Project description:We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories.
Project description:Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant growth, health, and production. To that end, it is necessary to develop methodologies that investigate the metabolic activities of the plant’s microbiome in orbit to enable rapid responses regarding the care of plants in space. In this study, we developed a protocol to characterize the endophytic and epiphytic microbial metatranscriptome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results showed that this enrichment approach was highly reproducible and effective for rapid on-site detection of microbial transcriptional activity. Taxonomic analysis based on 16S and 18S rRNA transcripts identified that the top five most abundant phyla in the lettuce microbiome were Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Ascomycota. The metatranscriptomic analysis identified the expression of genes involved in many metabolic pathways, including carbohydrate metabolism, energy metabolism, and signal transduction. Network analyses of the expression data show that, within the signal transduction pathway of the fungal community, the Mitogen-Activated Protein Kinase signaling pathway was tightly regulated across all samples and could be a potential driver for fungal proliferation. Our results demonstrated the feasibility of using MinION-based metatranscriptomics of enriched microbial RNA as a method for rapid, on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.
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:Untargeted proteomics approaches were shown to be suitable for composition and authenticity analyses of highly processed mixed food and feed products. In this work, a normal-flow tandem mass spectrometry-based proteomics method was setup for analysis and authentication of insect meal from five different species. Novel data acquired on a Q Exactive Orbitrap (QE) was compared with previously published data obtained on QTOF. Data from both proteomics workflows were compared using compareMS2 and a Trans-Proteomics Pipeline (TPP). The results obtained for species differentiation, peptides, and protein markers detection were comparable across both approaches. The collected mass spectrometry data from both instruments also were used to build spectral libraries for insect species and matching was performed successfully. Lastly, both datasets were screened for known allergens and arginine kinase and tropomyosin were consistently detected confirming the presence of potential allergenic risks associated with the consumption of edible insects.
Project description:This Project aims to screen for signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna)
Project description:Replacement of high-value fish species with cheaper varieties or mislabelling of food unfit for human consumption is a global problem violating both consumers’ rights and safety. For distinguishing fish species in pure samples, DNA approaches are available; however, authentication and quantification of fish species in mixtures remains a challenge. In the present study, a novel high-throughput shotgun DNA sequencing approach applying masked reference libraries was developed and used for authentication and abundance calculations of fish species in mixed samples. Results demonstrate that the analytical protocol presented here can discriminate and predict relative abundances of different fish species in mixed samples with high accuracy. In addition to DNA analyses, shotgun proteomics tools based on direct spectra comparisons were employed on the same mixture. Similar to the DNA approach, the identification of individual fish species and the estimation of their respective relative abundances in a mixed sample also were feasible. Furthermore, the data obtained indicated that DNA sequencing using masked libraries predicted species-composition of the fish mixture with higher specificity, while at a taxonomic family level, relative abundances of the different species in the fish mixture were predicted with slightly higher accuracy using proteomics tools. Taken together, the results demonstrate that both DNA and protein-based approaches presented here can be used to efficiently tackle current challenges in feed and food authentication analyses.
Project description:Food consumption is critical for animal survival and reproduction. The biomedical and economic consequences of metabolic diseases arising from excessive food intake, however, are a burden for human society. While the role of neuroendocrine feedback loops, food sensing modalities, and physiological state in regulating food intake are increasingly well understood, other genetic mechanisms remain elusive. Here, we applied ten generations of artificial selection for high and low food consumption in replicate populations of Drosophila melanogaster. The phenotypic response to selection was highly asymmetric, with efficient selection and an average realized heritability of 0.15 in the lines selected for high food consumption. To further nominate candidate genes contributing to response to selection for feeding behavior, we evaluated differences in genome wide gene expression between the selection lines using whole-fly RNA sequencing. We identified 1,631 differentially expressed genes in the analysis pooled across sexes, and 1,267 (2,321) differentially expressed genes in females (males).