the nanopore direct rna-seq dataset  (Biotechnology Information)

Journal: bioRxiv

Article Title: Direct RNA nanopore sequencing reveals rapid RNA modification changes following glucose stimulation of human pancreatic beta-cell lines

doi: 10.1101/2025.06.12.659352

Figure Lengend Snippet: (A) EndoC-BH1 (top) and EndoC-BH3 (bottom) were cultured in low glucose condition (2.8 mM, blue) or high glucose condition (15.0 mM, yellow). RNA was extracted from each culture and used to prepare native dRNA-seq libraries or in vitro transcribed (IVT) dRNA-seq libraries. (B) Insulin stimulation index (y-axis) across cell lines (x-axis) after one hour of glucose exposure. (C) Detected transcripts (x-axis) by biotype (y-axis).

Article Snippet: Direct RNA nanopore sequencing (dRNA-seq) by Oxford Nanopore Technologies (ONT) is a single-molecule, long-read sequencing method that reads native RNA molecules without creating complementary DNA (cDNA) .

Techniques: Cell Culture, In Vitro

Journal: bioRxiv

Article Title: An HIV-1 Reference Epitranscriptome

doi: 10.1101/2025.01.30.635805

Figure Lengend Snippet: ( A ) Nanopore modification-calling results for HIV-1 viral RNA extracted from Jurkat cell cultures treated with STM2457, a drug that inhibits METTL3 m 6 A modification activity. The position of each high frequency m 6 A nucleotide in the HIV-1 genome is indicated on the x-axis. ( B ) Comparison of modifications at DRACH motif sites where m 6 A was called at high frequency. The nucleotide position of the m 6 A modification within the DRACH motif is indicated on the x-axis. Upper plot: comparison of Jurkat cell samples infected with HIV-1 without (back row) or with (front row) 30 µM STM2457 treatment. Lower graph: comparison of Jurkat cell sample infected with HIV-1 before (front row) and after (back row) baseline correction. ( C-E ) Comparison of modification calling between NL4-3 from Jurkat cells (C) and two synthetic HIV-1 RNA fragments, one unmodified (D) and one bearing m 6 A (E) at two DRACH motifs. The nucleotide position corresponding to the NL4-3 genome is indicated on the x-axis. All modifications called in panel D are incorrect while modifications called in panel E, besides m 6 A at position 8975 and 8989, are incorrect.

Article Snippet: We also selected the Oxford Nanopore Technologies (ONT) nanopore-based direct RNA sequencing (dRNA-seq) platform.

Techniques: Modification, Activity Assay, Comparison, Infection

Journal: bioRxiv

Article Title: An HIV-1 Reference Epitranscriptome

doi: 10.1101/2025.01.30.635805

Figure Lengend Snippet: ( A ) Nanopore modification-calling results for HIV-1 RNA taken from Jurkats treated without (darker color) or with (lighter color) cART treatment. Error bars represent the standard deviation from three separate biological replicates. ( B ) Nanopore modification-calling results for HIV-1 RNA taken from Jurkat, primary CD4+ T cells infected in vitro , and CD4+ T cells from PLWH samples. In each nucleotide position cluster, the first column is Jurkat cell samples infected with HIV-1, the second column is CD4+ T cells from healthy donors and infected with HIV-1, and the third and fourth columns are samples taken from CD4+ T cells from PLWH donors. ( C ) Top: comparison of m 6 A modifications called from HIV-1 RNA from Jurkat cells against preservation of the DRACH motifs for these m 6 A modifications sequenced from two PLWH samples. Bottom: analysis of preservation of known m 6 A modification sites in a larger dataset of HIV-1 mutations. Positions containing m 6 A between nucleotide positions 8000 and 9171 were identified and the average and median events for these positions are shown. ( D ) Antisense nanopore modification-calling for HIV-1 viral RNA from Jurkat cells. m6A (blue), m5C (green), pseudouridine (yellow), and inosine (purple). Inset shows a close-up of the asp gene.

Article Snippet: We also selected the Oxford Nanopore Technologies (ONT) nanopore-based direct RNA sequencing (dRNA-seq) platform.

Techniques: Modification, Standard Deviation, Infection, In Vitro, Comparison, Preserving

Journal: bioRxiv

Article Title: RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling

doi: 10.1101/2024.11.07.622529

Figure Lengend Snippet: The RIBOSS transcriptome_assembly and operon_finder functions were used for this analysis. A total of 4672 transcripts were assembled by merging two independent transcriptome datasets with the reference gene annotation for Salmonella enterica serovar Typhimurium LT2. The datasets include a hybrid of Nanopore long-read direct RNA-seq and Illumina short-read RNA-seq metatranscriptome data (a cocktail of S. enterica serovar Enteritidis, Escherichia coli O157:H7, and Listeria monocytogenes ) and Nanopore cDNA sequencing data ( S. enterica ). The median length of the assembled transcripts is 861 nt (A) , where 1254 of these transcripts harbour two or more annotated ORFs (B) .

