Using TIF-Seq2 to investigate association between 5´ and 3´mRNA ends

Methods Enzymol. 2021:655:85-118. doi: 10.1016/bs.mie.2021.03.017. Epub 2021 May 28.


The development of high-throughput technologies has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization involving thousands of overlapping coding and non-coding RNA isoforms that challenge our traditional definitions of genes and functional regions of the genome. In this chapter, we discuss the application of an improved Transcript Isoform Sequencing approach (TIF-Seq2) able to concurrently determine the start and end sites of individual RNA molecules. We exemplify its use for the investigation of the human transcriptome and show how it is especially well suited to discriminate between overlapping molecules and accurately define their boundaries.

Keywords: Full-length isoform; Fusion gene; Overlapping transcriptome; Poly(A) site; Read-through transcript; Transcription complexity; Transcription start site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Profiling
  • Genome*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Protein Isoforms / genetics
  • RNA, Messenger / genetics
  • Sequence Analysis, RNA
  • Transcriptome*


  • Protein Isoforms
  • RNA, Messenger