High-throughput DNA Sequencing Research Articles

Immune and intestinal microbiota responses to heat stress in Chinese mitten crab (Eriocheir sinensis)

Temperature is an important environmental stressor that affects the metabolism and immunity of Chinese mitten crab. In this study, we investigated immune-related enzyme activity, metabolism-related substances in haemolymph and intestinal microbiota responses of Eriocheir sinensis assigned to the control (24 °C) and heat stress (32 °C) groups. Based on the data obtained, the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase in the heat stress group exhibited a trend of increasing first and then decreasing, and all peaked at 12 h or 24 h. The metabolism-related substances in the haemolymph, including glucose, total cholesterol, and total protein, all showed significant differences between the control and heat stress groups from the mid to late period (P < 0.05). Under heat stress, the levels of alkaline phosphatase and acid phosphatase were both significantly higher than those of the control group from 12 h to 24 h and significantly lower at 72 h and 96 h (P < 0.05). The T-AOC of the heat stress group was significantly lower than that of the control group from 48 h to 96 h (P < 0.05). High-throughput 16S ribosomal DNA (rDNA) sequencing revealed that heat stress altered the composition of intestinal microbes of Eriocheir sinensis. Specifically, the abundance of the beneficial bacteria Candidatus, Hepatoplasma and Marinifilum decreased significantly, while the abundance of the pathogenic bacteria Rhodococcus and Morganella increased significantly (P < 0.05). The results revealed that acute heat stress exposure affected immune function and intestinal microbial community in E. sinensis. This work can help to uncover the mechanisms of heat stress responses in this species.

The Parapoynx stagnalis Nucleopolyhedrovirus (PastNPV), a Divergent Member of the Alphabaculovirus Group I Clade, Encodes a Homolog of Ran GTPase.

We report the analysis of the genome of a novel Alphabaculovirus, Parapoynx stagnalis nucleopolyhedrovirus isolate 473 (PastNPV-473), from cadavers of the rice case bearer, Parapoynx stagnalis Zeller (Lepidoptera: Crambidae), collected in rice fields in Kerala, India. High-throughput sequencing of DNA from PastNPV occlusion bodies and assembly of the data yielded a circular genome-length contig of 114,833 bp with 126 annotated opening reading frames (ORFs) and six homologous regions (hrs). Phylogenetic inference based on baculovirus core gene amino acid sequence alignments indicated that PastNPV is a member of the group I clade of viruses in genus Alphabaculovirus, but different phylogenetic methods yielded different results with respect to the placement of PastNPV and four similarly divergent alphabaculoviruses in the group I clade. Branch lengths and Kimura-2-parameter pairwise nucleotide distances indicated that PastNPV-473 cannot be classified in any of the currently listed species in genus Alphabaculovirus. A unique feature of the PastNPV genome was the presence of an ORF encoding a homolog of Ran GTPase, a regulator of nucleocytoplasmic trafficking. PastNPV appears to have acquired a homolog of Ran relatively recently from a lepidopteran host via horizontal gene transfer.

Molecular landscape of immune pressure and escape in aplastic anemia.

Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.

Agricultural soil physico-chemical parameters and microbial abundance and diversity under long-run farming practices: A greenhouse study

The growth of agriculture led to indiscriminate use of synthetic pesticides or fertilizers and unsustainable crop management farming practices which can aggravate harmful impacts on the microbial population and physical and chemical characteristics of soil ecosystem. Based on this fact, the present study was planned to evaluate the effect of long run farming practices on different soil physico-chemical parameters and soil microbial abundance and diversity within different soil depth (0–20 cm and 20–40 cm) at Quzhou Experimental Station of China Agricultural University, Hebei, China during October and December, 2016. The effect of farming practices on soil microbial abundance and diversity was studied by phospho-lipid fatty acid (PLFA) and DNA high-throughput sequencing methods. The findings revealed that soil is neutral to slightly alkaline in nature with highest water content under organic farming (ORF) at 0–20 cm and least under conventional farming at 20–40 cm depth. It was found that the ORF significantly increased the contents of total organic carbon (TOC), total carbon (TC), ammonium nitrogen, available nitrogen (AN), total nitrogen (TN), total phosphorus (TP), and available phosphorus (AP) followed by low input and conventional farming modes in both October and December soil samples. The correlation analysis showed significantly (atp≤ 0.05 and 0.01) strong positive relationship within different physical and chemical properties of the soil under study. ANOVA and MANOVA analysis indicated significant effect of interaction between soil depth and farming modes on soil parameters. PCA analysis showed the most significant correlation between most of the bacterial types (G + bacteria, G− bacteria, actinomycetes) and soil AP, total available nitrogen, TOC and soil WC. Pearson correlation analysis revealed a significant correlation between microbial phylum groups (Proteobacteria, Bacteroidetes, andLatescibacteria) and microbial class group (Alphaproteobacteria, Sphingobacteriia, Flavobacteriia) with most of the soil physicochemical properties.