Article Snippet: We have used the transcriptome data from two independent studies. (i) A metatranscriptome data consisting of Nanopore long-read direct RNA sequencing (RNA-seq) and Illumina short-read RNA-seq data for a cocktail of S . enterica serovar Enteritidis, Escherichia coli O157:H7, and Listeria monocytogenes ( ).

Techniques: RNA Sequencing, Sequencing

Journal: BMC Genomics

Article Title: Sequencing accuracy and systematic errors of nanopore direct RNA sequencing

doi: 10.1186/s12864-024-10440-w

Figure Lengend Snippet: Adapter detection failure in Guppy. a A graphical illustration of the adapter trimming process. Polyadenylated RNA molecules are ligated with a DNA adapter at the 3’ end. The ONT basecaller will first perform adapter trimming before starting to basecall. b The length distribution of the soft-clipped bases at the 5’ and 3’ ends of reads, the latter of which shows a clear bimodal distribution, indicative of adapter detection failure, based on the native human sample. c A signal visualization of a selected read whose adapter failed to be detected by Guppy. The left y-axis shows the signal intensity value (in pA) and the right y-axis shows the quality scores per base. d The distribution of read Q-scores depending on whether the adapter was detected successfully, grouped by read length, based on the short RNA dataset. Adapter detection failure can reduce Q-score for short reads, leading to them being filtered at the conventional Q-score threshold of 7 (dashed line)

Article Snippet: Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths.

Techniques:

Journal: Nature

Article Title: In vitro production of cat-restricted Toxoplasma pre-sexual stages

doi: 10.1038/s41586-023-06821-y

Figure Lengend Snippet: a , IGB views of MORC and HDAC3 ChIP-seq enrichment at chromosomal ends following MORC or AP2XII-1/AP2XI-2 depletion. Read density is on the y-axis, with telomeric repeats (TTTAGGG) marked. b , Nanopore direct RNA sequencing (DRS) read alignment of initially suppressed non-coding RNAs, observable post-MORC knockdown via IAA on the subtelomeric ends of chromosome III and XII. The y-axis shows read-depth. Positive strand reads are colored in magenta while negative strand reads are colored in blue. c , Expression levels of MORC over time are presented through IFA on cells infected with RH MORC–mAID–HA. Cells were fixed, permeabilized, and probed with HA antibodies (green) and Hoechst DNA-specific dye.

Article Snippet: The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .

Techniques: ChIP-sequencing, RNA Sequencing, Knockdown, Expressing, Infection

Journal: Nature

Article Title: In vitro production of cat-restricted Toxoplasma pre-sexual stages

doi: 10.1038/s41586-023-06821-y

Figure Lengend Snippet: a , Principal Component Analysis (PCA) of mRNA sequencing data from biological triplicates of single KD or double KD parasites. Samples were collected from untreated conditions or after 24 or 48 h of IAA treatment. b , Venn diagram illustrating the overlapping genes that were upregulated in the three IAA-treated knockdown strains. Significant genes (FC > 8) were identified using DESeq2 with an independent-hypothesis-weighted approach and Benjamini–Hochberg false discovery rate (FDR) < 0.1. c , M-pileup representation of aligned Nanopore DRS reads at genes differentially expressed following IAA-induced knockdown of AP2XII-1 and AP2XI-2 individually or in combination. d , PCA illustrates the biological and technical variance between triplicate proteome samples extracted after 24-, 32-, and 48-hours post AP2XII-1/AP2XI-2 knockdown induction, juxtaposed with the untreated sample (UT). e , Histogram delineating the distribution of up- and down-regulated proteins (n = 276 and 285, respectively) post AP2XII-1 and AP2XI-2 knockdown, categorized by their life stage association. f , Representative vacuoles of ΔBFD1 /DD-BFD1-Ty parasites grown for 48 h with vehicle or 3 μM Shield-1, stained for ROP26 (green), DBA (red) and Hoechst DNA-specific dye (blue). g-h , Heat map showing hierarchical clustering analysis of selected SRS ( g ) and Family A ( h ) mRNA transcripts and their corresponding proteomic enrichments, which were significantly upregulated (Log2 FC > 2; P -value < 0.01) or downregulated (Log2 FC < −1; P -value < 0.01) following the simultaneous depletion of AP2XII-1 and AP2XI-2. The abundance of these transcripts is presented across different in vivo stages - merozoites, EES1-EES5 stages, tachyzoites, sporozoites, and cysts, as documented in prior studies , , . Analysis parameters are those of Fig. . g , Created with BioRender.com .

