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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 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)
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Fig4: 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)

Mentions: To explore the diversity of Dscam1 isoforms expressed in a biological sample, we prepared a Dscam1 library from RNA isolated from D. melanogaster heads prepared from mixed male and female adults using 25 cycles of PCR and sequenced it for 12 h on the MinION nanopore sequencer obtaining a total of 159,948 reads of which 78,097 were template reads, 48,474 were complement reads, and 33,377 were 2D reads (Fig. 4a). We aligned the reads individually to the exon 4, 6, and 9 variants using LAST. A total of 28,971 reads could be uniquely or preferentially aligned to a single variant in all three clusters. For further analysis, we used all 16,419 2D read alignments and 31 1D reads when both template and complement aligned to same variant exons (not all reads with both a template and complement yield a 2D read). The remaining 12,521 aligned reads were 1D reads where there was either only a template or complement read, or when the template and complement reads disagreed with one another and were therefore not used further. We observed 92 of the 93 potential exon 4, 6, or 9 variants – only exon 6.11 was not observed in any read (Fig. 4f). To assess the accuracy of the results we performed RT-PCR using primers in the flanking constitutive exons that contained Illumina sequencing primers to separately amplify the Dscam1 exon 4, 6, and 9 clusters from the same RNA used to prepare the MinION libraries, and sequenced the amplicons on an Illumina MiSeq. The frequency of variable exon use in each cluster was extremely consistent between the two methods (R2 = 0.95, Fig. 5a).Fig. 4


Determining exon connectivity in complex mRNAs by nanopore sequencing.

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

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)
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Related In: Results  -  Collection

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Fig4: 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)
Mentions: To explore the diversity of Dscam1 isoforms expressed in a biological sample, we prepared a Dscam1 library from RNA isolated from D. melanogaster heads prepared from mixed male and female adults using 25 cycles of PCR and sequenced it for 12 h on the MinION nanopore sequencer obtaining a total of 159,948 reads of which 78,097 were template reads, 48,474 were complement reads, and 33,377 were 2D reads (Fig. 4a). We aligned the reads individually to the exon 4, 6, and 9 variants using LAST. A total of 28,971 reads could be uniquely or preferentially aligned to a single variant in all three clusters. For further analysis, we used all 16,419 2D read alignments and 31 1D reads when both template and complement aligned to same variant exons (not all reads with both a template and complement yield a 2D read). The remaining 12,521 aligned reads were 1D reads where there was either only a template or complement read, or when the template and complement reads disagreed with one another and were therefore not used further. We observed 92 of the 93 potential exon 4, 6, or 9 variants – only exon 6.11 was not observed in any read (Fig. 4f). To assess the accuracy of the results we performed RT-PCR using primers in the flanking constitutive exons that contained Illumina sequencing primers to separately amplify the Dscam1 exon 4, 6, and 9 clusters from the same RNA used to prepare the MinION libraries, and sequenced the amplicons on an Illumina MiSeq. The frequency of variable exon use in each cluster was extremely consistent between the two methods (R2 = 0.95, Fig. 5a).Fig. 4

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.