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P.Gangadhara RaoIARI Ph.DVEGETABLE SCIENCE

Introduction Sequencing (DNA sequencing) is used to

determine the order of the nucleotide bases (ATGC) in a molecule of DNA.

(Ansorge et al., 2009)

Cont…

DNA sequence represents a single format onto which a broad range of biological phenomena can be projected for high throughput data collection

(Stenzel et al., 2008)

First generation sequencing

(Robertson et al., 2007)

Sanger method

(Sanger et al., 1977)

Sanger method (Helmut Kae et al., 2011)

Maxam and Gilbert method

• Chemical modification in the DNA

• Here purified DNA is used

• The fragments are radioactively labeled at 5’ end

• Chemical treatment generates the nucleotide bases

• DNA is cleaved by hot piperidine at modified base

• Conc. modifying chemicals is applied to the DNA

• Generates radiolabeled DNA fragments

• Electrophoresis and visualized in X-rays films (Ordway et al., 2007)

(www.molecularstation.com/...techniques/maxam-Gilbert-sequencing/2009)

Dye-terminator sequencing

(www.nslc.wustl.edu/elgin/genomics/tour/pdf/pcr.pdf/ 2008)

Capillary electrophoresis

Sequence ladder by radioactive sequencing compared to fluorescent peaks

(www.nslc.wustl.edu/elgin/genomics/tour/pdf/pcr.pdf 2008)

Cont…

Amplification and clonal selection

(BraslavskyI et al., 2003)

Next generation sequencing

(Metzker, 2010)

Next (2nd) generation sequencing platforms

(Metzker, 2010)

(Nature biotechnology, 2008)

(http://www.454.com)

Roche/454’s GS FLX Titanium

(http://www.454.com.)

454 Sequencing technology

(Genome Analyzer brochure, http://www.solexa.com)

(http://www.solexa.com)

11/10/2010 Advanced Centre for Biotechnology

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Fluorescent Signal Detection

Laser Energy

CC

D

Electrophoresis

+

-

dye

• Laser beam generated by single argon laser split to form dual pathway

• Split beam allows simultaneous illumination of 16 (4) capillaries from both sides of array at detection cell

• Fluorescence signal emitted from DNA fragments at the detection cell are collected on CCD camera

SOLiD DNA sequencing platform

(http://www.appliedbiosystems.com)

SOLiD DNA sequencing platform

(http://www.appliedbiosystems.com)

(Schuster et al., 2008)

Sequencing-by-ligation using the SOLiD DNA sequencing platform

(Johnson, 2009)

(Schuster et al., 2008)

Sequence determination by the SOLiD DNA sequencing platform

Cont…

(Johnson, 2009)

High-throughput shotgun Sanger sequencing

(Nardis et al., 2011)

( Nardis et al., 2011)

High-throughput shotgun Sanger sequencing

( Nardis et al., 2011)

Shotgun sequencing with cyclic-array methods

( Nardis et al., 2011)

(Nardis et al., 2011)

(Nardis et al., 2011)

Comparison of next (2nd) generation sequencing platforms

Platforms Lib/ template preparation

Prospects Constrains Biological applications

Roche/ 454’s GS FLX Titanium

Frag/ emPCR Longer reads improve mapping in the same regions; fast run times

High reagent cost; high error rate occurrence

Bacterial and insect genome assemblies

Illumina/ Solexa’s GA II

Frag/ solid- phase

Currently the most widely used platforms

Low samples reads

Gene discovery in metagenomics

Life/APG’s SOLid 3

Frag/ emPCR Error correction is possible

Long time runs Gene discovery in metagenomics

Helicos BioSciences HeliScope

Frag/single molecule

Genome and sequenced based applications

High error rate occurrence

Sequenced based methods

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Shotgun Sequence of the Melon Genome

(Jordi et al., 2011)

In total, more than 22 million reads from a mixture of shotgun and 3,820 kb paired-end reads representing 17.6x of the melon genome have been sequenced and assembled.

The current melon assembly contents 361Mbp(80%) of expected genome sequence of double haploid line.

The melon genome assembly that its quality is high and has 47,837 scaffolds and 1.1 Mb obtained using pure shotgun reads.

Sanger sequencing is more accurate but more cost whereas Roche method is less accuracy and lower in cost.

This studies have also been done in watermelon, cucumber.

