This Title All WIREs
How to cite this WIREs title:
WIREs Syst Biol Med
Impact Factor: 4.192

Multiplex parallel pair‐end‐ditag sequencing approaches in system biology

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Abstract Characterization of all the functional components constituted in human genome relies in our ability to completely elucidate the genetic/epigenetic regulatory networks, chromatin states, nuclear architectures, and genome variations. Such endeavors demand for the development of robust and effective genomic technologies. In the past few years, the availability of disruptive next generation DNA sequencing technologies has offered new promise for whole genome interrogation. However, despite the massive parallel and ultra‐high throughput capacity, the common nature of short read lengths found within these platforms limits their applications for many types of whole genome‐based analyses. To overcome such constrain, pair end ditag (PET) based sequencing concept was conceived as an immediate solution to expand the information content and extend the linear coverage. By sequencing paired end signatures from any desired DNA fragment and mapping them to the reference genome, PET strategy allows the accurate demarcation of target DNA boundaries and defines their locations on the genomic landscape. Furthermore, the ability to delineate relationship between two ends of a DNA molecule enables the full scale discovery of unconventional gene products, genome rearrangements, and chromatin interactions. Coupling with the massively parallel and ultra‐high throughput sequencing platforms, such unique features of PET strategy have the potential to revolutionize the approaches used to decipher regulatory networks in system biology, define the genome organizations, and characterize genome variations; which ultimately leads to the development of strategies for personalized medicine. Copyright © 2009 John Wiley & Sons, Inc. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods

This WIREs title offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images

(a) The concept of pair end ditag (PET) analysis. Paired tags from both 5′ and 3′ ends of the DNA molecule are simultaneously extracted and covalently linked as a unit. Upon sequencing and mapping analysis, the mapping coordinates on the reference genome define the identity of target DNA and its location. (b) Methods to generate PET sequencing templates. Adaptor containing type IIs restriction enzyme is added to each end of DNA. The adapter‐ligated DNA ends are either connected by cloning vector or self‐circularized followed by enzymatic digestion to release the PETs. Depending on the enzymes used, the tag length can be varied from 20 to 27 bp. (c) PET sequencing strategy. PET structures can be sequenced by all the commercially available sequencing platforms. PET can be concatenated as 10–20 units for capillary electrophoresis (CE) sequencing, 2–4 units for pyrosequencing or directly used as single unit in the pair‐end‐tag sequencing protocol by GA or SOLiD.

[ Normal View | Magnified View ]

ChIA‐PET analysis.The chromatin conformations tethered by specific protein factor are fixed by cross‐linking and enriched by ChIP. These interactions are captured by proximity‐based ligation facilitated by the linker adaptor. PETs connecting the interacting DNA of remote origins are extracted and sequenced. Depending on the nature of the ligating PETs (self‐ligation, intrachromosomal, or interchromosomal ligations), the PET mapping locations will reveal both the protein binding sites and long‐range interactions.

[ Normal View | Magnified View ]

ChIP‐PET anlaysis. The in vivo transcription factors and their target DNA interactions are cross‐linked by formaldehyde. With the antibody specific to the TF of interest, the TF‐target interactions can be enriched by chromatin immuno‐precipitation. The enriched DNA is subjected to PET analysis and the TFBS can be inferred by multiple overlapping PET mapping fragments.

[ Normal View | Magnified View ]

Structure variations detected by g‐PET analysis. (a) Genomic DNA of specific sizes (2, 5, or 10 Kb) is selected and self‐circularized. PETs are extracted and undergo sequence analysis. Rearranged genomic regions can be uncovered by comparing the directionality and span of g‐PETs mapping coordinates on reference genome. (b) Different types of structural variations detected by comparison of g‐PET mapping between the test and reference genome.

[ Normal View | Magnified View ]

Gene Identification Signature by Pair End diTag (GIS‐PET) analysis for gene identification and genome annotation. (a) PET is extracted from full length cDNA and mapped to reference genome. The mapping coordinates can demarcate the boundaries of expressed full length transcripts, define transcriptional units, and annotate genome for new genes and promoters. (b) An example of GIS‐PET structure and its location on chr.1:148040294–148053009. The numbers on the left represent the frequency of this cluster of PETs annotated to PSMD4 gene and tracks below show the same genes represented by Transfrag, transcription associated regions (TARs), cap analysis of gene expression (CAGE) and promoter assays.

[ Normal View | Magnified View ]

Related Articles

Genome‐wide DNA methylation profiling
DNA methylation and gene expression
Integrative Bioinformatics

Browse by Topic

Laboratory Methods and Technologies > Genetic/Genomic Methods

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts