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scnanoseq: an nf-core pipeline for Oxford Nanopore single-cell RNA-sequencing.

MOTIVATION: Recent advancements in long-read single-cell RNA sequencing (scRNA-seq) have facilitated the quantification of full-length transcripts and isoforms at the single-cell level. Historically, long-read data would need to be complemented with short-read single-cell data in order to overcome the higher sequencing errors to correctly identify cellular barcodes and unique molecular identifiers. Improvements in Oxford Nanopore sequencing, and development of novel computational methods have removed this requirement. Though these methods now exist, the limited availability of modular and portable workflows remains a challenge. RESULTS: Here, we present, nf-core/scnanoseq, a secondary analysis pipeline for long-read single-cell and single-nuclei RNA that delivers gene and transcript-level quantification. The scnanoseq pipeline is implemented using Nextflow and is built upon the nf-core framework, enabling portability across computational environments, scalability and reproducibility of results across pipeline runs. The nf-core/scnanoseq workflow follows best practices for analyzing single-cell and single-nuclei data, performing barcode detection and correction, genome and transcriptome read alignment, unique molecular identifier deduplication, gene and transcript quantification, and extensive quality control reporting. AVAILABILITY AND IMPLEMENTATION: The source code, and detailed documentation are freely available at https://github.com/nf-core/scnanoseq and https://nf-co.re/scnanoseq under the MIT License. Documentation for the version of nf-core/scnanoseq used for this paper, including default parameters and descriptions of output files are available at https://nf-co.re/scnanoseq/1.1.0.

Single-Cell Analysis↗

Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

Colonic Neoplasms↗

The minimal genome concept.

Complete genome sequences are becoming available for a large number of diverse species. Quantification of gene content, of gene family expansion, of orthologous gene conservation, as well as their displacement, are now possible - laying the ground for the estimation of the minimal set of proteins sufficient for cellular life. The consensus of computational results suggests a set close to 300 genes. These predictions will be evaluated by engineering of small bacterial genomes.

Bacterial Proteins↗

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work.

Experiments were performed to evaluate the Amplitype PolyMarker DNA typing system for application to forensic casework. DNA extraction using chelex was compared with phenol-chloroform extraction for various biological materials including postmortem blood, blood samples used for alcohol quantification, fresh urine, envelopes and cigarette butts. Different amounts of genomic DNA were amplified to test the sensitivity of the Amplitype PM. Mixed samples of two different bloods were typed to determine the dilution at which mixtures could be detected. Different storage conditions were evaluated using urine samples. Postmortem blood samples were typed during 4 months to determine the effects of natural degradation. A population sample of 105 unrelated individuals from South-West Switzerland was analyzed and the genotype frequencies were compared with those reported by others. Finally, practical usefulness of the Amplitype PM system is illustrated by analysing casework samples. The results of this validation proved the great usefulness and sensitivity of the Amplitype PM system using the appropriate extraction and typing method. However, mixed samples had to be interpreted with caution owing to the possibility of non-specific alleles with stored material such as urine and postmortem blood.

Alleles↗

A new method for analysis of mitochondrial DNA point mutations and assess levels of heteroplasmy.

Determination of mitochondrial DNA (mtDNA) heteroplasmy for the diagnosis of patients with mitochondrial disorders is a difficult task due to the coexistence of wild-type and mutant genomes. We have developed a new method for genotyping and quantification of heteroplasmic point mutations in mtDNA based on the SNaPshot technology. We compared the data of this method with the widely used "last hot-cycle" PCR-RFLP method by studying 15 patients carrying mtDNA mutations. We showed that SNaPshot is an accurate, reproducible, and sensitive technique for the determination of heteroplasmic mtDNA mutations in different tissues from patients, and it is a promising system to be used in prenatal and postnatal diagnosis of mtDNA-associated disorders.

Cell Fusion↗

Imaging and speciation of trace elements in biological environment.

Mineral elements, often at the trace level, play a considerable role in physiology and pathology of biological systems. Metallogenomics, metalloproteomics, and metallomics are among the emerging disciplines which are critically dependent on spatially resolved concentration maps of trace elements in a cell or tissue, on information on chemical speciation, and on that on metal-binding coordination sites. The mini-review discusses recent progress in analytical techniques for element profiling on the genome scale, biological trace element imaging, and probing, identification and quantification of chemical species in the biological environment. Imaging of the element distribution in cells and tissue sections is becoming possible with sub-micrometer spatial resolution and picogram-level sensitivity owing to advances in laser ablation MS, ion beam and synchrotron radiation X-ray fluorescence microprobes. Progress in nanoflow chromatography and capillary electrophoresis coupled with element specific ICP MS and molecule-specific electrospray MS/MS and MALDI enables speciation of elements in microsamples in a complex biological environment. Laser ablation ICP MS, micro-SXRF, and micro-PIXE allow mapping of trace element distribution in 1D and 2D proteomics gels. The increasing sensitivity of EXAFS and XANES owing to the use of more intense synchrotron beams and efficient focusing optics provide information about oxidation state, fingerprint speciation of metal sites and metal-site structures.

