DNA Sequencing

(1)

1

DNA Sequencing

(2)

2

DNA Sequencing

Description of Module Subject Name

Paper Name

Module Name/Title DNA Sequencing

Dr. Vijaya Khader Dr. MC Varadaraj

(3)

3

DNA Sequencing

1. Objectives

1. DNA sequencing Introduction 2. Types of methods available 3. Next generation sequencing

2. Lay Out

(4)

4

DNA Sequencing

3 Introduction

Sequencing

Maxam Gilbert Sanger's Next generation

(5)

5

DNA Sequencing

In the mid 1970s when molecular cloning techniques in general were rapidly improving, simple methods were also developed to determine the nucleotide sequence of DNA. These advances laid the foundation for the detailed analysis of the structure and function of the large number of genes. Other fields which utilize DNA sequencing include diagnostic and forensic biology. With the invention of DNA sequencing, research and discovery of new genes is increased many folds.

Using this technique whole genomes of many animal (human), plant, and microbial genomes have been sequenced. The first attempts to sequence DNA mirrored techniques developed in 1960s to sequence RNA. These involved :

a) Specific cleavage to the DNA in to smaller fragments by enzymatic digestion or chemical digestion

b) Nearest neighbour analysis c) Wandering spot method.

Indeed in some studies the DNA was transcribed in to RNA with E.coli RNA polymerase and then sequenced as RNA

So there were several questions unanswered;

 How many base pairs (bp) are there in a human genome?(~3 billion (haploid))

 How much did it cost to sequence the first human genome? ~$2.7 billion

 How long did it take to sequence the first human genome?(~13 years)

 When was the first human genome sequence complete? (2000-2003) Goal

figuring the order of nucleotides across a genome Problem

Current DNA sequencing methods can handle only

short stretches of DNA at once (<1-2Kbp) Solution

(6)

6

DNA Sequencing

Sequence and then use computers to assemble the small pieces 3.1 Maxam-Gilbert sequencing

Also known as chemical sequencing, here there is use radioactive labeling at one 5' end of the DNA fragment which is to be sequenced.

This method grew out of studies of the interaction between Lac repressor and lac operator in vitro. In this procedure DNA is radiolabelled at one end is partially cleaved in five different chemical reactions, and these are specific for each base. This generates five polulations of radiolabelled molecules that extend from a common point to the site of terminal cleavage. Each population consists of mixture of different molecules and their length is determined by how the bases are located in DNA. These populations are then resolved by electrophoresis through polyacrlamide gels and the end labelled molecules are detected by autoradiography. This method has not changed in any form or technique in later years, its same as it was devloped. The chemical degradation approach has one advantage over the chain termination sequencing: that the DNA sequence is obtained from the original DNA molecule and it is not an enzymatic copy. Therefore with this method one can sequence synthetic nucleotides, analyze DNA modifications such as DNA methylation and study both DNA secondary structure and interaction of proteins wth DNA be chemical protection or modification interference experiments.

(7)

7

DNA Sequencing

Figure: Maxam Gilbert Sequencing Method

3.2 Sanger’s Sequencing

Frederick Sanger and his colleagues developed the method of sequencing known as chain termination method.

This method soon became popular and common method of sequencing used by molecular biologists because of its ease to use and reliability. In this there is less use of toxic chemicals and also less amount of radioactivity is used in comparison to Maxam method. Sanger’s method was automated and it became the commonly used method of DNA sequencing. Next generation sequencing methods are also based on Sanger sequencing method. This method first described the use of: the use of specific primer for extension by DNA polymerase , base specific chain termination, use of polyacrylamide gels to discriminate between single stranded DNA chains differing in length by a single nucleotide. Despite these advances the method was too inaccurate to gain

(8)

8

DNA Sequencing

general acceptance and it was not until the introduction of chain terminating dideoxynucleoside triphosphates ddNTPs that enzymatic methods of DNA sequencing were used extensively.

2’, 3’ddNTPs differs from conventional DNTPs because of the absence of a hydroxyl residue at the 3’position of deoxyribose. These ddNTPs can be incorporated in to the DNA chain by DNA polymerases by their 5’triphosphate groups. The absence of a 3’hydroxyl residue will not allow to form the phosphodiester bond with the use of DNPT. It leads to prevention of extension of the growing DNA chain . When ddNTP is added with DNTPs, it set in the race between chain extension and termination of chain. The resultant are number of chains of oligonucleotide whose lengths is inferred by the distance between the end of the primer used to start DNA synthesis and the sites of premature termination. In a separate reactions there is use of four separate ddNTPs, number of oligonucleotides are generated which lead to termination at positions of A, C, G or T in the template strand.

Steps involved :

Mix DNA with dNTPs and ddNTPs Amplify

Run in Gel

Fragments migrate distance that is proportional to their size

(9)

9

DNA Sequencing

The 3′-OH group is missing in ddNTPs.which is important for the phosphodiester bond formation

 labelled primer and template are used in sequencing reaction mixture

 Dideoxynucleotides are added to all tubes .

Fluorescent dyes

 Multicyclic molecules which at different wavelengths absorb and emit light.

 Commonly sued are derivatives of fluorescein and rhodamine.

 In sequencing, these fluorescent molecules attached to the nucleotides covalently

 Separate color is used for four ddNTP.

 Because we are using a color dye so no need for separate tubes

(10)

10

DNA Sequencing

Figure: Diagrammatic representation of method

(11)

11

DNA Sequencing

Figure- Computerized visualization from a single lane of an automated sequencer.

Method uses non-radioactive fluorescent labelling.

Figure: Chromatogram

(12)

12

DNA Sequencing

Sanger Sequencing Advantages

 Long reads (~900bps)

 Suitable for small projects Disadvantages

 Low throughput

 Expensive

4. Next Generation Sequencing

Next-generation sequencing is term which applies to genome sequencing, genome resequencing, transcriptome profiling, protein-DNA interactions. Resequencing is important to study genome variations which is not possible by studying single genome. There is increasing demand for low cost sequencing and this has led to invention of next-generation sequencing or high-throughput sequencing technologies that can produce thousands or millions of sequences simultaneously.

NGS is divided further into polony-sequencing based technologies in which there is requirement to amplify DNA before sequencing, and single molecule sequencing which does not require amplification. By this method full E. coli genome was studied in 2005.

Applications of NGS include gene expression analysis and re-sequencing. And of course – advancements in chemistry, microscopy and other related technologies enabled the new sequencing technologies.

NGS Platforms

 Roche 454

 Illumina Solexa

 Applied Biosystems SOLiD

(13)

13

DNA Sequencing

4.1 454 pyrosequencing

In pyrosequencing sequence-based detection technology is used which enables fast and error free quantification of variation in a sequence. It is based on the principle of "sequencing by synthesis." Here chain termination method is not used rather in this technique when there is incorporation of nucleotide there is detection of pyrophosphate release. This technique employs emulsion polymerase chain reaction. DNA is amplified in water droplets in oil emulsion and each droplet contains DNA attached to beads which are coated with primers. The sequencing equipment contains many wells of picoliter-volume with each well containing a single bead and sequencing enzymes. Luciferase enzyme is used in this technique for the generation of light to detect single nucleotides added to nascent DNA. Pooled data is used to generate read outs of sequence.

(14)

14

DNA Sequencing

4.2 Illumina (Solexa) sequencing

Shankar Balasubramanian and David Klenerman co-founded Solexa in 1988 (it is part of Illumina). Their method of sequencing was based on reversible dye terminator technology and engineered DNA polymerase enzymes.

Here primers with DNA template are fixed to slide or flow cell and then amplification is done with polymerase

(15)

15

DNA Sequencing

which generate clonal DNA colonies or clusters. Four types of reversible terminator bases of four types are used to evaluate the sequence. Image of fluorescent labeled nucleotide is captured by camera.

4.3 SOLiD sequencing

Sequencing by ligation technique is being used in SOLiD sequencing. Mixture of oligonucleotides of known and fixed length are labeled according to the sequenced position. The oligonucleotides are first annealed and then ligated. DNA ligase will preferably ligate to matching sequences and will lead to signal corresponding to that nucleotide at that position. Emulsion PCR is used for amplification of DNA prior to sequencing. The resulting

(16)

16

DNA Sequencing

beads, each of which contain single copies of the similar DNA molecule, are being deposited on a glass slide.