A graph-based approach for the visualisation and analysis of bacterial pangenomes

BackgroundThe advent of low cost, high throughput DNA sequencing has led to the availability of thousands of complete genome sequences for a wide variety of bacterial species. Examining and interpreting genetic variation on this scale represents a significant challenge to existing methods of data analysis and visualisation.ResultsStarting with the output of standard pangenome analysis tools, we describe the generation and analysis of interactive, 3D network graphs to explore the structure of bacterial populations, the distribution of genes across a population, and the syntenic order in which those genes occur, in the new open-source network analysis platform, Graphia. Both the analysis and the visualisation are scalable to datasets of thousands of genome sequences.ConclusionsWe anticipate that the approaches presented here will be of great utility to the microbial research community, allowing faster, more intuitive, and flexible interaction with pangenome datasets, thereby enhancing interpretation of these complex data.

Human papillomavirus-related neoplasia of the ocular adnexa.

Human papillomaviruses (HPV) are involved in approximately 5% of solid cancers worldwide. The mucosotropic genotypes infect the stratified epithelium of various locations, where persistent infection may lead to invasive carcinomas. While the causative role of HPV in certain anogenital and head and neck carcinomas is well established, the role of HPV in carcinomas arising in the mucosal membranes of the ocular adnexal tissue (the lacrimal drainage system and the conjunctiva) has been a topic of great uncertainty. Therefore, we conducted a series of studies to assess the correlation between HPV and carcinomas arising in the mucosa of the ocular adnexal tissue and characterize the clinical, histopathological, and genomic features of the tumors in the context of HPV status in a Danish nationwide cohort. We collected clinical and histopathological data and tumor specimens from patients with carcinomas of the conjunctiva and the lacrimal drainage system, and their potential precursors, identified in Danish nationwide registries. The HPV status of the tumors was determined by the combined use of HPV DNA polymerase chain reaction (PCR), HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic profile was investigated by high-throughput DNA sequencing targeting 523 cancer-relevant genes. The literature to date on carcinomas of the lacrimal drainage system and the conjunctiva was summarized. In the Danish cohort, 67% of all carcinomas of the lacrimal drainage system and 21% of all conjunctival carcinomas were HPV-positive. HPV16 was the most frequently implicated genotype. A full-thickness expression of the viral oncogenes E6 and E7 was evident in almost all HPV DNA-positive cases. The HPV-positive carcinomas of the conjunctiva and the lacrimal drainage system shared histopathological and genomic features distinct from their HPV-negative counterparts. The HPV-positive carcinomas were characterized by a non-keratinizing morphology, p16 overexpression, high transcriptional activity of HPV E6/E7, and frequent pathogenic variants in the PI3K-AKT signaling cascade. In contrast, the HPV-negative carcinomas were characterized by a keratinizing morphology, lack of p16 and E6/E7 expression, and frequent somatic pathogenic variants in TP53, CDKN2A, and RB1. Among the patients with conjunctival tumors, HPV positivity was associated with a younger age at diagnosis and a higher risk of recurrence. In conclusion, the results support an etiological role of HPV in a subset of conjunctival and LDS carcinomas and their precursor lesions. Our investigations have shown that the HPV-positive carcinomas of the ocular adnexa share genomic and phenotypic characteristics with HPV-positive carcinomas of other anatomical locations. Therefore, these patients may be eligible for inclusion in future basket trials and future treatment regimens tailored to the more frequently occurring HPV-positive carcinomas of other locations. Future research will further elucidate the diagnostic, prognostic, and predictive role of HPV in these carcinomas.

Will epigenetics be a key player in crop breeding?

If food and feed production are to keep up with world demand in the face of climate change, continued progress in understanding and utilizing both genetic and epigenetic sources of crop variation is necessary. Progress in plant breeding has traditionally been thought to be due to selection for spontaneous DNA sequence mutations that impart desirable phenotypes. These spontaneous mutations can expand phenotypic diversity, from which breeders can select agronomically useful traits. However, it has become clear that phenotypic diversity can be generated even when the genome sequence is unaltered. Epigenetic gene regulation is a mechanism by which genome expression is regulated without altering the DNA sequence. With the development of high throughput DNA sequencers, it has become possible to analyze the epigenetic state of the whole genome, which is termed the epigenome. These techniques enable us to identify spontaneous epigenetic mutations (epimutations) with high throughput and identify the epimutations that lead to increased phenotypic diversity. These epimutations can create new phenotypes and the causative epimutations can be inherited over generations. There is evidence of selected agronomic traits being conditioned by heritable epimutations, and breeders may have historically selected for epiallele-conditioned agronomic traits. These results imply that not only DNA sequence diversity, but the diversity of epigenetic states can contribute to increased phenotypic diversity. However, since the modes of induction and transmission of epialleles and their stability differ from that of genetic alleles, the importance of inheritance as classically defined also differs. For example, there may be a difference between the types of epigenetic inheritance important to crop breeding and crop production. The former may depend more on longer-term inheritance whereas the latter may simply take advantage of shorter-term phenomena. With the advances in our understanding of epigenetics, epigenetics may bring new perspectives for crop improvement, such as the use of epigenetic variation or epigenome editing in breeding. In this review, we will introduce the role of epigenetic variation in plant breeding, largely focusing on DNA methylation, and conclude by asking to what extent new knowledge of epigenetics in crop breeding has led to documented cases of its successful use.

Distinct distribution patterns of planktonic ciliate communities along environmental gradients in a semi-enclosed bay

Jiaozhou Bay is a representative temperate coastal ecosystem and connects with the Yellow Sea by a narrow mouth. The eastern part of the Bay is more polluted than the western and outer parts due to the combined effects of urban sewage and weak water exchange between different water masses. However, the previous results from microscopy analyses did not show any distinct distribution pattern of planktonic ciliates, which are generally considered good bioindicators along environmental gradients. We hypothesize that ciliates may respond to environmental stress by regulating the community of rare taxa, which was not uncovered by morphological methods. The diversity and distribution patterns of planktonic ciliate communities were investigated through high-throughput DNA sequencing from 13 stations in the eastern, western and outer parts of Jiaozhou Bay. The alpha diversity indices showed an increasing trend from the east Bay to the west Bay to the outer Bay. Significant differences in planktonic ciliate communities from different zones of the Bay were mainly attributed to rare rather than abundant taxa. Correlation network analysis revealed that the rare taxa generally have positive correlations with other rare ones, while more negative correlations were found among abundant taxa. We further revealed the distributions of three dominant genera (Stenosemella, Tintinnopsis, and Strombidium) were significantly correlated with environmental variables, suggesting that they might be used as ecological indicators. Our results elucidate the bioindicator potential of ciliate along environmental gradients in a semi-enclosed bay.

Acute exposure to microplastics induces metabolic disturbances and gut dysbiosis in adult zebrafish (Danio rerio)

There is limited knowledge of the ecotoxicological impacts of MPs at the environmentally relevant concentration on freshwater animals, even though numerous studies have demonstrated the toxic effects of MPs on living organisms. In this study, zebrafish (Danio rerio) was used as a model organism to investigate the ecotoxicological effects of acute exposure of virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS) provided comprehensive insights into the metabolic responses of zebrafish exposed to PE (polyethylene) and PES (polyester) MPs. Statistical analysis of metabolomics data indicated that 39 and 27 metabolites, such as lysophosphatidylcholine, phosphocholine, phosphatidylserine, triglyceride, glycosphingolipid, psychosine, 8-amino-7-oxononanoate, cholesterol fatty acid ester, phosphatidylinositol, n-Triacontanol, were significantly altered in PE- and PES-exposed zebrafish, respectively. Furthermore, the enrichment pathway analysis unveiled the synthesis of the structural and functional lipids, signaling molecules, fatty alcohol metabolism, and amino acid metabolism, which was considerably perturbated in MPs-exposed zebrafish. In addition, high-throughput DNA sequencing was conducted to examine changes in gut microbiota in the MPs-treated zebrafish. The MPs exposure increased in the relative abundance of Fusobacteria and Proteobacteria, while the relative abundance of Firmicutes declined in MPs-treated zebrafish. Also, microbial diversity and linear discriminant analyses indicated microbiota dysbiosis, metabolomic dysregulation, and oxidative stress. Taken together, the acute exposure of MPs at environmentally relevant concentrations could disrupt the metabolic interaction via the microbiota-gut-liver-brain relationship, implying gastrointestinal and neurological/immune disorders in zebrafish.

