, measured as the duration between marriage formation and first birth. Our results support the hypothesis that sub-fertile couples, requiring more time to conceive the first time, show less seasonal variation in second births. The seasonal variations in second order births among couples with normal fecundity shows some similarities to traditional patterns as seen in agricultural and industrial societies of the past, with high numbers of births during the spring, and low numbers during the last quarter of the year. However, two important differences are notable. The characteristic Christmas peak in September has vanished, and the low birth rates in November and December come out much stronger than in the past. CONCLUSIONS: The birth seasonality among couples with normal fecundity are what we would expect if couples actively plan their births according to the cut-off date for Swedish pupils' school entry. We argue that our findings support the notion that scheduled childbirth is a reality in contemporary sociality.

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data.

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Parents' Preferences for Sex of Children in Sweden: Attitudes and Outcomes.

It has been argued that preferences for the sex of children would be small or non-existing in relatively gender equal societies. However, previous studies have suggested that a stronger preference for having daughter exists in Scandinavian countries, which are frequently noted for being among the most gender equal societies in the world. Combining new register data on birth rates by sex of the previous children and recent survey data on couples' stated preferences for the sex of children, we show that the preference for daughters has increased in Sweden over the last decade. In addition to the stronger preference for having daughters among two-child mothers documented in previous research, our findings show that during the previous decade this preference was noticeable also among one-child parents. Despite Swedish society being known for holding gender equal social norms, interviewed parents openly expressed some degree of preference for having daughters over sons.

SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population.

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.

Transcriptome sequencing in pediatric acute lymphoblastic leukemia identifies fusion genes associated with distinct DNA methylation profiles.

BACKGROUND: Structural chromosomal rearrangements that lead to expressed fusion genes are a hallmark of acute lymphoblastic leukemia (ALL). In this study, we performed transcriptome sequencing of 134 primary ALL patient samples to comprehensively detect fusion transcripts. METHODS: We combined fusion gene detection with genome-wide DNA methylation analysis, gene expression profiling, and targeted sequencing to determine molecular signatures of emerging ALL subtypes. RESULTS: We identified 64 unique fusion events distributed among 80 individual patients, of which over 50% have not previously been reported in ALL. Although the majority of the fusion genes were found only in a single patient, we identified several recurrent fusion gene families defined by promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5, and ZNF384, or recurrent fusion genes, such as DUX4-IGH. Our data show that patients harboring these fusion genes displayed characteristic genome-wide DNA methylation and gene expression signatures in addition to distinct patterns in single nucleotide variants and recurrent copy number alterations. CONCLUSION: Our study delineates the fusion gene landscape in pediatric ALL, including both known and novel fusion genes, and highlights fusion gene families with shared molecular etiologies, which may provide additional information for prognosis and therapeutic options in the future.

Recommendations on e-infrastructures for next-generation sequencing.

With ever-increasing amounts of data being produced by next-generation sequencing (NGS) experiments, the requirements placed on supporting e-infrastructures have grown. In this work, we provide recommendations based on the collective experiences from participants in the EU COST Action SeqAhead for the tasks of data preprocessing, upstream processing, data delivery, and downstream analysis, as well as long-term storage and archiving. We cover demands on computational and storage resources, networks, software stacks, automation of analysis, education, and also discuss emerging trends in the field. E-infrastructures for NGS require substantial effort to set up and maintain over time, and with sequencing technologies and best practices for data analysis evolving rapidly it is important to prioritize both processing capacity and e-infrastructure flexibility when making strategic decisions to support the data analysis demands of tomorrow. Due to increasingly demanding technical requirements we recommend that e-infrastructure development and maintenance be handled by a professional service unit, be it internal or external to the organization, and emphasis should be placed on collaboration between researchers and IT professionals.

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia.

BACKGROUND: We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL. RESULTS: We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations ('other' subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5. CONCLUSIONS: Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.

The mutational landscape in pediatric acute lymphoblastic leukemia deciphered by whole genome sequencing.

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

Deep amplicon sequencing of preselected isolates of Parascaris equorum in ß-tubulin codons associated with benzimidazole resistance in other nematodes.

BACKGROUND: The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the ß-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the ß-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed. METHODS: Two isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100,000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole. RESULTS: No SNPs were observed in codons 167, 198 or 200 of the ß-tubulin isotype 1 or 2 genes of P. equorum, even though 100,000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples. CONCLUSIONS: We concluded that fenbendazole does not exert selection pressure on the ß-tubulin genes of isotypes 1 and 2 in P. equorum. The fact that no pre-existing SNPs were found in codons 167, 198 and 200 in P. equorum also illustrates the difficulties in generalising about AR mechanisms between different taxonomic groups of nematodes.