Next-Generation DNA Sequencing of Paired-End Tags (PET) for Animal Transcriptomics and

Functional Genomics

Varij Nayan and Anuradha Bhardwaj

THE MAP OF HUMAN & ANIMAL GENOME

(Adapted from Fullwood M. 2009)

Promises: for huge improvements in human & animal healthcare and productionContemporary Study:

Genomes are read out as linear sequencesIn the cell- complex interactions and mechanisms to transduce DNA information into biological functionConventional DNA sequencing -explore genetic elements and structure

Practical Challenges:high sequencing costs and low throughputs limited in-depth analysis of genomic elements

Genomics

Next Generation Sequencers

Next (or 3rd) generation sequencers came onto the scene in the early 2000’s

General characteristics : Amplification of genetic material by PCR Ligation of amplified material to a solid

surface Sequence of the target genetic material is

determined using Sequence-by-Synthesis (using labelled nucleotides or pyrosequencing for detection) or Sequence by ligation

Sequencing done in a massively parallel fashion and sequence information is captured by a computer

Next Generation Sequencing

DNA is fragmented

Adaptors ligated to

fragments

Several possible protocols

yield array of PCR colonies

Emulsion PCR

Bridge PCR

Enyzmatic extension with

fluorescently tagged

nucleotides

Cyclic readout by imaging

the array

(Shendure, 2008)

Next-Generation Sequencing Workflow

Illumina, Roche 454 or ABI SOLiD?

Applications of Next-Generation Sequencing

Transforming the field of genomic science Read DNA templates in a highly parallel manner to generate massive amounts of sequencing dataRead length for each DNA template- short compared to Sanger capillary sequencing instrumentsMassively parallel and short read strategy of DNA sequencing

opens new ways for interrogating human and animal genomesshort read lengths- limitations in infering biological applications

Focus on overcoming the limitation of short tags for genome-wide analysis

Next Generation Sequencing Technology

“The paired-end tag (PET) sequencing is one such strategy for improving DNA sequencing efficiency and enabling biological functions”

Strategies for overcoming limitations of NGSTs

Sequencing-based methods for understanding genetic elements in genomes

(Fullwood et al., 2009)

Sequencing only a short stretch of DNA information, typically less than 100 bp.

The NGS platforms generate only short tag sequences, typically 16–50 bp.

Ideally in the future, tags would be 50–100 bp.

Short Tag Sequencing

ESTs are short DNA sequences corresponding to a fragment of a complimentary DNA (cDNA) molecule and which may be expressed in a cell at a particular given time

A short DNA sequence (a “tag”) from a cDNA clone (hence it is expressed)

EST (expressed sequence tag)

5’ 3’mRNA AAAA5’ UTR 3’ UTRProtein coding

Duplex inserts in cDNA clones

ESTs are sequences from each end of the cDNA inserts

Unigene cluster is an group of overlapping ESTs, likely from one gene

Short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known

Easily detected by PCR Useful for constructing genetic and physical maps

from sequence data When STS loci contain genetic polymorphisms

(e.g. SSLPs, SNPs), they become valuable genetic markers as microsatellites (SSRs, STMS or SSRPs), SCARs, CAPs, and ISSRs- loci can be used to distinguish individuals

Used in shotgun sequencing- to aid sequence assembly

Very helpful for detecting microdeletions in some genes

Sequence-tagged site (STS)

A method for preparation of short tags (13 bp), mostly for cDNA analysis to profile transcriptomes

Variants of SAGE includeLongSAGE (20 bp) SuperSAGE (25 bp)

SAGE (Serial Analysis of Gene Expression)

A short-tag approach similar to SAGE but using a ligation- based sequencing method to profile transcriptomesMeans of determining abundance of RNA species

Unique tags added to cDNAsTags hybridized to oligonucleotides on microbeads

MPSS (massive parallel signature sequencing)

For the identification of transcription start sites (TSSs) and their promoters, 5′ end–specific signature sequences are required for higher annotations of expression profiles.A method using the Cap-trapper method to retain 5’ intact transcripts and a SAGE-like approach to extract 5’ tags (20 bp)Variants include 5’ LongSAGE

CAGE (Cap-associated analysis of gene expression)

Schematic procedure of the CAGE protocol

(Shiraki et al., 2003)

Paired End vs. Unpaired Reads

Millions of reads are generated. Repetitive regions within the genome cause

the reads to be mapped to multiple locations.

