Project description:This study is a comparison of sequencing technologies and library preparations protocols performed on RNA extracted from Huh-7 cells, a human hepatocellular carcinoma (HCC) cell line, for which a few characteristic fusion transcripts have previously been reported. The sequencing platforms and library types include Oxford Nanopore Technologies (ONT) MinION direct-cDNA, ONT MinION direct-RNA and ONT MinION PCR-cDNA, PacBio Revio Kinnex sequencing. Additionally, this study includes matched short-read sequencing data from the Illumina NovaSeq platform.
Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
Project description:Rapidly increased studies by third-generation sequencing [Pacific Biosciences (Pacbio) and Oxford Nanopore Technologies (ONT)] have been used in all kinds of research areas. Among them, the plant full-length single-molecule transcriptome studies were most used by Pacbio while ONT was rarely used. Therefore, in this study, we developed ONT RNA-sequencing methods in plants. We performed a detailed evaluation of reads from Pacbio and Nanopore PCR cDNA (ONT Pc) sequencing in plants (Arabidopsis), including the characteristics of raw data and identification of transcripts. We aimed to provide a valuable reference for applications of ONT in plant transcriptome analysis.
Project description:We performed direct cDNA sequencing in HeLa GFP∆Promoter cells by Oxford Nanopore Technology (ONT) on a MinION device to detect EGFP RNA levels after DSB induction.
Project description:Whole Genome Bisulfite Sequencing (WGBS) has been the gold standard DNA methylation mapping and quantification for over a decade. Oxford Nanopore Technologies (ONT) sequencing directly measures nucleotide modifications. In this study, we have compared DNA methylation levels (5-methylcytosine) at CpG sites in the quail genome using WGBS and ONT. Samples were collected to investigate transgenerational DNA methylation changes in Japanese quail following ancestral exposure to a phytoestrogen. Blood samples from 24 third-generation (G3) individuals—descendants of either treated or untreated ancestors—were sequenced after bisulfite conversion. Both methods revealed broadly consistent methylation patterns. ONT reads covered more CpG sites and detected a higher number of differentially methylated cytosines (DMCs). Principal component analyses showed that both sex and ancestral treatment groups accounted for a portion of the observed epigenetic variation, for both technologies. Strong concordance between WGBS and ONT results supports the reliability of ONT sequencing for epigenomic research, including in quails. These data pave the way for further investigation into whether genistein induces epigenetic changes for several generations.
Project description:Investigation of transcriptome changes in four human cell lines (BJ, BJ-5ta, U2OS and HeLa) after treatment for 24 hours with nicotinamide adenine dinucleotide (NAD+). Cells were untreated as the control condition. Nanopore sequencing of cDNA was performed after library preparation with the ONT SQK-PCB109 kit.
Project description:The purpose was to investigate transcript and splicing changes in U2OS cells following siRNA-mediated depletion of MDC1 and PLRG1 or treatment with the splicing inhibitor pladienolide B. Cells were treated with control siRNA as control condition. Nanopore sequencing of cDNA was performed after library preparation with ONT kits SQK-PCS109 and SQK-PBK004.
Project description:One ONT-ULK sequencing run from the kidney of a single male CDMR (Bathyergus suillus) sample used to make the mBatSui1.1.primary genome assembly as an evolutionary comparator to our telomere-to-telomere naked mole-rat genome assembly. Specifically, we assembled a CDMR from a wild-derived sample in South African cape and sequenced in Toronto, Canada, using PacBio HiFi (89 Gb, read N50 = 18 Kb) and ONT-ULK (55 Gb, read N50 = 43 Kb) reads (contig N50 = 33 Mb, Compleasm S = 99%, QV = 71.0). This accession stores the ONT-ULK data for this assembly.
Project description:We compared the transcriptomes of ES cell derived erythroid progentor cells (ES-EP) and murine erythroleukemia (MEL) cells stably transfected with Gata-1 fused to ER. RNA was isolated from duplicate proliferating cultures of MEL and ES-EP using Affymetrix GeneChip Mouse Gene 1.0 ST.