Environmental Pollution↗

Quantitative PCR for hepatitis B virus with colorimetric detection.

A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.

Base Sequence↗

"Checkerboard" versus culture: a comparison between two methods for identification of subgingival microbiota.

The present study compared the "checkerboard" DNA-DNA hybridization methodology with culture techniques for the analysis of the composition of the subgingival microbiota. 70 subjects, presenting with a variety of periodontal conditions, contributed with a total of 283 subgingival plaque samples analyzed with respect to the following species: Porphyromonas gingivalis, Prevotella intermedia/Prevotella nigrescens, Fusobacterium nucleatum, Campylobacter rectus, Eikenella corrodens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, Streptococcus sanguis and Streptococcus mutans. Species identification and quantification was performed by (i) the checkerboard method, using whole genomic, digoxigenin labeled DNA probes; and (ii) culture, including non-selective and selective media in combination with routine biochemical testing using commercial test panels. We found that the checkerboard technology resulted in higher prevalence figures for half of the species tested when compared to culture data. If the latter were used as the reference, checkerboard detection sensitivities ranged from 0.17 to 0.86, specificities from 0.17 to 1.0, and diagnostic accuracies from 0.51 to 0.81, depending on bacterial species. The use of the checkerboard data as the reference resulted in detection sensitivities for the culture procedures between 0.24 and 1.0 and specificities between 0.21 and 0.87. The checkerboard methodology resulted in statistically significant higher bacterial counts for the majority of the species. It was further observed that, for most species, the higher the total number colony-forming units in the sample, the higher the discrepancy between the results obtained by the two techniques.

Adult↗

Asynchronous progression through the lytic cascade and variations in intracellular viral loads revealed by high-throughput single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth "delayed late" category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

Antigens, Viral↗

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence.

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.

Animals↗

Global disparities in COVID-19 vaccine coverage associated with trajectories of SARS-CoV-2 adaptation.

BACKGROUND: Vaccination serves as an effective intervention for health promotion and disease prevention across the socioecological systems and has played an important role during the COVID-19 pandemic. However, global disparities in vaccine coverage have increased uncertainty about the trajectories of viral adaptation, and the potential interplay between SARS-CoV-2 adaptation and vaccine rollout warrants further quantification. METHODS: Using over 13 million SARS-CoV-2 genomes across 86 countries from March 2020 to September 2022, we analyzed nonlinear associations between SARS-CoV-2 adaptation and vaccination coverage, considering public health and social measures, international travel, and infection dynamics, before and after the emergence of Omicron. Additionally, we examined the relationship between SARS-CoV-2 adaptation and COVID-19 mortality. RESULTS: During the pre-Omicron period, we found positive associations between nonsynonymous to synonymous divergence (dN/dS) ratios in the S1 subunit and medium levels of adjusted vaccine coverage (effect size: 0.96 [95% CI 0.47, 1.45]), while the association became insignificant at high levels (effect size: -1.89 [95% CI -4.20, 0.43]). However, no significant associations were found when Omicron dominated, possibly due to the immune escape ability of Omicron variants and the complex immune landscape shaped by mass hybrid immunity. Moreover, we observed evidence of dynamic interdependence and positive correlations between COVID-19 mortality and SARS-CoV-2 adaptation, with COVID-19 mortality interpreted as a proxy for uncontrolled viral spread. CONCLUSIONS: Our findings suggest a complex nonlinear relationship between vaccine-induced immunity and SARS-CoV-2 adaptation, with high vaccine coverage potentially linked to lower positive selection. We also observed directional coupling between COVID-19 mortality and SARS-CoV-2 adaptation. This may have implications for fair and fast vaccination in pandemic preparedness and response. CLINICAL TRIAL NUMBER: Not applicable.

Humans↗

Real-time PCR-based assay for quantitative determination of methylation status.