This results in sequences of quantities and lengths which are comparable to Illumina sequencing.

(17)

17

DNA Sequencing

Genome Sequencer 20 (454) Genome analyzer Solexa

4.4 Ion Torrent semiconductor sequencing

Life Technologies have invented a system which is based on general technique of sequencing but they have used a novel system based on semiconductor detection. This sequencing method is based on the detection of hydrogen ions which are released during the polymerisation of DNA, not like optical methods which are used in most of sequencing systems.

4.5 DNA nanoball sequencing

DNA nanoball sequencing can determine the full genome sequence. This sequencing technique uses RCR to amplify small genomic DNA fragments to DNA nanoballs. Sequencing by ligation is then used to see the nucleotide sequence. Here lafge number of DNA nanoballs can be sequenced per run and reagent cost is also very economic as compared to NGS methods which are available. There is one drawback that only small DNA sequences can be determined from each DNA nanoball which further makes interpretation of results difficult.

This technology is more successfully used in genome sequencing projects.

(18)

18

DNA Sequencing

Table: Comparative analysis of different methods of NGS

4.7 Sanger Sequencing: Advantages and disadvantages

Advantages Disadvantages

(19)

19

DNA Sequencing

Error rate is very low

Can read long length (~750 bp) Can target a primer

Cost is high per base

Takes long time for generation of data Cloning is prerequisite

4.8 454 Sequencing: Advantages and Disadvantages

Error rate is low

It can read medium length (~400-600 bp)

Cost per base is relatively high Need to be run at large scale Startup costs are medium to high

(20)

20

DNA Sequencing

4.9 Ion Torrent Sequencing: Advantages and disadvantages

Startup cost is low

Scalable (10 – 1000 Mb of data per run) Cost per base is medium to low

Error rate is less

Runs very fast (<3 hours)

It is New still developing technology Cost is high in comparisonto Illumina Read lengths only ~100-200 bp

4.10 IIlumina Sequencing: Advantages and disadvantages

Error rate is low

Its cost is lowest per base Can generate tons of data

Must run at very large scale Short read length

(50-75 bp)

Multiple days for runs Startup cost is very high De Novo assembly difficult

4.11 PacBio Sequencing: Advantages and disadvantages

(21)

21

DNA Sequencing

Single molecules can be used as template It is potential for very long reads (several kb+)

Error rate is very high (~10-15%) Cost per base is high

Startup costs are high

4.12 NGS Platforms Overview

• Differ in design and chemistries

• Fundamentally related-sequencing of thousands to millions of clonally amplified molecules in a massively parallel manner

• Orders of magnitude more information-will continue to evolve

• Attractive for clinical applications – individual sequencing assays costly and laborious- serial “gene by gene” analysis

(22)

22

DNA Sequencing

5. Sequencing: What when and why

• Sanger:

Small projects (less than 1Mbp)

• 454:

De-novo sequencing, metagenomics

• Solexa, SOLiD, Heliscope:

– Gene expression, protein-DNA interactions – Resequencing

• Significant improvements were made in Sanger Method but it could not bring down the cost

• Among many other technologies developed at that time, the one which became first became commercial in 2005 was the 454 Life Sciences sequencer.

• Sequencing costs have come down tremendously

• Large amount of data can be generated per run

(23)

23

DNA Sequencing

Figure: Graph showing fall in cost of sequencing

6. Applications of Next Generation Sequencing

• Detection of mutation

• Detection of foreign DNA

• Non invasive diagnosis aneuplody

• Characterization of population

• Cancer genetics

• Analysis of expression

(24)

24

DNA Sequencing

• Transcription binding

• Chromosomal interaction

6. Applications of DNA Sequencing(In general)

Forensic: To know the identity of person killed/or any unknown person, or to identify criminals by leftover items on the crime scene

Medicine: Genes responsible for up-regulating or down-regulating due to some diseases can be identified by comparing healthy and diseased.

Agriculture: Development of transgenic plants for increased yield or virus resistant plant development.