Journal: Molecular cell

Article Title: Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores

doi: 10.1016/j.molcel.2019.11.017

Figure Lengend Snippet: (A) Schematic of the nano-COP protocol with nascent RNA purification and direct RNA nanopore sequencing. Cells are labeled with 4-thiouridine (4sU); cellular fractionation is performed to isolate chromatin-associated RNA; and 4sU labeled chromatin-associated RNA is selected through biotinylation and affinity purification. Subsequently, a poly(A) or poly(I) tail is added to the purified RNA, represented here as a string of N’s. The sample is subjected to direct RNA library preparation, including 3’ end adapter ligation, followed by nanopore sequencing. (B) Splicing index, which represents the proportion of spliced transcripts in Illumina sequencing datasets, is plotted within different percentiles of total RNA gene expression. The distribution of 4sU labeled chromatin-associated RNA (4sU+chr) differs significantly (t-test p-value < 0.05) between chromatin-associated RNA (chr) and 4sU labeled RNA (4sU) at all gene expression levels. Gene expression percentiles are based on total RNA expression levels out of 9659 genes that have at least 25 reads spanning splice junctions in all datasets. (C) Representative nano-COP reads aligned to the GSTP1 gene in human K562 cells. The gene structure is represented from the transcription start site (TSS) to the poly(A) site, with black boxes representing exons and lines representing introns. Within the reads, blue boxes represent read coverage, black lines represent skipped coverage due to splicing and the start of the read (3’ end of RNA) is represented with an arrow. Dashed lines represent reads that continue beyond the region displayed. (D–E) Distribution of nano-COP 3’ ends by nanopore sequencing in (D) human K562 cells with enzymatic poly(A) tail addition and (E) human K562 cells in the absence of enzymatic poly(A) tail addition. See Methods for descriptions of 3’ end alignment categories. (F) The length of poly(A) tails for sequenced RNAs with enzymatic poly(A) tail addition was estimated using nanopolish-polyA (Loman et al., 2015; Workman et al., 2018). Estimated tail lengths were plotted for RNAs in each sample that have 3’ ends aligning within gene bodies (exon, intron, or splice site), at poly(A) sites, or just downstream of poly(A) sites (*** signifies t-test p-value < 1 × 10−30).

Article Snippet: Direct RNA sequencing samples with poly(A) selected RNA from immortalized human B-lymphocyte cells (GM12878) by the Oxford Nanopore RNA Consortium (Hopkins run 1 and UCSC run 1) ( Workman et al., 2018 ) were accessed from https://s3.amazonaws.com/nanopore-human-wgs/rna/fastq/Hopkins_Run1_20170928_DirectRNA.pass.dedup.fastq and https://s3.amazonaws.com/nanopore-human-wgs/rna/fastq/UCSC_Run1_20170907_DirectRNA.pass.dedup.fastq .

Techniques: Purification, Nanopore Sequencing, Labeling, Cell Fractionation, Affinity Purification, RNA Library Preparation, Adapter Ligation, Illumina Sequencing, Gene Expression, RNA Expression

Journal: Molecular cell

Article Title: Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores

doi: 10.1016/j.molcel.2019.11.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Direct RNA sequencing samples with poly(A) selected RNA from immortalized human B-lymphocyte cells (GM12878) by the Oxford Nanopore RNA Consortium (Hopkins run 1 and UCSC run 1) ( Workman et al., 2018 ) were accessed from https://s3.amazonaws.com/nanopore-human-wgs/rna/fastq/Hopkins_Run1_20170928_DirectRNA.pass.dedup.fastq and https://s3.amazonaws.com/nanopore-human-wgs/rna/fastq/UCSC_Run1_20170907_DirectRNA.pass.dedup.fastq .

Techniques: Recombinant, Selection, RNA Sequencing, Expressing, Software

Journal: BMC Genomics

Article Title: Poly(a) selection introduces bias and undue noise in direct RNA-sequencing

doi: 10.1186/s12864-022-08762-8

Figure Lengend Snippet: Workflow used in omission of poly(A) selection in ONT dRNA-seq protocol. Outline of procedures used for “selected” and “unselected” libraries analyzed in this study. Procedures detailed in the methods section. Figure created with BioRender.com

Article Snippet: Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing.

Techniques: Selection

Journal: BMC Genomics

Article Title: Poly(a) selection introduces bias and undue noise in direct RNA-sequencing

doi: 10.1186/s12864-022-08762-8

Figure Lengend Snippet: Omission of poly(A) selection does not significantly alter read lengths from ONT direct RNA-seq. A Cumulative distribution function of basecalled read lengths from three poly(A)-selected libraries, and two libraries in which poly(A) selection was omitted. This plot contains all basecalled reads, including those which were not mapped via MiniMap2. Later plots only include mapped reads. B Genes with only a single annotated transcript were binned based on their CDS length, then reads mapping to that gene were denoted as “spanning-CDS’ or “not-spanning-CDS” based on the location of the 5′ and 3′ read ends. Finally the percentage of “spanning-CDS” reads were calculated and plotted for each gene. C Plot comparing mean read lengths of each gene between a poly(A)-selected library (Y-axis) and an unselected library (X-axis) (Spearman R: 0.9713)

Article Snippet: Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing.

Techniques: Selection, RNA Sequencing