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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.


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)
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Fig8: 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)

Mentions: To extend this approach to other genes with complex splicing patterns, we focused on Rdl, MRP, and Mhc which have the potential to generate four, 16, and 180 isoforms, respectively. We prepared libraries for each of these genes by RT-PCR using primers in the constitutive exons flanking the most distal alternative exons using 25 cycles of PCR, pooled the three libraries and sequenced them together on the MinION nanopore sequencer for 12 h obtaining a total of 22,962 reads. The input libraries for Rdl, MRP, and Mhc were 567 bp, 1,769-1,772 bp, and 3,824 bp, respectively. The raw reads were aligned independently to LAST indexes of each cluster of variable exons. The alignment results were then used to assign reads to their respective libraries, identify reads that mapped to all variable exon clusters for each gene, and the exon with the best alignment score within each cluster. In total, we obtained 301, 337, and 112 full length reads for Rdl (Fig. 6), MRP (Fig. 7), and Mhc (Fig. 8), respectively. For Rdl, both variable exons in each cluster was observed, and accordingly all four possible isoforms were observed, though in each case the first exon was observed at a much higher frequency than the second exon (Fig. 6d). Interestingly, the ratio of isoforms containing the first versus second exon in the second cluster is similar for isoforms containing either the first exon or the second exon in the first cluster indicating that the splicing of these two clusters may be independent. For MRP, both exons in the first cluster were observed and all but one of the exons in the second cluster (exon B) were observed, though the frequency at which the exons in both clusters were used varied dramatically (Fig. 7d). For example, within the first cluster, exon B was observed 333 times while exon A was observed only four times. Similarly, in the second cluster, exon A was observed 157 times whereas exons B, E, F, and G were observed 0 times, thrice, once, and twice, respectively, and exons D, E, and H were observed between 40 and 76 times. As a result, we observed only nine MRP isoforms. For Mhc, we again observed strong biases in the exons observed in each of the five clusters (Fig. 8d). In the first cluster, exon B was observed more frequently than exon A. In the second cluster, 109 of the reads corresponded to exon A, while exons B and C were observed by only two and one read, respectively. In the third cluster, exon A was not observed at all while exons B and C were observed in roughly 80 % and 20 % of reads, respectively. In the fourth cluster, exon A was observed only once, exons B and C were not observed at all, exon E was observed 13 times while exon D was present in all of the remaining reads. Finally, in the fifth cluster, only exon B was observed. As with MRP, these strong biases and near or complete absences of exons in some of the clusters severely reduces the number of possible isoforms that can be observed. In fact, of the 180 potential isoforms encoded by Mhc, we observed only 12 isoforms. Various Mhc isoforms are known to be expressed in striking spatial and temporally restricted patterns [14] and thus it is likely that other Mhc isoforms that we did not observe, could be observed by sequencing other tissue samples.Fig. 6


Determining exon connectivity in complex mRNAs by nanopore sequencing.

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

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)
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Related In: Results  -  Collection

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Fig8: 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)
Mentions: To extend this approach to other genes with complex splicing patterns, we focused on Rdl, MRP, and Mhc which have the potential to generate four, 16, and 180 isoforms, respectively. We prepared libraries for each of these genes by RT-PCR using primers in the constitutive exons flanking the most distal alternative exons using 25 cycles of PCR, pooled the three libraries and sequenced them together on the MinION nanopore sequencer for 12 h obtaining a total of 22,962 reads. The input libraries for Rdl, MRP, and Mhc were 567 bp, 1,769-1,772 bp, and 3,824 bp, respectively. The raw reads were aligned independently to LAST indexes of each cluster of variable exons. The alignment results were then used to assign reads to their respective libraries, identify reads that mapped to all variable exon clusters for each gene, and the exon with the best alignment score within each cluster. In total, we obtained 301, 337, and 112 full length reads for Rdl (Fig. 6), MRP (Fig. 7), and Mhc (Fig. 8), respectively. For Rdl, both variable exons in each cluster was observed, and accordingly all four possible isoforms were observed, though in each case the first exon was observed at a much higher frequency than the second exon (Fig. 6d). Interestingly, the ratio of isoforms containing the first versus second exon in the second cluster is similar for isoforms containing either the first exon or the second exon in the first cluster indicating that the splicing of these two clusters may be independent. For MRP, both exons in the first cluster were observed and all but one of the exons in the second cluster (exon B) were observed, though the frequency at which the exons in both clusters were used varied dramatically (Fig. 7d). For example, within the first cluster, exon B was observed 333 times while exon A was observed only four times. Similarly, in the second cluster, exon A was observed 157 times whereas exons B, E, F, and G were observed 0 times, thrice, once, and twice, respectively, and exons D, E, and H were observed between 40 and 76 times. As a result, we observed only nine MRP isoforms. For Mhc, we again observed strong biases in the exons observed in each of the five clusters (Fig. 8d). In the first cluster, exon B was observed more frequently than exon A. In the second cluster, 109 of the reads corresponded to exon A, while exons B and C were observed by only two and one read, respectively. In the third cluster, exon A was not observed at all while exons B and C were observed in roughly 80 % and 20 % of reads, respectively. In the fourth cluster, exon A was observed only once, exons B and C were not observed at all, exon E was observed 13 times while exon D was present in all of the remaining reads. Finally, in the fifth cluster, only exon B was observed. As with MRP, these strong biases and near or complete absences of exons in some of the clusters severely reduces the number of possible isoforms that can be observed. In fact, of the 180 potential isoforms encoded by Mhc, we observed only 12 isoforms. Various Mhc isoforms are known to be expressed in striking spatial and temporally restricted patterns [14] and thus it is likely that other Mhc isoforms that we did not observe, could be observed by sequencing other tissue samples.Fig. 6

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.