RESUMEN
Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host-pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples.
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COVID-19 , Transcriptoma , Humanos , Fijación del Tejido , Formaldehído , SARS-CoV-2RESUMEN
The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogeneous repertoire of effector and memory cells. How individual T cell clones contribute to this heterogeneity throughout immune responses remains largely unknown. In this study, we longitudinally track human CD8+ T cell clones expanding in response to yellow fever virus (YFV) vaccination at the single-cell level. We observed a drop in clonal diversity in blood from the acute to memory phase, suggesting that clonal selection shapes the circulating memory repertoire. Clones in the memory phase display biased differentiation trajectories along a gradient from stem cell to terminally differentiated effector memory fates. In secondary responses, YFV- and influenza-specific CD8+ T cell clones are poised to recapitulate skewed differentiation trajectories. Collectively, we show that the sum of distinct clonal phenotypes results in the multifaceted human T cell response to acute viral infections.
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Linfocitos T CD8-positivos/inmunología , Virosis/virología , Fiebre Amarilla/virología , Enfermedad Aguda , Diferenciación Celular , Células Cultivadas , HumanosRESUMEN
The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.
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Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Código de Barras del ADN Taxonómico , Visualización de Datos , Biblioteca de Genes , Genoma Humano , Haplotipos , HumanosRESUMEN
Accurate variant calling and genotyping represent major limiting factors for downstream applications of single-cell genomics. Here, we report Conbase for the identification of somatic mutations in single-cell DNA sequencing data. Conbase leverages phased read data from multiple samples in a dataset to achieve increased confidence in somatic variant calls and genotype predictions. Comparing the performance of Conbase to three other methods, we find that Conbase performs best in terms of false discovery rate and specificity and provides superior robustness on simulated data, in vitro expanded fibroblasts and clonal lymphocyte populations isolated directly from a healthy human donor.
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Mutación , Análisis de la Célula Individual , Programas Informáticos , Linfocitos T CD8-positivos , Fibroblastos , Humanos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.
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Código de Barras del ADN Taxonómico/métodos , Haplotipos , Alelos , Biblioteca de Genes , Antígenos HLA-A/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. RESULTS: The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. DISCUSSION: In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.
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Exoma/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de la Célula Individual/métodos , Humanos , Análisis de Secuencia de ADN/métodosRESUMEN
Because human white adipocytes display a high turnover throughout adulthood, a continuous supply of precursor cells is required to maintain adipogenesis. Bone marrow (BM)-derived progenitor cells may contribute to mammalian adipogenesis; however, results in animal models are conflicting. Here we demonstrate in 65 subjects who underwent allogeneic BM or peripheral blood stem cell (PBSC) transplantation that, over the entire lifespan, BM/PBSC-derived progenitor cells contribute â¼10% to the subcutaneous adipocyte population. While this is independent of gender, age, and different transplantation-related parameters, body fat mass exerts a strong influence, with up to 2.5-fold increased donor cell contribution in obese individuals. Exome and whole-genome sequencing of single adipocytes suggests that BM/PBSC-derived progenitors contribute to adipose tissue via both differentiation and cell fusion. Thus, at least in the setting of transplantation, BM serves as a reservoir for adipocyte progenitors, particularly in obese subjects.
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Adipocitos/citología , Adipogénesis , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Obesidad , Trasplante de Células Madre de Sangre Periférica , Adipocitos/metabolismo , Adolescente , Adulto , Anciano , Células de la Médula Ósea/metabolismo , Niño , Preescolar , ADN/análisis , ADN/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Obesidad/metabolismo , Grasa Subcutánea/citología , Grasa Subcutánea/metabolismo , Trasplante Homólogo , Adulto JovenRESUMEN
High-throughput sequencing platforms mainly produce short-read data, resulting in a loss of phasing information for many of the genetic variants analysed. For certain applications, it is vital to know which variant alleles are connected to each individual DNA molecule. Here we demonstrate a method for massively parallel barcoding and phasing of single DNA molecules. First, a primer library with millions of uniquely barcoded beads is generated. When compartmentalized with single DNA molecules, the beads can be used to amplify and tag any target sequences of interest, enabling coupling of the biological information from multiple loci. We apply the assay to bacterial 16S sequencing and up to 94% of the hypothesized phasing events are shown to originate from single molecules. The method enables use of widely available short-read-sequencing platforms to study long single molecules within a complex sample, without losing phase information.
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Código de Barras del ADN Taxonómico , ADN/químicaRESUMEN
AIM: Lactate dehydrogenase (LDH) increases in several critical conditions that cause cell damage and could potentially be used for early detection of serious illness in the newborn. Our aim was to investigate the relationship between the early clinical course of NICU infants and LDH in plasma at admission. METHODS: LDH was measured in a cohort of patients consecutively admitted to a major NICU in Hanoi. The infants were classified as 'obviously needing intensive care during the first week' (n = 83) or 'not receiving intensive care measures during the first week' (n = 260) by a senior neonatologist blinded to the LDH and lactate activity. RESULTS: LDH differed significantly between the groups in infants born after 32 gestational weeks. LDH differed with the vitality of the patient (F = 26.25, p < 0.0001) at admittance and correlated with lactate (R = 0.496, p < 0.0001). Also, the predictive value for obvious need of intensive care was higher for LDH than for lactate assessed by area under the curve calculated with ROC-curves [0.82 (0.77-0.88) vs. 0.67 (0.60-0.75)]. CONCLUSION: There is a strong relationship between bad clinical condition of infants during first week of life and elevated plasma LDH. The results suggest that LDH might be a valuable support in decision making in the neonatal period.
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Enfermedades del Recién Nacido/sangre , Recién Nacido/sangre , Unidades de Cuidado Intensivo Neonatal , L-Lactato Deshidrogenasa/sangre , Índice de Severidad de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , MasculinoRESUMEN
BACKGROUND: Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. METHODOLOGY/PRINCIPAL FINDINGS: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions. CONCLUSIONS/SIGNIFICANCE: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.