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. 2020 Jan 14;9:e49658. doi: 10.7554/eLife.49658

Figure 1. Diverse Arabidopsis RNAs are detected by nanopore DRS.

(A) Nanopore DRS 12.7 kb read alignment at AT1G48090, comprising 63 exons. Blue, nanopore DRS read alignments; black, Araport11 annotation. (B) Nanopore DRS read alignments at the snoRNA gene U3B (AT5G53902). Blue, sample of nanopore DRS read alignments; black, Araport11 annotation. (C) PIN4 (AT2G01420) antisense RNAs detected using nanopore DRS. Blue, Col-0 PIN4 sense Illumina RNAseq coverage and sample of nanopore PIN4 sense read alignments; orange, Col-0 PIN4 antisense Illumina RNAseq coverage and nanopore PIN4 antisense read alignments; green, hen2-2 mutant PIN4 sense Illumina RNAseq coverage; purple, hen2-2 mutant PIN4 antisense Illumina RNAseq coverage; black, PIN4 sense RNA isoforms found in Araport11; grey, PIN4 antisense differentially expressed regions detected in Illumina RNAseq with DERfinder.

Figure 1—source data 1. hen2-2 vs Col-0 differentially expressed regions from Ilumina RNAseq –Figure 1C.

Figure 1.

Figure 1—figure supplement 1. Properties of nanopore DRS sequencing data.

Figure 1—figure supplement 1.

(A) Nanopore DRS identified a 12.8 kb transcript generated from the AT1G67120 gene that includes 58 exons. Blue, nanopore DRS isoform; black, Araport11 annotation. (B) Synthetic ERCC RNA spike-in mixes are detected in a quantitative manner. Absolute concentrations of spike-ins are plotted against counts per million (CPM) reads in log10 scale. Blue, ERCC RNA spike-in mix 1; orange, ERCC RNA spike-in mix 2. (C) Overview of the sequencing and alignment characteristics of nanopore DRS data for ERCC RNA spike-ins. Left, distribution of the length fraction of each sequenced read that aligns to the ERCC RNA spike-in reference; centre, distribution of fraction of identity that matches between the sequence of the read and the ERCC RNA spike-in reference for the aligned portion of each read; right, distributions of the occurrence of insertions (black), substitutions (orange) and deletions (blue) as a proportion of the number of aligned bases in each read. (D) Substitution preference for each nucleotide (left to right: adenine [A], uracil [U], guanine [G], cytosine [C]). When substituted, G is replaced with A in more than 63% of its substitutions, while C is replaced by U in 73%. Conversely, U is rarely replaced with G (12%) and A is rarely substituted with C (16%). (E) Nucleotide representation within the ERCC RNA Spike-In reference sequences (black dots) compared with nucleotide representation within four categories from the nanopore DRS reads. Identity matches between the sequence of the read and the ERCC RNA spike-in reference (green crosses), insertions (blue pentagons), deletions (yellow stars) and substitutions (purple diamonds).G is under-represented and U is over-represented for all three categories of error (insertion, deletion and substitution) relative to the reference nucleotide distribution. C is over-represented in deletions and substitutions. A is over-represented in insertions and deletions and under-represented in substitutions. (F) Signals originating from the RH3 transcripts are susceptible to systematic over-splitting around exons 7–9 (highlighted using a purple dashed box), resulting in reads with apparently novel 5′ or 3′ positions. This appears only to occur at high frequency in datasets collected after May 2018 (Supplementary file 1) and may result from an update to the MinKNOW software. (G) PIN7 antisense RNAs detected using nanopore DRS. Blue, Col-0 PIN7 sense Illumina RNAseq coverage and nanopore PIN7 sense read alignments; orange, Col-0 PIN7 antisense Illumina RNAseq coverage and nanopore PIN7 antisense read alignments; green, hen2–two mutant PIN7 sense Illumina RNAseq coverage; purple, hen2–two mutant PIN7 antisense Illumina RNAseq coverage; black, PIN7 sense RNA isoforms found in Araport11 annotation; grey, PIN7 antisense differentially expressed regions detected with DERfinder.