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Determining exon connectivity in complex mRNAs by nanopore sequencing.

Bolisetty MT, Rajadinakaran G, Graveley BR - Genome Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut Health Center, Farmington, CT, 06030, USA.

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.

No MeSH data available.


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. cMhc 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
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Fig1: 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. cMhc 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

Mentions: We were interested in determining the feasibility of using the MinION nanopore sequencer to characterize the connectivity of distantly located exons in the mRNAs expressed from genes with complex splicing patterns. For the purposes of these experiments, we have focused on four Drosophila genes with increasingly complex patterns of alternative splicing (Fig. 1). Resistant to dieldrin (Rdl) contains two clusters, each containing two mutually exclusive exons and therefore has the potential to generate four different isoforms (Fig. 1a). Multidrug-Resistance like Protein 1 (MRP) contains two mutually exclusive exons in cluster 1 and eight mutually exclusive exons in cluster 2, and can generate 16 possible isoforms (Fig. 1b). Myosin heavy chain (Mhc) can potentially generate 180 isoforms due to five clusters of mutually exclusive exons – clusters 1 and 5 contain two exons, clusters 2 and 3 each contain three exons, and cluster 4 contains five exons. Finally, Dscam1 contains 12 exon 4 variants, 48 exon 6 variants, 33 exon 9 variants (Fig. 1d), and two exon 17 variants (not shown) and can potentially express 38,016 isoforms. For this study, however, we have focused only on the exon 3 through exon 10 region of Dscam1, which encompasses the 93 exon 4, 6, and 9 variants, and 19,008 potential isoforms (Fig. 1d).Fig. 1


Determining exon connectivity in complex mRNAs by nanopore sequencing.

Bolisetty MT, Rajadinakaran G, Graveley BR - Genome Biol. (2015)

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. cMhc 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4588896&req=5

Fig1: 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. cMhc 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
Mentions: We were interested in determining the feasibility of using the MinION nanopore sequencer to characterize the connectivity of distantly located exons in the mRNAs expressed from genes with complex splicing patterns. For the purposes of these experiments, we have focused on four Drosophila genes with increasingly complex patterns of alternative splicing (Fig. 1). Resistant to dieldrin (Rdl) contains two clusters, each containing two mutually exclusive exons and therefore has the potential to generate four different isoforms (Fig. 1a). Multidrug-Resistance like Protein 1 (MRP) contains two mutually exclusive exons in cluster 1 and eight mutually exclusive exons in cluster 2, and can generate 16 possible isoforms (Fig. 1b). Myosin heavy chain (Mhc) can potentially generate 180 isoforms due to five clusters of mutually exclusive exons – clusters 1 and 5 contain two exons, clusters 2 and 3 each contain three exons, and cluster 4 contains five exons. Finally, Dscam1 contains 12 exon 4 variants, 48 exon 6 variants, 33 exon 9 variants (Fig. 1d), and two exon 17 variants (not shown) and can potentially express 38,016 isoforms. For this study, however, we have focused only on the exon 3 through exon 10 region of Dscam1, which encompasses the 93 exon 4, 6, and 9 variants, and 19,008 potential isoforms (Fig. 1d).Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut Health Center, Farmington, CT, 06030, USA.

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.

No MeSH data available.