Paired‐End diTagging for Transcriptome and Genome Analysis

Abstract

The Paired-End diTagging (PET) procedure enables one to obtain sequence information from both termini of any contiguous DNA fragment. This is achieved by a series of enzymatic manipulations that introduce MmeI sites directly flanking each DNA insert during the construction of a plasmid library. Subsequent MmeI digestion and self-ligation results in the production of covalently-linked paired-end ditags (PETs) that can be extracted and then concatenated for efficient sequencing. By mapping the PET sequences to assembled genomes, the original DNA fragments from which the PETs were derived can be precisely localized. This unit details two applications of PET technology. In GIS-PET, ditagging of mRNA converted to full-length cDNA enables whole-transcriptome analysis, including novel gene identification, gene prediction validation, and gene expression studies. In ChIP-PET, ditagging of chromatin immunoprecipitation-enriched genomic DNA fragments enables the global mapping of transcription factor binding sites. A recent innovation (Multiplex Sequencing of Paired-End ditags; MS-PET) enables PETs to be sequenced using high-throughput 454 sequencing, greatly increasing the amount of data that can be collected in each run.