Journal: bioRxiv

Article Title: N 6 -methyladenosine modification of HIV-1 RNA evades RIG-I-mediated sensing to suppresses type-I interferon induction in monocytic cells

doi: 10.1101/2020.11.04.368712

Figure Lengend Snippet: ( A ) HIV-1 5’ UTR (nt. 235–281) RNA oligo 1 (50 ng) with (m 6 A) or without (control, Ctrl) m 6 A modification were subjected to m 6 A dot-blot analysis. MB, methylene blue staining (an RNA loading control). ( B ) and ( C ) RNA oligo 1 (250 ng) were transfected into PMA-differentiated U937 cells. After 16 h, IFN-α and IFN-β mRNA levels were measured by RT-qPCR. Data shown are means ± S.D. of three independent experiments. Mann-Whitney t-test was used for statistical analysis. ( D ) HIV-1 5’ UTR (nt. 176–217) RNA oligo 2 (200 ng) with (m 6 A) or without (Ctrl) m 6 A modification were subjected to m 6 A dot-blot analysis. (E) and (F) RNA oligo 2 (250 ng) were transfected into PMA-differentiated U937 cells. After 16 h, IFN-α and IFN-β mRNA levels were measured by RT-qPCR. Data shown are means ± S.D. of three independent experiments. Un-paired t-test was used for statistical analysis. ** P < 0.005, compared with Ctrl samples.

Article Snippet: Four RNA oligo sequences are from the 5’ UTR of HIV-1 genomic RNA (NL4-3 strain) with or without a single m 6 A site [ ], which were commercially synthesized (Integrated DNA Technologies).

Techniques: Modification, Dot Blot, Staining, Transfection, Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: N 6 -methyladenosine modification of HIV-1 RNA evades RIG-I-mediated sensing to suppresses type-I interferon induction in monocytic cells

doi: 10.1101/2020.11.04.368712

Figure Lengend Snippet: (A) RIG-I expression levels in control (Con) and RIG-I knockout (sgRIG-I) U937 cells were measured by Western blotting. (B) Con and RIG-I KO U937 cells were transfected with 250 ng of poly(I:C). At 16 h post-transfection, cells were collected for the analysis of IFN-α and IFN-β mRNA levels by RT-qPCR. The results are shown as means ± S.D. of three repeats with similar result. ** P < 0.005, **** P < 0.0001. (C) and (D) PMA-differentiated Con and RIG-I KO U937 cells were transfected with 250 ng of RNA oligo 1 (C) or oligo 2 (D) . After 16 h, cells were collected for the analysis of IFN-α and IFN-β mRNA levels by RT-qPCR. The results are shown as means ± S.D. of three repeated experiments. * P < 0.05, ** P < 0.005, **** P < 0.0001. Un-paired t-test was used for statistical analysis. ns, not significant.

Article Snippet: Four RNA oligo sequences are from the 5’ UTR of HIV-1 genomic RNA (NL4-3 strain) with or without a single m 6 A site [ ], which were commercially synthesized (Integrated DNA Technologies).

Techniques: Expressing, Knock-Out, Western Blot, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: N 6 -methyladenosine modification of HIV-1 RNA evades RIG-I-mediated sensing to suppresses type-I interferon induction in monocytic cells

doi: 10.1101/2020.11.04.368712

Figure Lengend Snippet: (A) MDA5 expression levels were measured by Western blotting using control (shCon) and stable MDA5 knockdown (shMDA5) U937 cells. (B) shCon and shMDA5 U937 cells were transfected with poly(I:C). At 16 h post-transfection, cells were collected for the analysis of IFN-α and IFN-β mRNA levels by RT-qPCR. The results are shown as means ± S.D. of three repeats with similar result. * P < 0.05, ** P < 0.005. (C) and (D) PMA-differentiated shCon and shMDA5 U937 cells were transfected with 250 ng of RNA oligo 1 (C) or oligo 2 (D) . At 16 h post-transfection, cells were collected for the analysis of IFN-α and IFN-β mRNA levels by RT-qPCR. The results are shown as means ± S.D. of three repeated experiments.

Article Snippet: Four RNA oligo sequences are from the 5’ UTR of HIV-1 genomic RNA (NL4-3 strain) with or without a single m 6 A site [ ], which were commercially synthesized (Integrated DNA Technologies).