(Jordi et al., 2011)

Whole genome shotgun sequencing of Brassica oleraceae and its application to gene discovery and annotation in

Arabidopsis

Comparative studies of A. thaliana and close related species of Brassica oleraceae has been identified and represent as a functional sequence.

A total of 454,274 whole genome shotgun sequences covering 283 Mb of the estimated 650Mb Brassica genome were searched against Arabidopsis genome

(Ayele et al., 2011)

Cont..

229,735 conserved genes 167,375 genes intersected with Arabidopsis gene model

After removal of the matching known proteins, the genome was chained together as a comprising portions of same functional units.

(Ayele et al., 2011)

This resulted in 27,347 chains of 15,686 genes were distinct from existing gene expression

By Rapid amplification method of cDNA ends was used to obtain potential full length transcripts region of 58 genes

This resulting sequence led to creation of 21 gene models and 17 new overlapping genes against Arabidopsis genome

Collectively this resulted data sets suggests that several thousands of new Brassica genes has to be identified

(Ayele et al., 2011)

Comparison between Brassica sp and Arabidopsis thaliana

Genome of the Cucumber, Cucumis sativus L.

Nature genetics 41, 1275- 1281(2009)

Published online: 1 November 2009 I doi:10.1038/ng.475

The genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next- generation Illumina GA sequencing technologies to obtain 72.2- fold genome coverage.

The absence of recent whole-genome duplication, alone with the presence of few tandem duplications, explains the small number of genes in the cucumber

(Nature genetics, 2009)

Cont…

The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and fresh green odor.

Identified 686 gene clusters related to phloem functions and also evolution of vascular system

(Nature genetics, 2009)

Potato Genome Sequencing Consortium (PGSC) aims to elucidate the complete genome sequence of potato.

This strategy is being complemented by whole genome shotgun sequencing approaches using both 454 GS FLX and illumina GA2 platforms for BAC-by-BAC sequencing.

This allows the parallel sequencing of several BACs in one sequencing of several BACs tagged with Multiplex Identifiers increasing the sequencing the speed and reducing costs

(Visser et al., 2009)

The strategy for this is to combine anchored BAC- by-BAC based sequenced data with the random short read sequence data that can be generated by the Illumina GA2 and the ROCHE GS FLX platforms.

56 BAC were sequenced by traditional Sanger's method.

Among them 24 BAC contiguous DNA stretch sizes have been predicted by pulsed field gel electrophoresis (PFGE).

Then 8 BACs were sequenced followed by 48 BACs in two consecutive runs, using two reaction chambers and was predicted using PFGE (Visser et al., 2009)

Category Examples of applications

Complete genome resequencing Comprehensive polymorphism and mutation discovery in individual human genomes

Reduced representation sequencing

Large scale polymorphism discovery (tomato)

Targeted genomic resequencing Targeted polymorphism and mutation discovery (tomato)

Paired end sequencing Discovery of inherited and acquired structural variation

Metagenomic sequencing Discovery of infectious and commensal flora (potato)

Applications of next (2nd) generation sequencing

Category Examples of applications

Transcriptome sequencing Quantification of gene expression and alternative splicing; transcript annotation; discovery of transcribed SNPs or somatic mutations (cucumber, tomato)

Small RNA sequencing Micro RNA profiling (tomato)

Chromatin immunoprecipitation- sequencing (ChIP Seq)

Genome-wide mapping of protein-DNA interactions (cucumber)

Nuclease fragmentation and sequencing

Nucleosome positioning

Molecular barcoding Multiplex sequencing of samples from multiple individuals (Brassica sp)

Third generation sequencing technologies

• SBS technologies in which single molecules of DNA polymerase are observed as they synthesize a single molecule of DNA

• Nanopore-sequencing technologies- molecules of DNA are threaded through a nanopore and individual bases are detected

• Direct imaging sequencing of individual DNA molecules using advanced microscopy techniques

• Hence the above technologies are currently in pipeline

(http://www.pacificbiosciences.com/oxfordjournals.org)

Conclusion Future generation sequencing

Requires only a smaller space area to work

To determine the length of the DNA, gel electrophoresis is not needed

Hundreds of reactions can be run at any one time

It requires short time to sequence a DNA molecule

It is cost effective

Overall view of the 1st and 2nd generation sequencing