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Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes.

Farkas MH, Grant GR, White JA, Sousa ME, Consugar MB, Pierce EA - BMC Genomics (2013)

Bottom Line: These data represent a significant addition to the annotated human transcriptome.Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected.In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA. eric_pierce@meei.harvard.edu.

ABSTRACT

Background: The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease.

Results: We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3' and 5' alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products.

Conclusions: To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

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Related in: MedlinePlus

The ratio of novel to annotated isoform abundance as determined by reads crossing splice junctions. The number of reads crossing a novel splice junction relative to the corresponding annotated splice junction were used to calculate the ratio. This data demonstrates that a relatively significant portion (~15%) of novel isoforms in the human retinal transcriptome are at least as abundant or more abundant than their annotated counterparts. The green bars represent the percentage of total junctions in which the novel isoform was more abundant than the annotated. The blue-green bar represents the percentage of total junctions where the novel and annotated isoforms are equal (< 2-fold change). The blue bars represent the percentage of total junctions in which the annotated isoforms are more abundant than the novel.
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Figure 2: The ratio of novel to annotated isoform abundance as determined by reads crossing splice junctions. The number of reads crossing a novel splice junction relative to the corresponding annotated splice junction were used to calculate the ratio. This data demonstrates that a relatively significant portion (~15%) of novel isoforms in the human retinal transcriptome are at least as abundant or more abundant than their annotated counterparts. The green bars represent the percentage of total junctions in which the novel isoform was more abundant than the annotated. The blue-green bar represents the percentage of total junctions where the novel and annotated isoforms are equal (< 2-fold change). The blue bars represent the percentage of total junctions in which the annotated isoforms are more abundant than the novel.

Mentions: Greater than 100-fold more reads cross annotated splice junctions, relative to those reads crossing novel splice junctions. However, nearly 4% of the novel splice junctions in our dataset have more reads crossing them than reads crossing their annotated counterpart (Figure 2). Roughly 9% of the novel and the corresponding annotated splice junction have an equal number of reads crossing each splice junction. Over 87% of the annotated splice junctions have more reads crossing them relative to the corresponding novel splice junction. A novel exon in MLL2 (ENSG00000167548) is a clear example where the novel junctions form a major transcript isoform of the gene (Figure 3).


Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes.

Farkas MH, Grant GR, White JA, Sousa ME, Consugar MB, Pierce EA - BMC Genomics (2013)

The ratio of novel to annotated isoform abundance as determined by reads crossing splice junctions. The number of reads crossing a novel splice junction relative to the corresponding annotated splice junction were used to calculate the ratio. This data demonstrates that a relatively significant portion (~15%) of novel isoforms in the human retinal transcriptome are at least as abundant or more abundant than their annotated counterparts. The green bars represent the percentage of total junctions in which the novel isoform was more abundant than the annotated. The blue-green bar represents the percentage of total junctions where the novel and annotated isoforms are equal (< 2-fold change). The blue bars represent the percentage of total junctions in which the annotated isoforms are more abundant than the novel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924432&req=5

Figure 2: The ratio of novel to annotated isoform abundance as determined by reads crossing splice junctions. The number of reads crossing a novel splice junction relative to the corresponding annotated splice junction were used to calculate the ratio. This data demonstrates that a relatively significant portion (~15%) of novel isoforms in the human retinal transcriptome are at least as abundant or more abundant than their annotated counterparts. The green bars represent the percentage of total junctions in which the novel isoform was more abundant than the annotated. The blue-green bar represents the percentage of total junctions where the novel and annotated isoforms are equal (< 2-fold change). The blue bars represent the percentage of total junctions in which the annotated isoforms are more abundant than the novel.
Mentions: Greater than 100-fold more reads cross annotated splice junctions, relative to those reads crossing novel splice junctions. However, nearly 4% of the novel splice junctions in our dataset have more reads crossing them than reads crossing their annotated counterpart (Figure 2). Roughly 9% of the novel and the corresponding annotated splice junction have an equal number of reads crossing each splice junction. Over 87% of the annotated splice junctions have more reads crossing them relative to the corresponding novel splice junction. A novel exon in MLL2 (ENSG00000167548) is a clear example where the novel junctions form a major transcript isoform of the gene (Figure 3).

Bottom Line: These data represent a significant addition to the annotated human transcriptome.Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected.In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA. eric_pierce@meei.harvard.edu.

ABSTRACT

Background: The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease.

Results: We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3' and 5' alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products.

Conclusions: To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

Show MeSH
Related in: MedlinePlus