TERRA ONTseq: a long read-based sequencing pipeline to study the human telomeric transcriptome

The long noncoding RNA TERRA is transcribed from chromosome ends and supports telomere integrity. In humans, TERRA transcription is thought to be driven in part by promoters comprising CpG dinucleotide-rich tandem repeats of 29 base pairs (bp) present in roughly half of the subtelomeres. However, TERRA transcripts originate also from chromosome ends devoid of those 29 bp repeats. Systematic studies of TERRA molecules have been constrained by their low abundance and repetitive nature; hence, a complete picture of the TERRA transcriptome and its chromosomal origin is still lacking. Here, we used Oxford Nanopore (ONT) sequencing to generate a novel experimental pipeline to sequence and analyze human TERRA (TERRA ONTseq). By applying TERRA ONTseq to different human cell lines, we show that the vast majority of human telomeres produce TERRA transcripts and that different chromosome ends generate variable amounts of TERRA. Moreover, based on our TERRA ONTseq data, we isolated a novel CpG-rich promoter devoid of 29 bp repeats from the subtelomere of the long arm of chromosome 7, which appear to be the most highly transcribed human chromosome end. With the development of TERRA ONTseq, we produced the first unambiguous and complete picture of the human TERRA transcriptome and we provide the scientific community with an invaluable tool for future studies.