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:Antimicrobial resistance (AMR) arises from complex genetic and regulatory changes, including single mutations, gene acquisitions or cumulative effects. Advancements in genomics and proteomics facilitate more comprehensive understanding of the mechanisms behind antimicrobial resistance. In this study, 74 clinically obtained Klebsiella pneumoniae isolates with increased meropenem and/or imipenem MICs were characterized by broth microdilution and PCR to check for the presence of carbapenemase genes. Subsequently, a representative subset of 15 isolates was selected for whole genome sequencing (WGS) by Illumina and Nanopore sequencing, and proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the mechanisms underlying the differences in carbapenem susceptibility of Klebsiella pneumoniae isolates. Identical techniques were applied to characterize 4 mutants obtained after sequential meropenem exposure. We demonstrated that in clinically obtained isolates, increased copy numbers of blaOXA-48 containing plasmids, combined with OmpK36 loss, contributed to high carbapenem MICs without involvement of OmpK35 or other porins or efflux systems. In the meropenem exposed mutants, increased copy numbers of blaCTX-M-15 or blaOXA-48 containing plasmids, combined with OmpK36 loss was demonstrated. The OmpK36 loss resulted from the insertion of IS1 transposable elements or partial deletion of the ompK36 gene. Additionally, we identified two mutations, C59A and C58A, in the DNA coding the copA antisense RNA of IncFII plasmids and multiple mutations of an IncR plasmid, associated with increased plasmid copy numbers. This study demonstrates that by combining WGS and LC-MS/MS, the effect of genomic changes on protein expression related to antibiotic resistance and the mechanisms behind antibiotic resistance can be elucidated.
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 applied direct RNA long read sequencing for characterization of transcripts from constructs inserted into HEK293T mammalian cells with different promoters. Direct RNA sequencing was performed on an Oxford Nanopore GridION device using the Direct Sequencing Kit (SQK-RNA004, date accessed 15 May 2024), MinION RNA flow cell (FLO-MIN00RA), and data pre-processing was performed with MinKNOW (v24.06.10).