Journal: Scientific Reports

Article Title: Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons

doi: 10.1038/s41598-023-37588-x

Figure Lengend Snippet: SARS-CoV-2 ONT sequencing workflow, from the RNA sample to loading to sequence analysis. A schematic illustration of the tiling PCR strategy for the whole-genome sequencing of SARS-CoV-2 is shown. First-strand cDNA synthesis using the Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers (24 primers, from the only reverse direction from the pool B: B2-B48) was located approximately 1.2 kb apart. Two separate multiplexed PCR reactions (Pools A and B) were generated using the Phusion Hot Start II DNA Polymerase. These two pools of primers contain 12 primer pairs each that generate the odd- or even-numbered tiled amplicons for the 2.4 kb set with about 550 bp overlap of amplicons. The PCR products in pool A and B reactions for the same samples were combined and purified with AMPure XP and quantified with Qubit Broad Range Kit on SpectraMax. Followed by rapid barcoding using the Rapid Barcoding kit and SPRI bead DNA clean-up. The Oxford Nanopore Rapid Barcoding library kit generates barcoded sequencing libraries from pooled amplicons, the transposase approach simultaneously cleaves amplicons and attaches barcoded tags to the cleaved ends. These amplicons are then subjected to Oxford Nanopore library preparation, using methods that directly add adapters to the amplicons. Samples were pooled for sequencing in one flow cell.

Article Snippet: We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing.

Techniques: Sequencing, cDNA Synthesis, Reverse Transcription, Generated, Purification

Journal: Plant Physiology

Article Title: Seed Dormancy in Arabidopsis Is Controlled by Alternative Polyadenylation of DOG1 ¹

doi: 10.1104/pp.15.01483

Figure Lengend Snippet: Arabidopsis mutants in RNA 3′ processing complexes display weak seed dormancy, unchanged DOG1 expression, and a low DOG1 protein level. A, Germination efficiency (%) of freshly harvested seeds of fy-2 mutant. The graph shows the mean values from at least four biological replicates, with error bars representing SD. Each experiment was repeated at least three times. dog1-3 mutant shown as positive control. B, Germination efficiency (%) of freshly harvested seeds of RNA 3′ processing mutants. The graph shows the mean values from at least four biological replicates, with error bars representing SD. Each experiment was repeated at least three times. C, Relative expression level of DOG1 in freshly harvested seeds from Col-0, dog1-3, and fy-2 plants determined by RT-qPCR. Values were normalized to the level of UBC mRNA and are the means of at least three biological replicates, with error bars representing SD. D, The fy-2 mutant exhibits a decreased level of DOG1 protein in seeds. The DOG1 protein level was determined by western blotting in Col-0, fy-2, dog1-3, and dog1-4 mutant seed extracts using a polyclonal DOG1 antibody. The Coomassie blue-stained gel is shown as a loading control. Four biological replicates were analyzed for Col-0 and fy-2 plants. E, 3′ RACE initiated within DOG1 exon 3 reveals no clear defects in transcription termination. RACE was performed using RNA isolated from freshly harvested seeds of Col-0 and fy-2 lines. The alignment shows the 3′ end sequence of individual clones with the number of adenines (A) attached for each clone.

Article Snippet: Total RNA from seedlings was ligated to 3′ RACE oligo adapter (Gene Racer kit; Invitrogen) using T4 RNA Ligase 1 (New England Biolabs).

Techniques: Expressing, Mutagenesis, Positive Control, Quantitative RT-PCR, Western Blot, Staining, Isolation, Sequencing, Clone Assay