ARJUN DESAI INNOVATIVE WORLDTECH PVT LTD

DNA SQUENCING R & D PROJECT

BY Dr. SHRIKANTH DESAI

ADIWPL  #703,2nd floor,3rd block, BEL layout, vidhyranyapura, Bengaluru.
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Contents

PREFACE................................................................................................................................................................................ 2
INTRODUCTION..................................................................................................................................................................... 3
NEXT GENERATION SEQUENCING.........................................................................................................................................4
The DNA Sequencer..............................................................................................................................................................9
Flow chart........................................................................................................................................................................... 11
Attachments: - PSD. Pdf file

Graphical pictures of design and fabrication of

a) quantum well chip

b) metallic coating on gap/ bridge of quantum well chip.
c) ) photo diode chip with ball grid array
d) LED Chip connected to ball grid array
e) Droplet generator and filter apertures.
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PREFACE

This refers to the biotechnological project discussion about the development and fabrication of an innovative
bio/nano chip for next generation whole genome sequencing.

Proposed below is a brief introduction of the DNA sequencing procedure with the help of nanochip and its
chip reader or Genome sequencer instrument Design and fabrication method.

The design model of the sequencer is also proposed for considering to be developed and manufactured

We are indebt to your kind endeavors and support towards the development and progress of our project
/product and accessories their bye.

Thank you,

Yours truly.

Dr. Shrikanth Desai

ADIWPL, Director
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INTRODUCTION

Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous
applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics.
Comparing healthy and mutated DNA sequences can diagnose different diseases including various
cancers, characterize antibody repertoire, and can be used to guide patient treatment. Having a quick way to
sequence DNA allows for faster and more individualized medical care to be administered, and for more
organisms to be identified and cataloged.

The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in
the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including
the human genome and other complete DNA sequences of many animal, plant, and microbial species.

Importance

DNA sequencing played a pivotal role in mapping out the human genome, completed in 2003, and is an
essential tool for many basic and applied research applications today. It has for example provided an
important tool for determining the thousands of nucleotide variations associated with specific genetic
diseases, like Huntington's, which may help to better understand these diseases and advance treatment.
DNA sequencing also underpins pharmacogenomics. This is a relatively new field which is leading the way to
more personalized medicine. Pharmacogenomics looks at how a person's individual genome variations affect
their response to a drug. Such data is being used to determine which drug gives the best outcome in particular
patients. Over 140 drugs approved by the FDA now include pharmacogenomic information in their labelling.
Such labelling is not only important in terms of matching patients to their most appropriate drug, but also for
working out what their drug dose should be and their level of risk in terms of adverse events. Individual
genetic profiling is already being used routinely to prescribe therapies for patients with HIV, breast cancer,
lymphoblastic leukemia and colon cancer and in the future will be used to tailor treatments for cardiovascular
disease, cancer, asthma, Alzheimer's disease and depression. Drug developers are also using
pharmacogenomic data to design drugs which can be targeted at subgroups of patients with specific genetic
profiles.
Sequence data analysis has become a very important aspect in the field of genomics. Bioinformatics has made
the task of analysis much easier for biologists, by providing different software solutions and saving all the
tedious manual work.
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NEXT GENERATION SEQUENCING

1. What is sequencing??

• Deciphering the code hidden in biological sequences like DNA, polypeptides etc.

• Method and technologies that enables us to determine the order of nucleotides and amino acids in DNA and
Polypeptide respectively.

Traditional methods of Sequencing and its limitations

• Maxam-Gilbert Method

 Use of radioactive labels.

 Sanger Method
 It utilizes the fluorescent dye for labeling.
 separation of extended fragments of DNA

with the addition of di-deoxynucleotides (lack a 3’-OH group) Thus, chain termination. Limitation Slow
High cost per run.

Automated Sanger method

1. Bacterial cloning or PCR template purification

2. labelling of DNA fragments using the chain termination method with energy transfer

3. dye-labelled di-de ox nucleotides and a DNA polymerase

4. capillary electrophoresis

5. fluorescence detection that provides four-color plots to reveal the DNA sequence.

NEXT GENERATION SEQUENCING

Also known as

 High throughput sequencing or

 ultra-deep sequencing or
 massively parallel sequencing.

What is next generation sequencing??

• Automated Sanger method (1st generation)

• Technologies developed after that are known as next generation sequencing.

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• NGS enables the sequencing of biological codes at a very rapid pace with low cost per operation.

• This is the primary advantage over conventional methods.

• For example, Billions of short reads can be sequenced in one operation.

Major Platforms for NGS

•454 (By Roche)

•Solid (By Applied Biosystems)

•Solexa (By Illumina)

Above mentioned platform varies in strategies, application and type of data generated.

• However, all technologies are common in

 That they generate sequences on an unprecedented scale

 DNA cloning is not required
 and very low operation cost.

What NGS Consists of Next generation technologies for sequencing is combination of strategies for

• template preparation

• sequencing and imaging

• genome alignment

• assembly methods

Template preparation as even most sensitive imaging technique is not able to detect single molecule,
amplification of templates is inevitable.