SSNIP-seq: A simple and rapid method for isolation of single-sperm nucleic acid for high-throughput sequencing.

We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.

Metagenome analysis using the Kraken software suite.

Metagenomic experiments expose the wide range of microscopic organisms in any microbial environment through high-throughput DNA sequencing. The computational analysis of the sequencing data is critical for the accurate and complete characterization of the microbial community. To facilitate efficient and reproducible metagenomic analysis, we introduce a step-by-step protocol for the Kraken suite, an end-to-end pipeline for the classification, quantification and visualization of metagenomic datasets. Our protocol describes the execution of the Kraken programs, via a sequence of easy-to-use scripts, in two scenarios: (1) quantification of the species in a given metagenomics sample; and (2) detection of a pathogenic agent from a clinical sample taken from a human patient. The protocol, which is executed within 1-2 h, is targeted to biologists and clinicians working in microbiome or metagenomics analysis who are familiar with the Unix command-line environment.

Network-based gut microbiome analysis in dogs

A growing number of studies in the last decade described the microbial populations in different niches of the organism thank to High-throughput DNA sequencing techniques, that are easily accessible to researchers. Furthermore, network analysis allows characterisation of bacteria that are indirectly linked to outcomes such as disease, diet and host sex through their association with other taxa. The present work on the gut microbiome follows previous research in which data were collected from 8 in-house dietary intervention studies in healthy dogs. Animals were divided according to diet and sex. The process of sample collection, storage, DNA extraction and sequencing, bioinformatic and statistical analysis followed a tailored, internally defined pipeline. The extracted DNA was prepared for sequencing of the V3 and V4 regions of the 16 rRNA gene. Correlation network analysis was performed by calculating pairwise relationships between taxa using the Sparse Correlations for Compositional Data (SparCC) algorithm. First, we identified candidate bacteria that were highly abundant in the microbial community, and second, we examined taxa that were directly related to diet and sex factors. In summary, this study underpins the network structure of the gut microbiome of dogs categorised by diet and sex and provides a better explanation for the interactions between bacteria that led to the grouping of dogs based on environmental or genetic factors. Specifically, this study has provided the basis for understanding how the bacterial community in the gut is interconnected and functions as a function of diet composition and sex. Highlights Network analysis could allow indirect association between bacteria composing the gut microbiome and outcomes, such as disease, diet and sex of the host. This research put the basis on the definition of enterotypes also in dogs, a concept already applied in humans. Positively correlated bacteria, belonging to modules could help to modulate diets and therapies for the treatment of enteropathies or gut-related pathologies linked to a dysbiosis status of the host.

Ancient DNA from the extinct New Zealand grayling (Prototroctes oxyrhynchus) reveals evidence for Miocene marine dispersal

Abstract The evolutionary history of Southern Hemisphere graylings (Retropinnidae) in New Zealand (NZ), including their relationship to the Australian grayling, is poorly understood. The NZ grayling (Prototroctes oxyrhynchus) is the only known fish in NZ to have gone extinct since human arrival there. Despite its historical abundance, only 23 wet and dried, formalin-fixed specimens exist in museums. We used high-throughput DNA sequencing to generate mitogenomes from formalin-fixed P. oxyrhynchus specimens, and analysed these in a temporal phylogenetic framework of retropinnids and osmerids. We recovered a strong sister-relationship between NZ and Australian grayling (P. mareana), with a common ancestor ~13.8 Mya [95% highest posterior density (HPD): 6.1–23.2 Mya], after the height of Oligocene marine inundation in NZ. Our temporal phylogenetic analysis suggests a single marine dispersal between NZ and Australia, although the direction of dispersal is equivocal, followed by divergence into genetically and morphologically distinguishable species through isolation by distance. This study provides further insights into the possible extinction drivers of the NZ grayling, informs discussion regarding reintroduction of Prototroctes to NZ and highlights how advances in palaeogenetics can be used to test evolutionary hypotheses in fish, which, until relatively recently, have been comparatively neglected in ancient-DNA research.

Does filter pore size introduce bias in DNA sequence-based plankton community studies?

