SOLEXA aka: Sequencing by Synthesis

Slides:

Advertisements

Similar presentations

Lecture 1 Problem: From an E. coli cell extract, you assay enzyme activity for beta-galactosidase. You divide the extract into two samples, one of which.

Advertisements

RNA-Seq An alternative to microarray. Steps Grow cells or isolate tissue (brain, liver, muscle) Isolate total RNA Isolate mRNA from total RNA (poly.

What Is Genomics? Genomics is the study of how the entire genome of a species functions as a unit and evolves over time. It is the study of life’s blueprint,

Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome Jay Shendure, Gregory J. Porreca, Nikos B. Reppas, Xiaoxia Lin, John P. McCutcheon.

Proprietary Bead Deposition Load DNA & Enzyme beads into PicoTiter™ Plate.

RNA-Seq An alternative to microarray. Steps Grow cells or isolate tissue (brain, liver, muscle) Isolate total RNA Isolate mRNA from total RNA (poly.

Chapter 10 DNA Sequencing.

Emily Buckhouse. Nitrogenous Bases Nucleosides  Base linked to a 2-deoxy-D-ribose at 1’ carbon Nucleotides Nucleosides with a phosphate at 5’ carbon.

7.1 cont’d: Sanger Sequencing SBI4UP MRS. FRANKLIN.

CS 6293 Advanced Topics: Current Bioinformatics

NEXT GENERATION SEQUENCING Technologies on Biomedical Research

Update on Next-Generation Sequencing

The impact of next-generation sequencing technology of genetics Elaine R. Mardis – 11 February Washington School of Medicine, Genome Sequencing Center.

DNA Sequencing Today, laboratories routinely sequence the order of nucleotides in DNA. DNA sequencing is done to: Confirm the identity of genes isolated.

MCB 7200: Molecular Biology

1.) DNA Extraction Follow Kit Grind sample Mix with solution and spin Bind, Wash, Elute.

SOLEXA aka: Sequencing by Synthesis 2) Attach each adaptor to a flow cell to create ssDNA templates. After each round of amplification, the strands are.

High Throughput Sequencing Methods and Concepts

NEXT – GEN SEQUENCING TECHNIQUES

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Chapter 3 Fundamentals of Mapping and Sequencing Basic principles.

Pyromania USAFSAM “EPI Lab” MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11 1 Distribution Statement A: Approved for Public release;

High Throughput Sequencing Methods and Concepts Cedric Notredame adapted from S.M Brown.

CHAPTER 7 DNA SEQUENCING - INTRODUCTION - SANGER DIDEOXY METHOD - AUTOMATED SEQUENCING - NEXT GENERATION OF SEQUENCING METHODS MISS NUR SHALENA SOFIAN.

Stratton Nature 45: 719, 2009 Evolution of DNA sequencing technologies to present day DNA SEQUENCING & ASSEMBLY.

Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX

6.3 Advanced Molecular Biological Techniques 1. Polymerase chain reaction (PCR) 2. Restriction fragment length polymorphism (RFLP) 3. DNA sequencing.

Sequencing tutorial Peter HANTZ EMBL Heidelberg.

Sanger or Dideoxy DNA Sequencing

Biotechnology and Genetic Engineering PBIO 450/550 Characterization of DNA clones including: Restriction Enzyme (RE) mapping Subcloning Southerns Northerns*

Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels.

Semiconservative DNA replication Each strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized.

핵산 염기서열 분석(DNA SEQUENCING)

Introduction to Illumina Sequencing

DNA Sequencing First generation techniques

Next-generation sequencing technology

6.3 – Manipulating genomes

DNA Sequencing Second generation techniques

Next generation sequencing

Next Generation Sequencing

DNA Sequencing.

Sequencing Introduction

DNA Sequencing -sayed Mohammad Amin Nourion -A’Kia Buford

SNPs in forensic genetics: a review on SNP typing methodologies

Chapter 7 Recombinant DNA Technology and Genomics

Next-generation sequencing technology

DNA Sequencing Techniques

NGS technologies.

DNA Sequencing.

Sequencing Technologies

AMPLIFYING AND ANALYZING DNA.

B3- Olympic High School Bioinformatics

DNA Sequence Determination (Sanger)

Polymerase Chain Reaction (PCR)

CISC 667 Intro to Bioinformatics (Spring 2007) Molecular Biology Tools

DNA Sequencing The DNA from the genome is chopped into bits- whole chromosomes are too large to deal with, so the DNA is broken into manageably-sized overlapping.

DNA Sequencing A population of DNA fragments is generated.

Introduction to Bioinformatics II

A B - deoxynucleotide (dNTP) dideoxynucleotide (ddNTP)

ULTRASEQUENCING. Next Generation Sequencing: methods and applications.

The impact of next-generation sequencing technology on genetics

Sequencing techniques

Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine

Molecular Biology lecture -Putnoky

Sequencing DNA – the Sanger Method – the most-popular way (one of many methods) A primer binds to one of the DNA strands at a specific location (such as.

High-Throughput Sequencing Technologies

High-Throughput Sequencing Technologies

Comparison of GenFlex Tag Array and Pyrosequencing in SNP Genotyping

Standard (Sanger) sequencing

SBI4U0 Biotechnology.

xTAG liquid-phase microarray.

Presentation transcript:

SOLEXA aka: Sequencing by Synthesis 1) Randomly cut DNA fragments and ligate adaptors to both ends http://www.big.ac.cn/news/images/news070430.jpg 2) Attach each adaptor to a flow cell to create ssDNA templates. After each round of amplification, the strands are denatured to create more templates.

Add four labeled reversible terminators, primers, and DNA polymerase. Wash off all of the unbound components, excite the laser, and read the emitted fluorescence of the first base for each cluster. The blocked 3’ terminus and fluorophore has to be removed before repeating to determine the next base http://www.channelwolf.com/lvv/sem6/index_files/image9651.gif

454 Sequencing Capable of sequencing 400-600 megabases of DNA in a 10 hour run Applications: Whole genome sequencing Amplicon sequencing Transcriptome sequencing Metagenomics Advantages: low cost, longer reads and higher accuracy than the Sanger chain-termination method

454 Sequencing Concept Large-scale parallel pyrosequencing, “sequencing by synthesis”: Each nucleotide is added in turn, producing PPi Sulfurylase uses PPi to produce ATP, that is used by luciferase to produce light Apyrase denatures the remaining dNTP at each step The light produced with the addition of each nucleotide can be plotted to form a sequence Each incorporation releases PPi Sulfurylase uses PPi to produce ATP, which luciferase can use to generate light The detection of light by a camera when a new nucleotide is added indicates which nucleotide was next in the sequence Since ATP is used by luciferase as part of the detection mechanism, dATP-alphaS is used for sequencing (deoxyadenosine alpha-thio triphosphate) that cannot be used by luciferase but still can be incorporated into a sequence like ATP image: http://www.biotagebio.com/DynPage.aspx?id=7454 additional info: (pdf format) http://www.biotagebio.com/graphics/3019.pdf

454 Sequencing Technology DNA is fractionated, 5’ biotin labeled and attached to streptavidin coated beads. The non-labeled strand is released. The DNA on each bead is amplified onto the bead One bead is deposited in each well Pyrosequencing takes place in the Genome Sequencer FLX instrument

Applied Biosystems SOLiD System © Copyright 2008 Applied Biosystems. All Rights Reserved.

© Copyright 2008 Applied Biosystems. All Rights Reserved.