SRA

De novo transcriptome construction from short-read RNA-seq is a common method for reconstructing mRNA transcripts within a given sample. However, the precision of this process is unclear as it is difficult to obtain a ground-truth measure of transcript expression. With advances in third generation sequencing, full length transcripts of whole transcriptomes can be accurately sequenced to generate a ground-truth transcriptome. We generated long-read PacBio and short-read Illumina RNA-seq data from a human induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) cell line. We use the long-read data to identify simple metrics for assessing de novo transcriptome construction and optimizing a short-read based de novo transcriptome construction pipeline. We apply this pipeline to construct transcriptomes for 340 publicly available short-read RNA-seq samples originating from healthy adult and fetal human retina, cornea, and RPE and identify hundreds of novel gene isoforms and examine their significance in the context of ocular development and disease.