Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms

Abstract

Studying isoform expression at the microscopic level has always been a challenging task. A classical example is kidney, where glomerular and tubulo-interstitial compartments carry out drastically different physiological functions and thus presumably their isoform expression also differs. We aim at developing an experimental and computational pipeline for identifying isoforms at microscopic structure-level. We microdissected glomerular and tubulo-interstitial compartments from healthy human kidney tissues from two cohorts. The two compartments were separately sequenced with the PacBio RS II platform. These transcripts were then validated using transcripts of the same samples by the traditional Illumina RNA-Seq protocol, distinct Illumina RNA-Seq short reads from European Renal cDNA Bank (ERCB) samples, and annotated GENCODE transcript list, thus identifying novel transcripts. We identified 14,739 and 14,259 annotated transcripts, and 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Of note, relying solely on either short or long reads would have resulted in many erroneous identifications. We identified distinct pathways involved in glomerular and tubulo-interstitial compartments at the isoform level, creating an important experimental and computational resource for the kidney research community. Author summary Gene expression is closely associated with functions of diverse tissues and mechanisms underlying human diseases. The regulation of cellular and physiological functions is further complicated by alternative splicing, which enables a single mRNA to be translated into multiple different protein isoforms. The information from the human genome largely increases during this process. Therefore, it is important to study tissue-specific gene and isoform expression at the microscopic level. In this work, we focused on the kidney, a model tissue that has two compartments with different physiological functions and isoform expressions. Chronic kidney disease is prevalent in 14.5% of the US population. We develop an experimental and computational pipeline for identifying isoforms in glomerular and tubulo-interstitial compartments at the microscopic level. Through analyzing multiple kidney samples by two types of RNA sequencing techniques, we integrated both short reads and long reads to identify transcripts and validate results. We identified a total of 14,739 and 14,259 known transcripts, as well as 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Distinct functional pathways were further revealed in these two compartments.