The cell size of microbial eukaryotic plankton normally ranges from 0.2 to 200 μm. During the past decade, high-throughput sequencing of DNA has been revolutionizing their study on an unprecedented scale. Nonetheless, it is currently unclear whether we can accurately, effectively, and quantitatively depict the microbial eukaryotic plankton community using size-fractionated filtration combined with environmental DNA (eDNA) molecular methods. Here we assessed the microbial eukaryotic plankton communities with two filtering strategies from two subtropical reservoirs, that is one-step filtration (0.2–200 μm) and size-fractionated filtration (0.2–3 and 3–200 μm). The difference of 18S rRNA gene copy abundance between the two filtering treatments was less than 50% of the 0.2–200 μm microbial eukaryotic community for 95% of the total samples. Although the microbial eukaryotic plankton communities within the 0.2–200 μm and the 0.2–3 and 3–200 μm size fractions had approximately identical 18S rRNA gene copies, there were significant differences in their community composition. Furthermore, our results demonstrate that the systemic bias introduced by size-fractionation filtration has more influence on unique OTUs than shared OTUs, and the significant differences in abundance between the two eukaryotic plankton communities largely occurred in low-abundance OTUs in specific seasons. This work provides new insights into the use of size-fractionation in molecular studies of microbial eukaryotes populating the plankton.

Host association, environment, and geography underlie genomic differentiation in a major forest pest.

Diverse geographic, environmental, and ecological factors affect gene flow and adaptive genomic variation within species. With recent advances in landscape ecological modelling and high-throughput DNA sequencing, it is now possible to effectively quantify and partition their relative contributions. Here, we use landscape genomics to identify determinants of genomic differentiation in the forest tent caterpillar, Malacosoma disstria, a widespread and irruptive pest of numerous deciduous tree species in North America. We collected larvae from multiple populations across Eastern Canada, where the species experiences a diversity of environmental gradients and feeds on a number of different host tree species, including trembling aspen (Populus tremuloides), sugar maple (Acer saccharum), red oak (Quercus rubra), and white birch (Betula papyrifera). Using a combination of reciprocal causal modelling (RCM) and distance-based redundancy analyses (dbRDA), we show that differentiation of thousands of genome-wide single nucleotide polymorphisms (SNPs) among individuals is best explained by a combination of isolation by distance, isolation by environment (spatial variation in summer temperatures and length of the growing season), and differences in host association. Configuration of suitable habitat inferred from ecological niche models was not significantly related to genomic differentiation, suggesting that M.disstria dispersal is agnostic with respect to habitat quality. Although population structure was not discretely related to host association, our modelling framework provides the first molecular evidence of host-associated differentiation in M.disstria, congruent with previous documentation of reduced growth and survival of larvae moved between natal host species. We conclude that ecologically mediated selection is contributing to variation within M.disstria, and that divergent adaptation related to both environmental conditions and host association should be considered in ongoing research and management of this important forest pest.

A meta-analysis of acetogenic and methanogenic microbiomes in microbial electrosynthesis

A meta-analysis approach was used, to study the microbiomes of biofilms and planktonic communities underpinning microbial electrosynthesis (MES) cells. High-throughput DNA sequencing of 16S rRNA gene amplicons has been increasingly applied to understand MES systems. In this meta-analysis of 22 studies, we find that acetogenic and methanogenic MES cells share 80% of a cathodic core microbiome, and that different inoculum pre-treatments strongly affect community composition. Oxygen scavengers were more abundant in planktonic communities, and several key organisms were associated with operating parameters and good cell performance. We suggest Desulfovibrio sp. play a role in initiating early biofilm development and shaping microbial communities by catalysing H2 production, to sustain either Acetobacterium sp. or Methanobacterium sp. Microbial community assembly became more stochastic over time, causing diversification of the biofilm (cathodic) community in acetogenic cells and leading to re-establishment of methanogens, despite inoculum pre-treatments. This suggests that repeated interventions may be required to suppress methanogenesis.

Evaluation of the control effect of bighead carp and silver carp on cyanobacterial blooms based on the analysis of differences in algal digestion processes