Article Snippet: The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .

Techniques: Sequencing, Knockdown, Staining, In Vivo

Journal: Nature

Article Title: In vitro production of cat-restricted Toxoplasma pre-sexual stages

doi: 10.1038/s41586-023-06821-y

Figure Lengend Snippet: a , HOMER analysis reveals global distribution of significant Tn5 accessibility peaks across all genomic features in the AP2XII-1/AP2XI-1 double knockdown strain. Similar profiles were observed between untreated and treated conditions. b , Comparison profile of ChIP-seq summed occupancies over a bin size of 10 for AP2XI-2 (HA) and Tn5 accessibility density at all gene loci centered at TSS (± 3 kb) in the AP2XII-1/AP2XI-2 double knockdown strain without auxin treatment. (c-e) , IGB screenshots illustrate representative genomic regions containing merozoite genes, displaying ChIP-seq signal occupancy, ATAC-seq chromatin accessibility profiles, and nanopore DRS data, similar to Fig. .

Article Snippet: The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .

Techniques: Knockdown, Comparison, ChIP-sequencing

Journal: Nature

Article Title: In vitro production of cat-restricted Toxoplasma pre-sexual stages

doi: 10.1038/s41586-023-06821-y

Figure Lengend Snippet: a , AP2XII-1 (HA) and AP2XI-2 (MYC) expression levels and subcellular localization were assessed using IFA following AP2XII-1 depletion. Hoechst DNA-specific dye was used for counterstaining. b , Heatmap and profile analysis of APXII-1 (HA) and AP2XI-2 (MYC) ChIP-seq mean occupancy over a bin size of 10 in APXII-1 KD parasites with AP2XI-2-MYC knock-in (KI), comparing untreated (UT) and co-depleted (24 h post-IAA) conditions. Average signal profiles centered at TSS ( ± 8 kb) and heat maps of peak density display signal intensity. c , IGB screenshots depict the enrichment of AP2XII-1 and AP2XI-2 at the GRA80 and GRA81 loci in the context of AP2XII-1 single knockdown. d , M-pileup representation of aligned Nanopore DRS reads at genes up-regulated following IAA-induced knockdown of AP2XII-1 and AP2XI-2 individually or in combination. e , IGB screenshot highlighting the genomic region of the merozoite-specific purine nucleoside phosphorylase (PNP) gene, which is up-regulated upon IAA treatment independently of MORC and HDAC3. f , IGB screenshots displaying the genomic regions of the rhoptry genes (ROP16, ROP18) and the microneme gene (MIC1), demonstrating their repression in IAA-treated parasites. g , IGB screenshots showcasing the AMA1 gene family. ( e-g ) ChIP-seq signal occupancy, ATAC-seq chromatin accessibility profiles, and nanopore DRS are visualized in a manner consistent with Fig. .

Article Snippet: The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .

Techniques: Expressing, ChIP-sequencing, Knock-In, Knockdown

Journal: Nature

Article Title: In vitro production of cat-restricted Toxoplasma pre-sexual stages

doi: 10.1038/s41586-023-06821-y

Figure Lengend Snippet: a , IGB screenshots displaying the genomic regions of the rhoptry genes (RON4, ROP39, ROP5) repressed in IAA-treated parasites in an AP2XII-1/AP2XI-2-independent manner. ChIP-seq signal occupancy, ATAC-seq chromatin accessibility profiles, and nanopore DRS are visualized in a manner consistent with Fig. . b , Heat map showing hierarchical mRNA clustering analysis (Pearson correlation) of AP2 TFs regulated by simultaneous depletion of AP2XII-1 and AP2XI-2. Shown is the abundance of their transcripts at different developmental stages, namely merozoites, EES, tachyzoites, and cysts. The color scale indicates the log2-transformed fold changes. c-d , IGB screenshots of genomic regions with secondary transcription factors, including C2H2 Zinc Finger and AP2s, exhibiting activated expression in IAA-treated parasites. Displayed are ChIP-seq signal occupancy, ATAC-seq chromatin accessibility profiles, and nanopore DRS data, following the same representation as in Fig. .

Article Snippet: The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .

Techniques: ChIP-sequencing, Transformation Assay, Expressing