Determining exon connectivity in complex mRNAs by nanopore sequencing

Abstract

Short-read high-throughput RNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 'full-length' isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be a powerful method for comprehensive transcriptome characterization.

Fig. 1

Schematic of the exon-intron structures of the genes examined in this study. a The Rdl gene contains two clusters (cluster one and two) which each contain two mutually exclusive exons. b The MRP gene contains contains two and eight mutually exclusive exons in clusters 1 and 2, respectively. c Mhc contains two mutually exclusive exons in clusters 1 and 5, three mutually exclusive exons in clusters 2 and 3, and five mutually exclusive exons in cluster 4. d The Dscam1 gene contains 12, 48, and 33 mutually exclusive exons in the exon 4, 6, and 9 clusters, respectively. For each gene, the constitutive exons are colored blue, while the variable exons are colored yellow, red, orange, green, or light blue

Fig. 2

Similarity distance between the variable alternative exons of MRP, Mhc, and Dscam1. a Violin plots of the LAST alignment scores of each variable exon within MRP cluster 1 and MRP cluster 2 to themselves and the second (2nd) best alignments. b Violin plots of the LAST alignment scores of each variable exon within each Mhc cluster to themselves and the second (2nd) best alignments. c Violin plots of the LAST alignment scores of each variable exon within each Dscam1 cluster to themselves (1st), and to the exons with the second (2nd), third (3rd) and fourth (4th) best alignments

Fig. 3

Optimized RT-PCR minimizes template-switching for MinION sequencing. a Histogram of read lengths from MinION sequencing of Dscam1 spike-ins from the library generated using 25 cycles of PCR. b Bar plot indicating the extent of template switching in Dscam1 spike-ins at different PCR cycles (left). The blue portions indicate the fraction of reads corresponding to input isoforms while the red portions correspond to the fraction of reads corresponding to template-switched isoforms. On the right, plots of the rank order versus number of reads (log10) for the 20, 25, and 30 cycle libraries. The blue dots indicate input isoforms while the red portions correspond to template-switched isoforms

Fig. 4

MinION sequencing of Dscam1 identified 7,874 isoforms. a Histogram of read length distribution for Drosophila head samples. b The total number of Dscam1 isoforms identified from MinION sequencing. c Cumulative distribution of Dscam1 isoforms with respect to expression. d Violin plot of the number of isoforms identified using 100 random pools of the indicated number of reads. e Plot of the estimated number of total isoforms present in the library using the capture-recapture method with two random pools of the indicated number of reads. The shaded blue area indicates the 95 % confidence interval. f Deconvoluted expression of Dscam1 exon cluster variants (top) and the isoform connectivity of two highly expressed Dscam1 isoforms (bottom)

Fig. 5

Accuracy of Dscam1 sequencing results. a Comparison of the frequency of variable exon inclusion for the Dscam1 exon 4 (yellow), 6 (red), and 9 (orange) clusters as determined by nanopore sequencing or by amplicon sequencing using an Illumina MiSeq. b Percent identities (left) or LAST alignment scores (right) of full-length template, complement, and two directions (sequencing both template and complements) nanopore read alignments

Fig. 6

MinION sequencing of Rdl identified four isoforms. a Histogram of read lengths. b The number of reads per isoform. c Cumulative distribution of isoforms with respect to expression. d The number of reads per alternative exon (top) and per isoform (below)

Fig. 7

MinION sequencing of MRP identified nine isoforms. a Histogram of read lengths. b The number of reads per isoform. c Cumulative distribution of isoforms with respect to expression. d The number of reads per alternative exon (top) and per isoform (below)

Fig. 8

MinION sequencing of Mhc identified 12 isoforms. a Histogram of read lengths. b The number of reads per isoform. c Cumulative distribution of isoforms with respect to expression. d The number of reads per alternative exon (top) and per isoform (below)