Project description:E18 mouse brain single cell profiling using the 10x Genomics Chromium instrument workflow with either Illumina short read sequencing for the standard gene profiling and Nanopore PromethION long read sequencing for isoform profiling.
Project description:Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct both short-read and long-read sequencing, thereby allowing users to recover more reads per cell that permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and multiple myeloma cell lines to evaluate differential isoform usage and Ewing’s sarcoma cells to demonstrate Ig fusion transcript analysis.
Project description:Environmental variation along the geographical space can shape populations by natural selection. In the context of global warming and changing precipitation regimes, it is crucial to understand the role of environmental heterogeneity in tropical trees adaptation, given their disproportional contribution to water and carbon biogeochemical cycles. Here, we investigated how heterogeneity in freshwater availability along tropical wetlands has influenced molecular variations of the black mangrove (Avicennia germinans). A total of 57 trees were sampled at seven sites differing markedly in precipitation regime and riverine freshwater inputs. Using 2,297 genome‐wide single nucleotide polymorphic markers, we found signatures of natural selection by the association between variations in allele frequencies and environmental variables, including the precipitation of the warmest quarter and the annual precipitation. Additionally, we found candidate loci for selection based on statistical deviations from neutral expectations of interpopulation differentiation. Most candidate loci within transcribed sequences were functionally associated with central aspects of drought tolerance or plant response to drought. Moreover, our results suggest the occurrence of the rapid evolution of a population, probably in response to sudden and persistent limitations in plant access to soil water, following a road construction in 1974. Observations supporting rapid evolution included the reduction in tree size and changes in allele frequencies and in transcript expression associated with increased drought tolerance through the accumulation of osmoprotectants and antioxidants, biosynthesis of cuticles, protection against protein degradation, stomatal closure, photorespiration and photosynthesis. We describe a major role of spatial heterogeneity in freshwater availability in the specialization of this typically tropical tree.
Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
Project description:Global climate change increasingly polarizes environments, presenting unprecedented challenges to many organisms (Smol, 2012). Polarization occurs not only in the spatial dimension, producing greater desert drought and tropical rainfall, for example, but also in the temporal dimension by making a local environment more variable over time. Many organisms survive these fluctuating environmental conditions by manifesting multiple distinct phenotypes through developmental processes that enable phenotypic plasticity (Pigliucci et al., 2006; Parsons et al., 2011). As with early development, these processes are expected to strictly regulate gene expression to canalize phenotype, despite the genetic diversity within populations (Alberch, 1982; Riska, 1986, Pigliucci et al., 1996). For plasticity to evolve, natural selection must act on genes that regulate trait variation, e.g, those conferring norms of reaction to a specific set of conditions. Despite the importance of these reaction norms for coping with environmental challenges, the genetic framework underlying phenotypic plasticity remains poorly defined, making it impossible to study how they function, differ among natural populations, and evolve. Here we used arsenic, a chemical inhibitor of salinity acclimation, to identify genes involved in transforming the gill from its freshwater to its seawater architecture in the euryhaline teleost Fundulus heteroclitus. Linear model interaction terms associated with the combined effect of arsenic and salinity challenge revealed an antagonistic relationship between arsenic exposure and salinity acclimation Exposure to arsenic during salinity acclimation yielded gene expression values similar to those observed in unexposed fish that remained in a stable environment, demonstrating that arsenic prevents changes in gene expression that normally enable osmotic plasticity. The gene sets defined by the interaction terms showed reduced inter-individual variation, suggesting unusually tight control, consistent with the hypothesis that they participate in a canalized developmental response. Evidence that natural selection acts to preserve their canalized gene expression was obtained by referencing three populations that differ in their adaptive tolerance to salinity changes (Whitehead et al., 2011). Specifically, populations adapted to withstand the widest salinity range showed both reduced transcriptional variation in genes enabling gill plasticity and an increased osmoregulatory capacity, highlighted by more stable plasma chloride concentrations in response to an osmotic challenge. Finally, we observed significantly fewer associations between genes underlying trait variation and their transcriptional regulators compared to genes that responded to only arsenic or salinity. Collectively, our results demonstrate that phenotypic plasticity converges on a molecular solution that parallels early development, in which the expression of phenotypic plasticity genes and phenotypes are canalized in part by reducing trans-regulatory complexity. 36 Sample comparisons with fish gills exposed to freshwater, freshwater to seawater for 1 hour, freshwater to seawater for 1 hour with arsenic, freshwater to seawater for 24 hours, freshwater to seawater for 24 hours with arsenic, and freshwater with arsenic for 48 hours
Project description:Using long-read nanopore sequencing, we obtained chromosome-wide phased methylomes of the active and inactive X in mouse placenta and neural stem cells (NSCs), overcoming the limitations if short-read bisulfite sequencing in allelic resolution. We also conducted quantitative analysis of methylation properties like symmetry and entropy, providing a more comprehensive view of epigenetic silencing in X chromosome inactivation. We also resolved the allele-specific genetics and epigenetics of structural macrosatellite Dxz4 and other repeats.
Project description:Environmental DNA metabarcoding uncovers environmental correlates of fish communities in spatially heterogeneous freshwater habitats
Project description:Freshwater salinization is an escalating global environmental issue that threatens freshwater biodiversity, including fish populations. This study aims to uncover the molecular basis of salinity physiological responses in a non-native minnow species (Phoxinus septimaniae x P. dragarum) exposed to saline effluents from potash mines in the Llobregat River, Barcelona, Spain. Employing high-throughput mRNA sequencing and differential gene expression analyses, brain, gills, and liver tissues collected from fish at two stations (upstream and downstream of saline effluent discharge) were examined. Salinization markedly influenced global gene expression profiles, with the brain exhibiting the most differentially expressed genes, emphasizing its unique sensitivity to salinity fluctuations. Pathway analyses revealed the expected enrichment of ion transport and osmoregulation pathways across all tissues. Furthermore, tissue-specific pathways associated with stress, reproduction, growth, immune responses, methylation, and neurological development were identified in the context of salinization. Rigorous validation of RNA-seq data through quantitative PCR (qPCR) underscored the robustness and consistency of our findings across platforms. This investigation unveils intricate molecular mechanisms steering salinity physiological response in non-native minnows confronting diverse environmental stressors. This comprehensive analysis sheds light on the underlying genetic and physiological mechanisms governing fish physiological response in salinity-stressed environments, offering essential knowledge for the conservation and management of freshwater ecosystems facing salinization.
Project description:Methylation differences between SIC 5 cocoa seedlings germinated in a common environment before growth for 74 days under semic controlled environmental conditions set to mimic the Malaysian or Brazilian temperature environment. Genomic DNA sonicated and the methylated fraction enriched using a 5-methylcytosine antibody before whole genome amplification and hybridisation to the array. Two-condition experiment, Methyl enriched gDNA Malaysian vs. Brazilian leaf samples. 7 Biological replicates independently grown and harvested. Environmental (temperature) effect