Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. ...Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.
Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we ...sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.
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•Single-cell RNA-seq enabled molecular profiling of rare human pancreatic cells•Subpopulations were identified within endocrine and exocrine cell types•Genes associated with obesity or diabetes had correlating expression with BMI•Transcriptional alterations found in type 2 diabetic individuals
Segerstolpe et al. use single-cell transcriptomics to generate transcriptional profiles of individual pancreatic endocrine and exocrine cells of healthy and type 2 diabetic donors. They revealed cell-type-specific gene expression and novel subpopulations, as well as gene correlations to BMI and gene expression alterations in diabetes.
Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an ...explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis.
Induction of trained immunity by Bacille-Calmette-Guérin (BCG) vaccination mediates beneficial heterologous effects, but the mechanisms underlying its persistence and magnitude remain elusive. In ...this study, we show that BCG vaccination in healthy human volunteers induces a persistent transcriptional program connected to myeloid cell development and function within the hematopoietic stem and progenitor cell (HSPC) compartment in the bone marrow. We identify hepatic nuclear factor (HNF) family members 1a and b as crucial regulators of this transcriptional shift. These findings are corroborated by higher granulocyte numbers in BCG-vaccinated infants, HNF1 SNP variants that correlate with trained immunity, and elevated serum concentrations of the HNF1 target alpha-1 antitrypsin. Additionally, transcriptomic HSPC remodeling was epigenetically conveyed to peripheral CD14+ monocytes, displaying an activated transcriptional signature three months after BCG vaccination. Taken together, transcriptomic, epigenomic, and functional reprogramming of HSPCs and peripheral monocytes is a hallmark of BCG-induced trained immunity in humans.
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•Human BCG vaccination induces a persistent innate immune training of CD14+ monocytes•BCG vaccination imprints a persistent transcriptomic myeloid bias on human HSPCs•Hepatic nuclear factors are regulators of BCG-induced trained immunity in HSPCs•BCG induces persistent epigenetic changes in peripheral CD14+ monocytes
Cirovic and de Bree et al. investigate the effects of BCG vaccination in humans and reveal the induction of a transcriptomic rewiring of human stem and progenitor cells toward the myeloid cell lineage, instructed by hepatic nuclear factors, resulting in epigenetic and functional changes within CD14+ peripheral monocytes.
Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, ...malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We ...present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform while retaining a high degree of flexibility, potentially also for other high-throughput applications.
Objective Colorectal cancer (CRC) has a substantial heritable component. Common genetic variation has been shown to contribute to CRC risk. A study was conducted in a large multi-population study to ...assess the feasibility of CRC risk prediction using common genetic variant data combined with other risk factors. A risk prediction model was built and applied to the Scottish population using available data. Design Nine populations of European descent were studied to develop and validate CRC risk prediction models. Binary logistic regression was used to assess the combined effect of age, gender, family history (FH) and genotypes at 10 susceptibility loci that individually only modestly influence CRC risk. Risk models were generated from case-control data incorporating genotypes alone (n=39 266) and in combination with gender, age and FH (n=11 324). Model discriminatory performance was assessed using 10-fold internal cross-validation and externally using 4187 independent samples. The 10-year absolute risk was estimated by modelling genotype and FH with age- and gender-specific population risks. Results The median number of risk alleles was greater in cases than controls (10 vs 9, p<2.2×10−16), confirmed in external validation sets (Sweden p=1.2×10−6, Finland p=2×10−5). The mean per-allele increase in risk was 9% (OR 1.09; 95% CI 1.05 to 1.13). Discriminative performance was poor across the risk spectrum (area under curve for genotypes alone 0.57; area under curve for genotype/age/gender/FH 0.59). However, modelling genotype data, FH, age and gender with Scottish population data shows the practicalities of identifying a subgroup with >5% predicted 10-year absolute risk. Conclusion Genotype data provide additional information that complements age, gender and FH as risk factors, but individualised genetic risk prediction is not currently feasible. Nonetheless, the modelling exercise suggests public health potential since it is possible to stratify the population into CRC risk categories, thereby informing targeted prevention and surveillance.
The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009 (Tang et al. Nat Methods 6:377-382, 2009). While the first methods analyzed ...just a handful of cells, the throughput and performance rapidly increased over a very short time span. However, it was not until the introduction of emulsion droplets methods, such as the well-known kits commercialized by 10x Genomics, that the robust and reproducible analysis of thousands of cells became feasible (Zheng et al Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049, 2017). Despite generating data at a speed and a cost per cell that remains unmatched for full-length protocols like Smart-seq (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708-714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096-1098, 2013), scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the entire transcriptome.Building upon the existing Smart-seq2/3 workflows (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708-714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096-1098, 2013), we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than previous versions, requiring limited hands-on time and with a great potential for customization (Hahaut et al. Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq. http://biorxiv.org/lookup/doi/10.1101/2021.07.14.452217. https://doi.org/10.1101/2021.07.14.452217, 2021). Here, we present three variants of the FS protocol.Standard FLASH-seq (FS), which builds upon Smart-seq2 developed in the past, is non-stranded and does not use unique molecular identifiers (UMIs) but still remains the easiest method to measure gene expression in a cell population.FLASH-seq low-amplification (FS-LA) represents the fastest method, which generates sequencing-ready libraries in 4.5 h, without sacrificing performance.FLASH-seq with UMIs (FS-UMI) builds upon the same principle as Smart-seq3 and introduces UMIs for molecule counting and isoform reconstruction. The newly designed template-switching oligonucleotide (TSO) contains a 5-bp spacer, which allows the generation of high-quality data while minimizing the amount of strand-invasion artifacts.
Abstract The genetic susceptibility to colorectal cancer (CRC) has been estimated to be around 35% and yet high-penetrance germline mutations found so far explain less than 5% of all cases. Much of ...the remaining variations could be due to the co-inheritance of multiple low penetrant variants. The identification of all the susceptibility alleles could have public health relevance in the near future. To test the hypothesis that what are considered polymorphisms in human CRC genes could constitute low-risk alleles, we selected eight common SNPs for a pilot association study in 1785 cases and 1722 controls. One SNP, rs3219489:G>C (MUTYH Q324H) seemed to confer an increased risk of rectal cancer in homozygous status (OR = 1.52; CI = 1.06–2.17). When the analysis was restricted to our ‘super-controls’, healthy individuals with no family history for cancer, also rs1799977:A>G (MLH1 I219V) was associated with an increased risk in both colon and rectum patients with an odds ratio of 1.28 (CI = 1.02–1.60) and 1.34 (CI = 1.05–1.72), respectively (under the dominant model); while 2 SNPs, rs1800932:A>G (MSH6 P92P) and rs459552:T>A (APC D1822V) seemed to confer a protective effect. The latter, in particular showed an odds ratio of 0.76 (CI = 0.60–0.97) among colon patients and 0.73 (CI = 0.56–0.95) among rectal patients. In conclusion, our study suggests that common variants in human CRC genes could constitute low-risk alleles.
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