Single-cell sequencing of tumor-associated macrophages in a Drosophila model.

Introduction: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. Methods: We use a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (RasV12) to study dysplastic growth during early tumor progression. We performed single-cell RNA-sequencing of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Results and discussion: We identified five distinct hemocyte clusters. In addition to RasV12 larvae, we included a tumor model where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, we provide evidence for transcript transfer between hemocytes and possibly other tissues. Taken together, our data support the usefulness of Drosophila to study the response against tumors at the organismic level.

scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut.

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. IMPORTANCE The malaria parasite Plasmodium falciparum causes more than half a million deaths per year. The current treatment regimen targets the symptom-causing blood stage inside the human host. However, recent incentives in the field call for novel interventions to block parasite transmission from humans to the mosquito vector. Therefore, we need to better understand the parasite biology during its development inside the mosquito, including a deeper understanding of the expression of genes controlling parasite progression during these stages. Here, we have generated single-cell transcriptome data, covering P. falciparum's development, from gamete to ookinete inside the mosquito midgut, uncovering previously untapped parasite biology, including a repertoire of novel biomarkers to be explored in future transmission-blocking efforts. We anticipate that our study provides an important resource, which can be further explored to improve our understanding of the parasite biology as well as aid in guiding future malaria intervention strategies.

Delineating Plasmodium liver infection across space and time.

The liver is a major entry point and gatekeeper for invasive pathogens. However, high-resolution, spatiotemporal transcriptomic analysis of host-pathogen interactions has remained challenging. Afriat et al. have deconvoluted Plasmodium berghei liver-stage maturation at an unprecedented scale and discovered molecular signatures of heterogeneity during pre-erythrocytic development of malarial parasites.

Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections.

The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19. IMPORTANCE Identification of host factors affecting individual SARS-CoV-2 susceptibility will provide a better understanding of the large variations in disease severity and will identify potential factors that can be used, or targeted, in antiviral drug development. With the use of an advanced lung cell model established from several human donors, we identified cellular protease inhibitors, serpins, as host factors that restrict SARS-CoV-2 infection. The antiviral mechanism was found to be mediated by the inhibition of a serine protease, TMPRSS2, which results in a blockage of viral entry into target cells. Potential treatments with these serpins would not only reduce the overall viral burden in the patients, but also block the infection at an early time point, reducing the risk for the hyperactive immune response common in patients with severe COVID-19.

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver.

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation.

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

The spatiotemporal distribution of historical malaria cases in Sweden: a climatic perspective.

BACKGROUND: Understanding of the impacts of climatic variability on human health remains poor despite a possibly increasing burden of vector-borne diseases under global warming. Numerous socioeconomic variables make such studies challenging during the modern period while studies of climate-disease relationships in historical times are constrained by a lack of long datasets. Previous studies have identified the occurrence of malaria vectors, and their dependence on climate variables, during historical times in northern Europe. Yet, malaria in Sweden in relation to climate variables is understudied and relationships have never been rigorously statistically established. This study seeks to examine the relationship between malaria and climate fluctuations, and to characterise the spatio-temporal variations at parish level during severe malaria years in Sweden 1749-1859. METHODS: Symptom-based annual malaria case/death data were obtained from nationwide parish records and military hospital records in Stockholm. Pearson (rp) and Spearman's rank (rs) correlation analyses were conducted to evaluate inter-annual relationship between malaria data and long meteorological series. The climate response to larger malaria events was further explored by Superposed Epoch Analysis, and through Geographic Information Systems analysis to map spatial variations of malaria deaths. RESULTS: The number of malaria deaths showed the most significant positive relationship with warm-season temperature of the preceding year. The strongest correlation was found between malaria deaths and the mean temperature of the preceding June-August (rs = 0.57, p < 0.01) during the 1756-1820 period. Only non-linear patterns can be found in response to precipitation variations. Most malaria hot-spots, during severe malaria years, concentrated in areas around big inland lakes and southern-most Sweden. CONCLUSIONS: Unusually warm and/or dry summers appear to have contributed to malaria epidemics due to both indoor winter transmission and the evidenced long incubation and relapse time of P. vivax, but the results also highlight the difficulties in modelling climate-malaria associations. The inter-annual spatial variation of malaria hot-spots further shows that malaria outbreaks were more pronounced in the southern-most region of Sweden in the first half of the nineteenth century compared to the second half of the eighteenth century.

A Histone Methyltransferase Inhibitor Can Reverse Epigenetically Acquired Drug Resistance in the Malaria Parasite Plasmodium falciparum.

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC50 concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, and reversed acquired resistance to the antimalarial compound blasticidin S that is transported through PSACs. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer an integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and inhibitory antibodies.

Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites.

The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.

A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection.

Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates.

BACKGROUND: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. RESULTS: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by ~100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. CONCLUSIONS: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

Sex: how malaria parasites get turned on.

The mechanisms underlying sexual stage switching in Plasmodium spp. have hitherto remained a mystery. However, two recent studies have revealed that an apicomplexan-specific DNA-binding protein is essential for the initiation of this cell fate decision, ultimately providing the malaria community with a novel and important tool in the battle to prevent malaria transmission.

Transcriptome profiling of Giardia intestinalis using strand-specific RNA-seq.

Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most of the genome was transcribed in trophozoites grown in vitro, but at vastly different levels. RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.

Common coinfections of Giardia intestinalis and Helicobacter pylori in non-symptomatic Ugandan children.

BACKGROUND: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections. METHODOLOGY/PRINCIPAL FINDINGS: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection. CONCLUSIONS/SIGNIFICANCE: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.

Allelic sequence heterozygosity in single Giardia parasites.

BACKGROUND: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? RESULTS: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. CONCLUSIONS: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens.

In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.