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

doi: 10.1038/s41467-024-48673-8

Figure Lengend Snippet: a RISER classifies RNA molecules as they commence sequencing by directly assessing raw nanopore signals, then sends an accept or reject decision to the sequencing hardware depending on the user-defined target RNA class and whether the user wants to enrich or deplete the target class (shown: target depletion). The accepted reads are sequenced to completion, while the rejected reads are truncated. b Percentage of reads in the training dataset ( y -axis) with raw signals long enough to be input to RISER for each candidate input signal length expressed in seconds ( x -axis). c – e Model performance on the test set for each candidate input signal length ( x -axes), color-coded by the three convolutional network architectures assessed: vanilla convolutional neural network (CNN) (cyan), residual network (ResNet) (dark blue), temporal convolutional network (TCN) (pink). We show the accuracy ( c ), the ratio of true positive rate (TPR) to false-positive rate (FPR) ( d ), and the mean prediction time per batch of signals, expressed in milliseconds ( e ). f Neural network architecture for the CNN model selected to implement RISER. Source data for b – e are provided as a Source Data file.

Article Snippet: RISER seamlessly integrates with nanopore direct RNA sequencing (DRS), building on Oxford Nanopore Technologies’ (ONT) read-until system, which allows the software to prematurely terminate the sequencing of individual molecules.

Techniques: Sequencing

Journal: Nature Communications

Article Title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

doi: 10.1038/s41467-024-48673-8

Figure Lengend Snippet: a , b Time in seconds ( y -axis, log10-scale) taken to classify fixed-length DRS signals ( n = 1000 DRS reads randomly sampled from the mRNA test set) by sequence-based adaptive sampling (AS) using basecalling and mapping to the protein-coding transcriptome (fuchsia) and by classification with RISER’s mRNA model (blue) using a GPU ( a ) or CPU ( b ). In the box plots, the lower and upper boundaries of the box are the first and third quartiles, with the median annotated with a line inside the box. The whiskers extend to the maximum and minimum values within 1.5 times the interquartile range. c Percentage of mRNA DRS signals classified as mRNA ( y -axis) within a given time ( x -axis) by AS (fuchsia) and RISER (blue). Source data are provided as a Source Data file.

Article Snippet: RISER seamlessly integrates with nanopore direct RNA sequencing (DRS), building on Oxford Nanopore Technologies’ (ONT) read-until system, which allows the software to prematurely terminate the sequencing of individual molecules.

Techniques: Sequencing, Sampling

Journal: Nature Communications

Article Title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

doi: 10.1038/s41467-024-48673-8

Figure Lengend Snippet: Performance in non-live independent experiments, using poly(A) + RNA from HeLa cells ( a – d ) and GM12878 cells ( e – h ). For the mRNA ( a , e ) and mtRNA ( c , g ) models in each experiment, we show overall accuracy, precision, true positive rate (TPR) and false positive rate (FPR). For the same mRNA ( b , f ) and mtRNA ( d , h ) models, we show the accuracy for each biotype, color-coded by whether the biotype belongs to the class targeted for RISER depletion (purple) or not (teal). Source data are provided as a Source Data file.

Article Snippet: RISER seamlessly integrates with nanopore direct RNA sequencing (DRS), building on Oxford Nanopore Technologies’ (ONT) read-until system, which allows the software to prematurely terminate the sequencing of individual molecules.

Techniques:

Journal: Nature Communications

Article Title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

doi: 10.1038/s41467-024-48673-8

Figure Lengend Snippet: RISER performance during live sequencing of poly(A) + RNA from HEK293 cells, using a MinION Mk1B flow cell split into two conditions: RISER targeting both mRNA and mtRNA for depletion (pink), and no RISER as a control (blue). a Distribution of read lengths ( y -axis, log10-scale) for each RNA class in each condition. In the box plots, the lower and upper boundaries of the box are the first and third quartiles, with the median annotated with a line inside the box. The whiskers extend to the maximum and minimum values within 1.5 times the interquartile range. Outliers were not included. The read lengths of each RNA class were compared in the control and deplete conditions using a one-tailed Wilcoxon rank sum test (H1: control > deplete). The probability of superiority (PS) is also shown above each comparison. PS is the probability that a randomly sampled read from the control condition is longer than a randomly sampled read from the deplete condition (i.e., PS close to 0.5 means the lengths are likely to be the same, whereas PS close to 1 means that the control lengths are highly likely to be larger) (mRNA: p -value p < 2.2E−308, test statistic U1 = 30493942144.5, PS = 0.87 and n = 376,048, mtRNA: p < 2.2E–308, U1 = 909552316.0, PS = 0.89 and n = 65,569, lncRNA: p = 2.3E−69, U1 = 13787658.0, PS = 0.60 and n = 9574). b Percentage of reads covering the first 1500 bases from the 3′ ends of the transcript ( y -axis) for an example mRNA (upper panel), mtRNA (middle panel) and lncRNA (lower panel). The reference positions ( x -axes) are ordered from 3′ to 5′. The vertical line indicates 280nt upstream of the 3′ end, which approximately corresponds to the maximum RISER input length of 4 s. c Density distributions of the transcript fraction ( x -axis) covered by the sequenced reads. d , e Distribution of the percent change in read ( d ) and nucleotide (nt) ( e ) counts ( y -axis), with respect to a control run, when RISER was used to deplete mRNA and mtRNA (orange) and for a separate control run (purple). In the RISER vs control comparison, lncRNA read counts increased from 602 (control) to 885 (depletion of mRNA and mtRNA by RISER). The box plots are defined the same as in ( a ). Outliers were not included. For the set of transcripts in each biotype, the percent change in nt or reads using RISER was compared to the (no-RISER) control using a paired one-tailed Wilcoxon signed rank test (H1 for mRNA and mtRNA: RISER vs control < between controls, H1 for lncRNA: RISER vs control > between controls). For this comparison, lncRNAs that did not overlap with coding exons from protein-coding transcripts were used. Test parameters and statistics are provided in Suppl. Table . Source data are provided as a Source Data file.

Article Snippet: RISER seamlessly integrates with nanopore direct RNA sequencing (DRS), building on Oxford Nanopore Technologies’ (ONT) read-until system, which allows the software to prematurely terminate the sequencing of individual molecules.

Techniques: Sequencing, One-tailed Test, Comparison

Journal: Nature Communications

Article Title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

doi: 10.1038/s41467-024-48673-8

Figure Lengend Snippet: a Proportion of reads originating from globin genes ( HBB , HBA1 , HBA2 ) or other genes in a standard DRS run of a whole blood sample. b Performance of the RISER model for the detection of globin mRNA using DRS reads from a non-live experiment of whole blood. We show the performance metrics of accuracy, precision, true positive rate (TPR) and false positive rate (FPR). c Accuracy of the globin mRNA model per biotype, color-coded by whether the biotype belongs to the class targeted for RISER depletion (purple) or not (teal). d Distribution of read lengths ( y -axis, log10-scale) for globin mRNAs and non-globin mRNAs in each condition. In the box plots, the lower and upper boundaries of the box are the first and third quartiles, with the median annotated with a line inside the box. The whiskers extend to the maximum and minimum values within 1.5 times the interquartile range. Outliers were not included. The read lengths of each RNA class were compared in the control and deplete conditions using a one-tailed Wilcoxon rank sum test (H1: control > deplete). The probability of superiority (PS) is also shown. PS is the probability that a randomly sampled read from the control condition is longer than a randomly sampled read from the deplete condition (PS close to 0.5 means the lengths are likely to be the same, whereas PS close to 1 means that control lengths are highly likely to be larger) (globin mRNA: p < 2.2E−308, U1 = 30726328836.5, PS = 0.91 and n = 391,906, non-globin mRNA: p = 0.99, U1 = 144441871.5, PS = 0.48 and n = 34,848). e Density distributions of the transcript fraction ( x -axis) covered by the sequenced reads for globin (upper panel) and for non-globin (lower panel) mRNA. f , g Distribution of the percent change in read ( d ) and nucleotide (nt) ( e ) counts ( y -axis), relative to a control run, when RISER was used to deplete globin mRNA (orange) and for a separate control run (purple). In the RISER vs control comparison, non-globin mRNA read counts increased from 6776 (control) to 7363 (depletion of globin mRNA by RISER). The box plots are defined the same as in ( d ). Outliers were not included. For the set of transcripts per biotype, the percent change in nt or reads using RISER was compared to the (no-RISER) control using a paired one-tailed Wilcoxon signed rank test (H1 for globin mRNA: RISER vs control < between controls, H1 for non-globin mRNA: RISER vs control > between controls). Test parameters and statistics are provided in Suppl. Table . Source data are provided as a Source Data file.

Article Snippet: RISER seamlessly integrates with nanopore direct RNA sequencing (DRS), building on Oxford Nanopore Technologies’ (ONT) read-until system, which allows the software to prematurely terminate the sequencing of individual molecules.

Techniques: One-tailed Test, Comparison