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:To investigate the mechanisms of endotoxin-induced acute kidney injury in mice, we performed Nanopore long-read RNA sequencing on bulk kidney tissues using the direct cDNA sequencing kit (SQK-DCS109) and R9.4.1 flow cells.
Project description:We cultured MCF10a-Snail-ER cells and induced EMT initiation with tamoxifen. A matched sequencing of their PolyA RNA was performed, using Illumina and direct RNA Oxford Nanopore sequencing technologies. Both generated datasets supported the development of hybrid bioinformatics tools.
Project description:In this study, based on Nanopore direct RNA-seq where native RNAs are sequenced directly as near full-length transcripts in the 3' to 5' direction, transcription units of the phytopathogen Dickeya dadantii 3937 were validated and transcriptional termination sites were determined. Briefly, D. dadantii cultures were grown in M63 medium supplemented with 0.2% glucose and 0.2% PGA, until the early exponential phase (A600nm = 0.2, condition 1), or the early stationary phase (A600nm = 1.8, condition 2). RNAs were extracted using a frozen acid-phenol method, as previously described (Hommais et al. 2008), and treated successively with Roche and Biolabs DNases. Two samples were prepared: 50 µg of RNAs from each condition were pulled into one sample (sample 1), whereas the other one contained 100 µg of RNAs from condition 2 (sample 2). Both samples were then supplied to Vertis Biotechnologie AG for Nanopore native RNA-seq: total RNA preparations were first examined by capillary electrophoresis. For sample 1, ribosomal RNA molecules were depleted using an in-house developed protocol (recovery rate = 84%), whereas no ribodepletion was performed for sample 2. 3' ends of RNA were then poly(A)-tailed using poly(A) polymerase, and the Direct RNA sequencing kit (SQK-RNA002) was used to prepare the library for 1D sequencing on the Oxford Nanopore sequencing device. The direct RNA libraries were sequenced on a MinION device (MIN-101B) using standard settings. Basecalling of the fast5 files was performed using Guppy (version 3.6.1) with the following settings: --flowcell FLO-MIN106 --kit SQK-RNA002 --cpu_threads_per_caller 12--compress_fastq --reverse_sequence true --trim_strategy rna. Reads smaller than 50 nt were removed. 466 393 and 556 850 reads were generated for sample 1 and 2, respectively.
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:We sequenced DNA from a bulk of Col x Ler F2 hybrid plants (WT and recq4) using Nanopore long-read sequencing and identified crossover sites with COmapper. For nanopore sequencing of gDNA from 1,000 pooled seedlings, 10-day-old seedlings were ground in liquid nitrogen using a mortar and pestle. The ground tissue was resuspended in four volumes of CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA) and incubated at 65°C for 30 min. Following chloroform extraction, isopropanol precipitation and removal of RNAs as above, the gDNA pellet was resuspended in 150 μl TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer and gDNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853). Nine micrograms of gDNA from pollen or seedlings was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).
Project description:We report the direct RNA sequencing of HEK293 and a primary human mammary epithelial cell (HMEC) line using Oxford Nanopore based sequencing. Using this data, we built an algorithm to detect m6A modifications within the DRACH motif context. Evaluation of m6A sites was carried out with HEK METTL3 knockdown and HMEC ALKBH5 over expression cell lines.