Polymorphism in a read can cause it to be mapped to a wrong location.

Discarding ambiguous reads can reduce coverage

“This method covalently links the 5’ tag and 3’ tag of a DNA fragment into a ditag structure for sequencing analysis, thus combining the benefits of the cost-effective SAGE and the linkage information from paired-end sequencing”

Paired End Ditag Method

Sequencing-based methods for understanding genetic elements in genomes

(Fullwood et al., 2009)

Paired End SequencingShort tag sequencing

PET Converged from 2 technological concepts

(Hong, 1981)

Schematic view of PET methodology

Mme I (type IIS RE)- 18/20 bp dsEcoP15I (type III RE)- 25/27 bp dsRecircularization of tag-vector-tag

tag-linker-tag

Nebulization: - Tags of 106 bp size- unbalanced with tags

under 15 bp

(Fullwood et al., 2009)

Cloning Method Cloning-free Method

Benefit: Preserves original flcDNA/ ChIP DNA fragments as library clones

- Straightforward- Avoid biases related

to cloningLimitation:- Construction

process long (2-4 weeks)

- Technically challenging

Applications of PET

(Fullwood et al., 2009)

Demarcation of 1st and Last ExonsDefine the TSSs and PASsConnectivity between two sites

Limitation: no information of internal exonsUnique Feature: Ability to detect unconventional fusion genesEarly version of RNA-PET was cloning based method i.e. GIS-PET analysis

RNA-PET for Transcriptome Studies

Early version of RNA-PET was cloning based method i.e. GIS (Gene Identification signature)-PET analysisIt has revealed its ability to high-throughput identification of fusion genesNow, Cloning-free methodsAlternatively, perform shotgun paired-end sequencing of cDNA templates

RNA-PET for Transcriptome Studies

Sequencing based approach to to characterize ChIP-enriched DNA fragments

ChIP-PET provides linked 5’ and 3’ sequences for ChIP-enriched DNA molecules, which are mapped to the reference genome such that the complete ChIP DNA fragment can be inferred from the genome sequence in between the 5’ and 3’ tags, and the enriched TFBS can be determined

ChIP-PET :identifying regulatory and epigenetic elements

Introduce a linker sequence in the junction of two DNA fragments during nuclear proximity ligation to build connectivity of DNA fragments that are tethered together by protein factors

All linker connected ligation products can be extracted for the tag–linker–tag constructs that can be analyzed by ultra-high-throughput PET sequencing

ChIA-PET : identifying chromatin interactions

When mapped to reference genome, the ChIA-PET sequences are read out to detect the relationship of two DNA fragments in chromatin interactions captured by chromatin proximity ligation

ChIA-PET has the potential to be an unbiased genome-wide approach for de novo detection of chromatin interactions

Ideal method for sequencing and assembling genomes as well as studying genome structural variationsFirst demonstrated in resequencing an evolved E. coli genome using the polony sequencing-by ligation methodApplied to map cancer genome rearrangementsDNA-PET can become a vital part in the concept of personal genomics for personal medicine

DNA-PET: Genome Structure Analysis

Detection of structural variations: insertions, deletions, duplications, inversions, translocations

Limitations:- require more starting material- involve more molecular

manipulations- certain DNA portions not

recoveredVersatile readily adapt to new sequencing technologyCouple methods for asking biological questions with NGS Future is bright……….

Future of PET Technology

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