Identification of Splice Isoforms in T Helper (TH) Cells Using Nanopore-based cDNA sequencing

Abstract

Naïve T cells are the precursors for the adaptive immune response against pathogens. They circulate the blood and secondary lymphoid organs until they are stimulated through a diverse pool of T cell receptors to become effector or memory cells. The differentiation of naïve T cells to effector subsets is regulated through a complex gene regulatory network resulting in transcriptomic alterations. Our understanding of how this process is controlled transcriptionally is limited. Nanopore-based RNA sequencing technology enables identification of the full length transcripts and alternatively spliced isoforms. We used this technique to identify the transcriptional signatures of differentiated T cell subtypes. Here, we present a single molecule transcriptome map of differentiated Treg cells obtained from mouse spleen to characterize full-length transcripts and alternatively spliced isoforms. We obtained 5,092,757 quality filtered sequence reads from cDNA sequencing of Treg cells with mean base quality of 9.2 and mean length of 876.4 bp. Preprocessing and filtering of the data set followed by alignment to the mouse genome (mmlO) resulted in 4,724,442 mappable reads (92.7% with 15% error allowance) corresponding to 29,716 transcripts and 15,645 annotated genes. Functional analysis of 212 transcript expressed at TPM > 0.25 resulted in the enrichment of ''Nonsense Mediated Decay (NMD)", "apoptotic signaling pathway", "Antigen processing" and other significant (<1% FDR) pathways. While splicing analysis revealed hundreds of genes exhibiting multiple splicing isoforms.