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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.


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
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Fig3: 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

Mentions: Template switching can occur frequently when libraries are prepared by PCR and can confound the interpretation of results [9, 10]. For example, CAM-Seq [11] and a similar method we independently developed called Triple-Read sequencing [12] to characterize Dscam1 isoforms, were found to have excessive template switching due to amplification during the library prep protocols. To assess template switching in our current study, we generated a spike-in mixture of in vitro transcribed RNAs representing six unique Dscam1 isoforms – Dscam14.2,6.32,9.31, Dscam14.1,6.46,9.30, Dscam14.3,6.33,9.9, Dscam14.12,6.44,9.32, Dscam14.7,6.8,9.15, and Dscam14.5,6.4,9.4. We used 10 pg of this control spike-in mixture and prepared libraries for MinION sequencing by amplifying the exon 3 through exon 10 region for 20, 25, or 30 cycles of RT-PCR. We then end-repaired and dA-tailed the fragments, ligated adapters, and sequenced the samples on a MinION (7.3) for 12 h each. We obtained 33,736, 8,961, and 7,511 base-called reads from the 20, 25, and 30 cycle libraries, respectively. Consistent with the size of the exon 3 to 10 cDNA fragment being 1,806–1,860 bp in length, depending on the precise combination of exons it contains, most reads we observed were in this size range (Fig. 3a). We used Poretools [13] to convert the raw output files into fasta format and then used LAST to align the reads to a LAST database containing each variable exon. From these alignments, we identified reads that mapped to all three exon clusters, as well as the exon with the best alignment score within each cluster. When examining the alignments to each cluster independently, we found that for these spike-in libraries, all reads mapped uniquely to the exons present in the input isoforms. Therefore, any observed isoforms that were not present in the input pool were a result of template switching during the RT-PCR and library prep protocol and not due to false alignments or sequencing errors.Fig. 3


Determining exon connectivity in complex mRNAs by nanopore sequencing.

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

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: 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
Mentions: Template switching can occur frequently when libraries are prepared by PCR and can confound the interpretation of results [9, 10]. For example, CAM-Seq [11] and a similar method we independently developed called Triple-Read sequencing [12] to characterize Dscam1 isoforms, were found to have excessive template switching due to amplification during the library prep protocols. To assess template switching in our current study, we generated a spike-in mixture of in vitro transcribed RNAs representing six unique Dscam1 isoforms – Dscam14.2,6.32,9.31, Dscam14.1,6.46,9.30, Dscam14.3,6.33,9.9, Dscam14.12,6.44,9.32, Dscam14.7,6.8,9.15, and Dscam14.5,6.4,9.4. We used 10 pg of this control spike-in mixture and prepared libraries for MinION sequencing by amplifying the exon 3 through exon 10 region for 20, 25, or 30 cycles of RT-PCR. We then end-repaired and dA-tailed the fragments, ligated adapters, and sequenced the samples on a MinION (7.3) for 12 h each. We obtained 33,736, 8,961, and 7,511 base-called reads from the 20, 25, and 30 cycle libraries, respectively. Consistent with the size of the exon 3 to 10 cDNA fragment being 1,806–1,860 bp in length, depending on the precise combination of exons it contains, most reads we observed were in this size range (Fig. 3a). We used Poretools [13] to convert the raw output files into fasta format and then used LAST to align the reads to a LAST database containing each variable exon. From these alignments, we identified reads that mapped to all three exon clusters, as well as the exon with the best alignment score within each cluster. When examining the alignments to each cluster independently, we found that for these spike-in libraries, all reads mapped uniquely to the exons present in the input isoforms. Therefore, any observed isoforms that were not present in the input pool were a result of template switching during the RT-PCR and library prep protocol and not due to false alignments or sequencing errors.Fig. 3

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.