Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet: A) Scheme for direct RNA nanopore sequencing. rRNAs containing native modifications are poly(A) tailed before ligation of adapters and RT. Ionic current blockage events are characteristic of the kmer sequence of RNA transiting through the pore constriction. (B) Read quality heatmap for IVT rRNA (left) and native rRNA (right) from E. coli . Dashed red lines indicated the expected lengths for 16S rRNA (1.5 kb) and 23S rRNA (2.9 kb) (C) Normalized native (red) and IVT (black) current signal alignment for 16S rRNA from E. coli spanning positions 1395–1415 performed using Tombo. Sites of known modifications within this window are highlighted in blue. (D) Positional Kolmogorov-Smirnov (KS) statistical testing of current signals across rRNA from E. coli and S. cerevisiae using both Tombo and Nanopolish. Modification positions described in the literature are indicated as black lines. (E) Median current and dwell KS statistic profiles separated by modification type (base [excluding Ψ ], blue; 2'- O -methyl, red; and Ψ , green) and aligned by modification position from both Tombo and Nanopolish. Colored, shaded regions represent the standard deviation of the KS statistic.

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Nanopore Sequencing, Ligation, Sequencing, Modification, Standard Deviation

Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet: (A) Scheme for 3' end modification, SHAPE probing, and 3' end tailing. (B) Normalized SHAPE-MaP reactivity (blue) and nanoSHAPE reactivity detected by changes in current (red) and dwell time (gray) for the pri-miR-17~92 RNA. (C) Heatmap of 1,000 nanopore reads of pri-miR-17~92 modified with 150 mM AcIm. Modifications were determined by per-nucleotide Student’s t test using Fisher’s method context of ±1 of the current signal. Per-nucleotide p values were corrected using the Benjamini-Hochberg procedure and binarized. Nucleotides scored as modified and unmodified are shown in black and teal, respectively; unmapped regions are gray. (D) Kernel density estimate of the number of called modifications per pri-miR-17~92 molecule as a function of AcIm concentration. Called modifications correspond to an upper limit. (E) Spearman’s rank-order correlation (rho) between nanoSHAPE and SHAPE-MaP as a function of the number of contributing pri-miR-17~92 molecules across the AcIm concentrations tested. (F) Spearman’s rho as a function of the distance from the 3' end of the pri-miR-17~92 RNA.

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Modification, Concentration Assay

Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet:

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Virus, Recombinant, Nanopore Sequencing, Sequencing, Modification, Software

Journal: Genes

Article Title: Deletion in the Bardet–Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs

doi: 10.3390/genes11091090

Figure Lengend Snippet: Full-length cDNA sequencing (ONT).

Article Snippet: We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet–Biedl syndrome with heterogeneous clinical signs.

Techniques: Sequencing, Produced

Journal: Genes

Article Title: Deletion in the Bardet–Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs

doi: 10.3390/genes11091090

Figure Lengend Snippet: Expression of retinal marker genes. ( a ) The average expression of marker genes for each retinal cell type in the unaffected BE02 ( a ) and GS01 ( b ) dogs as well as the affected GR02 ( c ), based on cDNA sequencing. ( d ) Relative mRNA expression levels by quantitative RT-qPCR in two different regions (exons 7–8 and exons 13–14) of the TTC8 gene, as well as the retinal marker genes GFAP (macroglial cells), OPN1LW and OPN1SW (cone photoreceptors), RHO (rod photoreceptors), RPE65 (RPE cells), and POU4F1 (retinal ganglion cells) expression in three unaffected dogs (BE02, LR02, GS01) and the affected GR02, normalized to GAPDH and ACTB gene expression. Rod: rod photoreceptors, cone: cone photoreceptors, RGC: retinal ganglion cells, AC: amacrine cells, HC: horizontal cells, BC: bipolar cells, MG: macroglial cells, RPE: retinal pigment epithelium, BE02: unaffected beagle, GS01: unaffected German shepherd, GR02: affected golden retriever, LR02: unaffected Labrador retriever.

Article Snippet: We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet–Biedl syndrome with heterogeneous clinical signs.

Techniques: Expressing, Marker, Sequencing, Quantitative RT-PCR, Gene Expression

Journal: Genome Biology and Evolution

Article Title: Natural Diversity of Heat-Induced Transcription of Retrotransposons in Arabidopsis thaliana

doi: 10.1093/gbe/evae242

Figure Lengend Snippet: Expression of ONSEN and Copia-35 . a) PCoA on gene expression in all sequenced samples. b to d) SalmonTE analysis with RNA-Seq data. Labeled consensus sequences in solid outlines represent candidates that have a base mean value that is greater than 100,000. Copia-35 consensus sequences in b) and c) are labeled in dashed boxes due to below-cutoff base mean. e) Annotation of ONSEN and Copia-35 full-length copies in the three accessions. Reference insertions (in Col-0 ) and TE insertion polymorphisms (TIPs) are marked. f and g) Expression of TE per copy measured using RNA-Seq (four replicates) and ONT direct cDNA sequencing, respectively. As seedling was pooled for mRNA extraction, replicates cannot be plotted for ONT data and barplots display expression level instead.

Article Snippet: In this context, the breakthrough recently brought by Oxford Nanopore Technologies’ (ONT) direct cDNA sequencing, which generates longer reads, has begun to drastically reduce alignment ambiguities, hereby facilitating the detection of TE expression at the single insertion level.

Techniques: Expressing, Gene Expression, RNA Sequencing, Labeling, Sequencing, Extraction