The best studied epigenetic modification in mammals is the methylation of cytosine. During the development and progression of malignant neoplasia, a global hypomethylation is often accompanied by a locus-specific increase in methylation. Also, during normal development specific alterations in DNA methylation patterns take place. In recent years it has become clear that in many situations only quantitative changes in methylation levels occur and that the pure qualitative detection of cytosine methylation misses important biological and pathophysiological information. Therefore, several protocols were developed for the quantitative detection of cytosine methylation. Here, we describe a real-time polymerase chain reaction-based assay for the sensitive and precise quantification of methylated and unmethylated alleles after bisulfite treatment of genomic DNA. In addition to providing quantitative methylation data, this methodology is suitable for high-throughput analysis.

Animals↗

Double-step PCR assay to quantify Epstein-Barr viral load in peripheral blood.

Posttransplant lymphoproliferative disorders (PTLD) are a severe complication arising in solid organ transplant patients. A strong correlation between Epstein-Barr virus (EBV) infection, the grade and type of immunosuppression, and the development of PTLD has been recognized. This article describes the development of a double-step polymerase chain reaction (PCR) assay for the quantification of EBV-deoxyribonucleic acid (DNA) to monitor EBV infection. Screening of samples containing >/=10(3) viral genomes/10(5) peripheral blood mononuclear cells (PBMC) or 100 micro L serum by a semiquantitative PCR assay is followed by quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Screening by semiquantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. Our double-step PCR assay can be employed in EBV viral load measurement in PBMC and serum samples to monitor transplanted patients at risk to develop PTLD.

Cells, Cultured↗

Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders.

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.

Calibration↗

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.

Alleles↗

Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression.

The moss Physcomitrella patens is an excellent tool to study plant gene-function relationships due to its high rate of homologous recombination (HR). It has also been shown to be very useful in the production of recombinant proteins which are secreted into a simple medium. Thus, there is a need for suitable promoters functional in this well established model organism. We isolated genomic flanking regions of the beta-tubulin gene family from Physcomitrella, concentrating on those family members showing high transcript abundance integrated over gametophytic tissues. Using a novel, fast and reliable quantification assay based on the transient expression and secretion of a recombinant human protein, three genomic upstream regions were characterised in serial deletion constructs. Expression rates were up to three times higher than those obtained with the 35S cauliflower mosaic virus (35S) promoter, which served as a reference.

5' Untranslated Regions↗

Quantification of Marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection.

A quantitative assay was developed for Marek's disease virus (MDV). The assay determines the numbers of viral genomes present in samples by polymerase chain reaction (PCR) amplification of a portion of the viral genome for a restricted number of cycles. Fluorescent-tagged primers are used for the PCR amplification which allows quantification of the fluorescent product. Previously, quantitation of Marek's disease virus has required plaque assays, which are laborious and potentially error-prone, and this had limited quantitative comparisons. The PCR assay is rapid, less laborious and can be applied to high levels of accuracy, since replicate assays can be carried out relatively easily. The PCR-based assay assesses the number of viral genomes present in the sample, rather than the numbers of infected cells measured in the plaque assay, however correlation between the two assays is high, suggesting viral copy number per cell may be rather uniform. In crosses between genetically resistant and susceptible animals the PCR-based assay was correlated significantly with subsequent development of disease, and was a better predictor than the plaque assay of the likelihood of development of pathological disease in the birds studied.

Animals↗

DNA base composition of Allium genomes with different chromosome numbers.

The present report examines whether the presence of an additional chromosome can be detected as modifying the nuclear DNA amount and base composition of the cell, determined here by flow cytometry of interphasic nuclei, using four monosomic additions (chromosomes 3C, 4C, 7C and 8C transmitted from Allium cepa to Allium fistulosum L.). A. cepa differs significantly from A. fistulosum in genome size (2C DNA = 33.2 pg in A. cepa and 23.3 pg in A. fistulosum) as well as in GC content (38.7% and 39.8%, respectively). The presence of an extra chromosome of A. cepa obviously increases the nuclear DNA amount above the A. fistulosum value but also alters the apparent mean GC content. By comparing the monosomic additions and the parental background, the DNA amount and base composition of each of the four single chromosomes were calculated to quantify the GC content per chromosome and therefore to deduce their initial contribution to the A. cepa genome. Taken individually, some chromosomes are atypical in terms of GC content: the single chromosome 3C is AT-rich, having only about only 25% GC. However, the three other chromosomes examined are typical of the A. cepa genome in base composition. Indeed, this biological panel gives access to chromosomal features via a cytometric assay of nuclei. It should facilitate quantification of GC-rich repetitive sequences forming heterochromatic domains located mainly at the telomeres in the monocotyledonous A. cepa genome.

Allium↗