GRANTA-519 Nanopore sequencing: Technical Assessment of Long-Read Nanopore Whole-Genome Sequencing for Detecting Large Chromosomal Alterations in Mantle Cell Lymphoma
Project description:Purpose: The goal of this study is to get a deeper insight of whole transcriptome changes and of signaling pathways involved in eliciting the DPN-mediated effects on Granta-519 tumor progression in vivo. Methods: Mantle cell lymphoma cell line Granta-519 cells were grafted to male NSG mice, followed by treated with ESR2 selective agonist DPN or vehicle. When tumor size reached around 1300 mm3, RNA was extracted with RNeasy Plus Mini Kit (Qiagen) for sequencing. Results: ESR2 selective agonist DPN could inhibit cell adhesion, EMT and angiogenesis in vivo which likely involves regulation of VEGFA, FOS, FOSB, CXCR4, MALAT1 and NEAT1. Conclusions: Our study represents the detailed analysis of Granta-519 transcriptomes in vivo experiment after DPN treatment, with biologic replicates, generated by RNA-seq technology.
Project description:Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.
Project description:The nanopore sequencing of short sequences, whose lengths are typically less than 0.3kb therefore comparable with Illumina sequencing techniques, has recently gained wide attention. Here, we design a scheme for training nanopore basecallers that are specialized for short biomolecules. With bioengineered RNA (BioRNA) molecules as examples, we demonstrate the superior accuracy of basecallers trained by our scheme.
Project description:Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity. Notably, this study elucidates additional functional sites regulating exonuclease activity (Asp43 and Glu45) and processivity using artificial nucleotides (Glu266, Gln283, Leu334, Glu335, Ser426, and Asp430). By disclosing the intricate dynamics in exonuclease activity, strand displacement, and artificial nucleotide-based processivity at specific functional sites, our findings not only advance the fundamental understanding of psychrophilic polymerases but also provide novel insights into polymerase engineering.
Project description:We investigated the relative expression levels of these miRs in a series of meningioma and normal meningeal tissues. The effects of miR-16 and miR-519 on cell growth and transcriptome were assessed in vitro using two human cell lines (Ben-Men-1 and IOMM-Lee). Both miR-16 and miR-519 were significantly downregulated in meningioma compared with normal meningeal tissue. Overexpression of either miR in IOMM-Lee and Ben-Men-1 cells significantly reduced cell growth. The transfection of miR-16 and miR-519 significantly downregulated HuR mRNA, and genes involved in various functions such as pre-replicative complex, mitotic recombination, S phase and M phase of cell cycle, and upregulated genes implicated in cell junction, and positive regulation of cell death. Cell-cycle-related genes associated cluster included HuR mRNA (ELAVL1), and was highly enriched with HuR gene targets.
Project description:BackgroundComplex chromosomal rearrangements (CCRs) are associated with high reproductive risk, infertility, abnormalities in offspring, and recurrent miscarriage in women. It is essential to accurately characterize apparently balanced chromosome rearrangements in unaffected individuals.MethodsA CCR young couple who suffered two spontaneous abortions and underwent labor induction due to fetal chromosomal abnormalities was studied using long-read sequencing(LRS), single-nucleotide polymorphism (SNP) array, G-banding karyotype analysis (550-band resolution), and Sanger sequencing.ResultsSNP analysis of the amniotic fluid cells during the third pregnancy revealed a 9.9-Mb duplication at 7q21.11q21.2 and a 24.8-Mb heterozygous deletion at 13q21.1q31.1. The unaffected female partner was a carrier of a three-way CCR [46,XX,? ins(7;13)(q21.1;q21.1q22)t(2;13)(p23;q22)]. Subsequent LRS analysis revealed the exact breakpoint locations on the derivative chromosomes and the specific method of chromosome rearrangement, indicating that the CCR carrier was a more complex structural rearrangement comprising five breakpoints. Furthermore, LRS detected an inserted fragment of chromosome 13 in chromosome 7.ConclusionsLRS is effective for analyzing the complex structural variations of the human genome and may be used to clarify the specific CCRs for effective genetic counseling and appropriate intervention.