The phytoplankton community in Qiandao Lake has typical seasonal succession characteristics, and Cyanophyta, especially the genus Microcystis, are dominant in summer. To evaluate the control effects of filter-feeding fish (silver carp and bighead carp) on cyanobacterial blooms, the changes in algal species composition along the fish intestine were analyzed by microscopic examination, chlorophyll fluorescence, high-throughput DNA sequencing and pigment ratios. The microscopic examination results showed that both silver carp and bighead carp have a certain control effect on bloom-forming cyanobacteria in spring. The digestion of silver carp in summer could still effectively remove cyanobacteria and reduce the dominance of Microcystis in the phytoplankton community, while the digestion of bighead carp seemed to have little effect on cyanobacteria. The results of the chlorophyll fluorescence method were similar to those of the microscopic analysis, which indicated that silver carp could reduce the cyanobacterial biomass in spring, while bighead carp could significantly promote the dominance of cyanobacteria (p < 0.001). High-throughput DNA sequencing showed that there were significant differences in filter-feeding plankton between silver carp and bighead carp. The midgut may be the main gathering site of intestinal microorganisms, and cyanobacterial digestion mainly occurs in the intestinal segments between the foregut and midgut. The analysis of pigment ratios indicated that selective digestion of phytoplankton by silver carp significantly reduced the dominance of Cyanophyta in its hindgut (p < 0.01), while bighead carp had the opposite effect (p < 0.05), especially in seasons other than spring. The results of the above four methods are basically the same, that is, the digestion effect of silver carp on cyanobacteria is better than that of bighead carp. Therefore, the stocking proportion of silver carp should be appropriately increased in some meso-eutrophic waterbodies.

Convergence of biofilm successional trajectories initiated during contrasting seasons.

Biofilm communities play a major role in explaining the temporal variation of biogeochemical conditions in freshwater ecosystems, and yet we know little about how these complex microbial communities change over time (aka succession), and from different initial conditions, in comparison to other stream communities. This has resulted in limited knowledge on how biofilm community structure and microbial colonization vary over relevant time scales to become mature biofilms capable of significant alteration of the freshwater environment in which they live. Here, we monitored successional trajectories of biofilm communities from summer and winter in a headwater stream and evaluated their structural state over time by DNA high-throughput sequencing. Significant differences in biofilm composition were observed when microbial colonization started in the summer vs. winter seasons, with higher percentage of algae (Bacillariophyta) and Bacteroidetes in winter-initiated samples but higher abundance of Proteobacteria (e.g., Rhizobiales, Rhodobacterales, Sphingomonadales, and Burkholderiales), Actinobacteria, and Chloroflexi in summer-initiated samples. Interestingly, results showed that despite seasonal effects on early biofilm succession, biofilm community structures converged after 70 days, suggesting the existence of a stable, mature community in the stream that is independent of the environmental conditions during biofilm colonization. Overall, our results show that algae are important in the early development of biofilm communities during winter, while heterotrophic bacteria play a more critical role during summer colonization and development of biofilms.

Sgcocaller and comapr: personalised haplotype assembly and comparative crossover map analysis using single-gamete sequencing data.

Profiling gametes of an individual enables the construction of personalised haplotypes and meiotic crossover landscapes, now achievable at larger scale than ever through the availability of high-throughput single-cell sequencing technologies. However, high-throughput single-gamete data commonly have low depth of coverage per gamete, which challenges existing gamete-based haplotype phasing methods. In addition, haplotyping a large number of single gametes from high-throughput single-cell DNA sequencing data and constructing meiotic crossover profiles using existing methods requires intensive processing. Here, we introduce efficient software tools for the essential tasks of generating personalised haplotypes and calling crossovers in gametes from single-gamete DNA sequencing data (sgcocaller), and constructing, visualising, and comparing individualised crossover landscapes from single gametes (comapr). With additional data pre-possessing, the tools can also be applied to bulk-sequenced samples. We demonstrate that sgcocaller is able to generate impeccable phasing results for high-coverage datasets, on which it is more accurate and stable than existing methods, and also performs well on low-coverage single-gamete sequencing datasets for which current methods fail. Our tools achieve highly accurate results with user-friendly installation, comprehensive documentation, efficient computation times and minimal memory usage.

The spatial organization of transcriptional control.

In animals, the sequences for controlling gene expression do not concentrate just at the transcription start site of genes, but are frequently thousands to millions of base pairs distal to it. The interaction of these sequences with one another and their transcription start sites is regulated by factors that shape the three-dimensional (3D) organization of the genome within the nucleus. Over the past decade, indirect tools exploiting high-throughput DNA sequencing have helped to map this 3D organization, have identified multiple key regulators of its structure and, in the process, have substantially reshaped our view of how 3D genome architecture regulates transcription. Now, new tools for high-throughput super-resolution imaging of chromatin have directly visualized the 3D chromatin organization, settling some debates left unresolved by earlier indirect methods, challenging some earlier models of regulatory specificity and creating hypotheses about the role of chromatin structure in transcriptional regulation.