• Clonally amplified templates

 By emulsion PCR (emPCR) e.g. 454 and Solid

 Solid phase amplification e.g. illumina

• Single-molecule templates

Template preparation: Traditional vs. NGS

• Immobilization of templates fragments over bead /glass plate allows billions of the sequencing reaction run
simultaneously.

sequencing and imaging

• Sequencing
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o cyclic reversible termination (CRT) e.g. illumina/solexa

o single-nucleotide addition (SNA) e.g. 454/roche
o real-time sequencing: R&D going on (pacific Bioscience)
o Sequencing by ligation (SBL) e.g. SOLiD

• Imaging

 measuring bioluminescent signals

 four-colour imaging of single molecular events e.g. illumina/solexa.

Genome alignment and assembly After NGS reads have been generated, they are aligned to either

• a known reference sequence or

• assembled de novo

454 (Pyrosequencing)

• DNA is fragmented, joined to adapters at either end of the fragmented DNA

• amplified in an emulsion PCR (includes agarose bead with complimentary adaptors to fragmented DNA)

• PCR amplified allowing up to 1 million identical fragments around one bead and finally dropped into a
PicoTitreTube (PTT)

PCR amplification Pico Titre Tube

• Adapter containing the universal priming site are ligated to target ends

• Same primer can be used for amplification

• In Pico titre tube reaction of fluorescence occurs with the addition of nucleotides Nucleotide addition

Nucleotide addition Output

SOLiD (support oligonucleotide ligation detection)

• Sequencing by Oligo/Ligation and Detection.

• Steps

▫ Library Preparation

 two types of libraries sequencing-fragment or mate-paired are prepared.

▫ Emulsion PCR/Bead Enrichment

 amplification of template fragments is done in same manner as 454.

▫ Bead Deposition Deposit 3’ modified beads onto a glass slide.

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Sequencing by Ligation

• Primers hybridize to the P1 adapter sequence on the templated beads

• The method uses two-base- encoded probes (4 probes), which has the primary advantage of improved
accuracy.

• Multiple cycles of ligation, detection and cleavage are performed.

• Extension product is removed and the template is reset with a primer complementary to the n-1 position for
a second round of ligation cycles.

Illumina

• Breaking up DNA

• Adding adaptors, but in this case attach not to a bead but to a slide

• Fold-back PCR is then used to amplify the fragmented DNA into a cluster

Sequential addition of nucleotides is added using a polymerase

NGS and Bioinformatics

• Alignment of sequence reads to a reference BLAST doesn’t blast here Short read aligners side-lines BLAST
Software

• Bowtie

• MAQ

• BWA

Above strategy works if reference genome exists.

• de novo assembly from paired or unpaired reads

• base-calling and/or polymorphism detection

• structural variant detection

• genome browsing.

Application of NGS

• Variants discovery in targeted region or whole genome by re-sequencing

• Reassembling genome of lower organism by de novo method.

• Cost-effective sequencing of complex samples at remarkable scale and speed.

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• Sequencing entire transcriptome.

• In Meta genomics: Sequencing genome of entire biological communities

• Replacing Chip-on-chip with Chip-seq in case of multicellular eukaryotes.

• Personalized genome for personalized medicine

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The DNA Sequencer

A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample

of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A
(adenine) and T (thymine). This is then reported as a text string, called a read. Some DNA sequencers can be
also considered optical instruments as they analyze light signals originating from fluorochromes attached
to nucleotides.

The Human Genome Project catalyzed the development of cheaper, high throughput and more accurate
platforms known as Next Generation Sequencers (NGS) to sequence the human genome. These include the
454, Solid and Illumina DNA sequencing platforms. Next generation sequencing machines have increased the
rate of DNA sequence substantially compared with previous Sanger methods. DNA samples can be prepared
automatically in as little as 90 mins,[5] while a human genome can be sequenced at 15 times coverage in a
matter of days.

Because of limitations in DNA sequencer technology these reads are short compared to the length of
a genome therefore the reads must be assembled into longer contigs.[7] The data may also contain errors,
caused by limitations in the DNA sequencing technique or by errors during PCR amplification. DNA sequencer
manufacturers use a number of different methods to detect which DNA bases are present. The specific
protocols applied in different sequencing platforms have an impact in the final data that is generated.
Therefore, comparing data quality and cost across different technologies can be a daunting task. Each
manufacturer provides their own ways to inform sequencing errors and scores. However, errors and scores
between different platforms cannot always be compared directly. Since these systems rely on different DNA
sequencing approaches, choosing the best DNA sequencer and method will typically depend on the
experiment objectives and available budget.
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Flow chart
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DESIGN OF BIO CHIP

1 sq mm = 1 million micrometers

Q-Well is 3 micrometer each

Gap/ bridge is 3 micrometer each

Both together = 6 micrometer

1 sq mm = 1000000/6 = 27,775 Q-Wells

1sq inch= 625 sq mm = 27775 x 625 = 17, 3, 59,375 (17 millions)

1 chip = 3x3= 9 sq inch =17359375 x 9 = 156, 2, 34,375 (156 millions)

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Design and fabrication of ‘Quantum-well’ Chip parameters

The Black holes indicate the 3 um Q-wells

While

The Yellow grid indicate the fabricated metallic coating

Useful for electro-wetting based displacement of single droplet to be deposited in the q-well
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Droplet generator Design

Tube or syringe with 3 um aperture for droplet dispensing technology.

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Quantum well chip fixed to Ball grid photo-diode chip

And
Ball grid photo-diode chip is connected to LED chip.
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