Longitudinal RNA-seq analysis in human platelets

Longitudinal studies are required to distinguish within versus between-individual variation in gene expression, and are uniquely positioned to decipher genetic signal from environmental noise. However, longitudinal analyses of gene expression in healthy individuals—especially with regards to alternative splicing—are lacking for most primary cell types. In this study, we sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examine within and between-individual variation in platelet RNA-expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression and exon skipping are generally stable between and within individuals over time. However, some genes and exon splicing events varied broadly between individuals, while remaining stable within individuals. We show that this repeatability in gene expression is predictive of the presence of cis-eQTLs, and demonstrate the use of repeatability as an effective strategy to prioritize genes for platelet eQTL and sQTL discovery. We identify rs6128 as a platelet splice QTL for SELP, and find an rs6128 dependent association between exon 14 skipping and race. In vitro, this single nucleotide variant directly affects exon 14 (transmembrane domain) skipping, and changes the ratio of transmembrane versus soluble P-selectin protein. This data establishes a link between SELP splicing in platelets and previous studies associating rs6128 with soluble P-selectin and disease. In summary, we have used longitudinal analysis to define the stability of the platelet transcriptome and identify genetic modifiers of gene expression and splicing. We believe our approach will be broadly applicable to QTL analysis in other cell types.