Project description:Using sRNA-Seq to provide small RNA status in fruit ripening stages in sweet orange DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared to immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes including genes involved in ABA responses. Our results suggest important roles of DNA methylation in orange fruit ripening.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual.
Project description:miRNAs-mediated gene silencing pathway plays vital roles in plant development, abiotic and biotic stress responses. Here, we carried out a high-throughput sequencing approach to identify miRNAs in leaves and flowers of sweet orange. Consequently we identified genome-wide 183 known miRNAs and 38 novel miRNAs. Small RNA sequencing of the leaves and flowers in sweet orange
Project description:Small RNAs (sRNAs) are emerging as important regulators of biological processes in plants. To characterize the small RNA species and expression changes in ‘Anliu’ sweet orange (wild type, WT) and its red-flesh mutant (MT) with lycopene accumulation, we used high-throughput pyrosequencing to identify and quantitatively profile sRNAs. We identified 112 known miRNAs belonging to 64 families from the sweet orange. Comparative analysis revealed that 63 of the 112 known miRNAs exhibited significant expression differences between WT and MT. We also identified 12 novel miRNAs and 9 miRNA candidates from sweet orange; the 12 novel miRNAs are in line with the biogenesis characteristics including the stem-loop structure of the pre-miRNAs and existence of the miRNA*s in the sRNA libraries. Comparative profiling revealed that 10 novel miRNAs and 8 miRNA candidates are differentially expressed between WT and MT. Potential targets of these differentially expressed miRNAs included several important genes that are involved in carotenoid biosynthesis, such as geranylgeranyl pyrophosphate synthase (GGPS) and lycopene β-cyclase (LYCb). Moreover, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. We proposed that miRNA-target genes are the hot-spots for generating sRNAs. This study provides the first large scale cloning and characterization of citrus miRNAs, which also lays a foundation for unraveling the mechanism of lycopene accumulation in the sweet orange mutant on post-transcription level. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. We combined bioinformatic and experimental analyses to improve the RT-LAMP assay performance for COVID-19 diagnosis. First, we developed an improved algorithm to design LAMP primers targeting the nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV-2. Next, we rigorously validated these new assays for their efficacy and specificity. Further, we demonstrated that multiplexed RT-LAMP assays could directly detect as low as a few copies of SARS-CoV-2 RNA in saliva, without the need of RNA isolation. Importantly, further testing using saliva samples from COVID-19 patients indicated that the new RT-LAMP assays were in total agreement in sensitivity and specificity with standard RT-qPCR. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.