Limits...
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.

Show MeSH

Related in: MedlinePlus

Depth of coverage plots for detected exons found in a non-redundant database created from the eight empirically determined human annotation tracks. This database included a total of 1,016,490 unique annotated exons. A) Depth of coverage determined from the combined set of annotation tracks by detecting the number of annotated exons with an average of 1 and 5 read coverage at every 10 million reads. As shown, the plots approach plateaus at 300 million reads, suggesting the transcriptome is nearly fully covered at this level of sequencing. B) Depth of coverage for the individual annotation tracks at 300 million reads. The lincRNA and CCDS annotation tracks have the lowest (35%) and highest (94%), respectively. On average, the other 6 annotation tracks are covered near 80%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924432&req=5

Figure 1: Depth of coverage plots for detected exons found in a non-redundant database created from the eight empirically determined human annotation tracks. This database included a total of 1,016,490 unique annotated exons. A) Depth of coverage determined from the combined set of annotation tracks by detecting the number of annotated exons with an average of 1 and 5 read coverage at every 10 million reads. As shown, the plots approach plateaus at 300 million reads, suggesting the transcriptome is nearly fully covered at this level of sequencing. B) Depth of coverage for the individual annotation tracks at 300 million reads. The lincRNA and CCDS annotation tracks have the lowest (35%) and highest (94%), respectively. On average, the other 6 annotation tracks are covered near 80%.

Mentions: At 314 million reads, we were able to detect 75% of all exons annotated in the reference transcriptome at an average read depth of 5 or greater (Figure 1A). Some additional exons can be observed at average read depth of 1 or greater, with 83% of all exons detected, corresponding to approximately 160,000 unique transcripts. Detection of annotated exons from the standpoint of the individual databases was relatively consistent, aside from the lincRNA and CCDS databases (Figure 1B). We detected only 35% of the annotated lincRNA exons, but 90% of the annotated CCDS exons, at an average read depth of 5. We detected between 77-82% of the annotated exons in the remaining 6 databases.


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)

Depth of coverage plots for detected exons found in a non-redundant database created from the eight empirically determined human annotation tracks. This database included a total of 1,016,490 unique annotated exons. A) Depth of coverage determined from the combined set of annotation tracks by detecting the number of annotated exons with an average of 1 and 5 read coverage at every 10 million reads. As shown, the plots approach plateaus at 300 million reads, suggesting the transcriptome is nearly fully covered at this level of sequencing. B) Depth of coverage for the individual annotation tracks at 300 million reads. The lincRNA and CCDS annotation tracks have the lowest (35%) and highest (94%), respectively. On average, the other 6 annotation tracks are covered near 80%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Depth of coverage plots for detected exons found in a non-redundant database created from the eight empirically determined human annotation tracks. This database included a total of 1,016,490 unique annotated exons. A) Depth of coverage determined from the combined set of annotation tracks by detecting the number of annotated exons with an average of 1 and 5 read coverage at every 10 million reads. As shown, the plots approach plateaus at 300 million reads, suggesting the transcriptome is nearly fully covered at this level of sequencing. B) Depth of coverage for the individual annotation tracks at 300 million reads. The lincRNA and CCDS annotation tracks have the lowest (35%) and highest (94%), respectively. On average, the other 6 annotation tracks are covered near 80%.
Mentions: At 314 million reads, we were able to detect 75% of all exons annotated in the reference transcriptome at an average read depth of 5 or greater (Figure 1A). Some additional exons can be observed at average read depth of 1 or greater, with 83% of all exons detected, corresponding to approximately 160,000 unique transcripts. Detection of annotated exons from the standpoint of the individual databases was relatively consistent, aside from the lincRNA and CCDS databases (Figure 1B). We detected only 35% of the annotated lincRNA exons, but 90% of the annotated CCDS exons, at an average read depth of 5. We detected between 77-82% of the annotated exons in the remaining 6 databases.

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