Sequencing technologies

past, current and next generation

•Introduction to Helicos, Illumina and Solid sequencing technology

•ApplicationsRobert Pinard

Some slides were adapted from Karen Staehling-Hampton

The Stowers Genome Center

Introduction

• Genomics research has entered a new age, in which deciphering the genome’s effect on biology and medicine requires not only the detection of mutations and sequence variation, but also understanding the dynamic nature of genome biology.

• The “Next Generation Sequencing” should be called the Current Generation Sequencing

Overview of three major next Generation Sequencing Technologies

• Illumina / GAII

• Helicos / Heliscope

• Life Tech / Solid

• (briefly Ion Torrent, PacBio

& Complete Genomics)

• The principle at the heart of all these technologies is similar

• (sequence by synthesis)

Sanger Sequencing

(Except Solid)

Detect stopped fluorescent fragments (using ddNTP spikes)

• It is similar with the next generation sequencers where the different platforms either detect the incorporation of a fluorescent nucleotide or a bi-product of the reaction like the PPi or the release of a proton by the DNA polymerase

Common Steps (to all platforms)

• Library Preparation

• Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Common Steps

• Library Preparation

• Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Shear

Modify ends/Adaptors Selection

AmplificationAttach

Sequence & Detection

Overall Steps

Library Preparation

Library Preparation Workflow (Illumina & Solid)

Helicos Library Preparation

Common Steps

• Library Preparation

• Selection/Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Shear

Modify ends/Adaptors Selection

AmplificationAttach

Sequence & Detection

Overall Steps

Library Preparation

PONDBAITs

ENRICHED POND

Selection Step (Principles)

Step A Region Selection: using Sure Select, capture region that we really want to look at (complete exomes), subset of genes

(Familial Cardiac Genes).

Step B some PCR involved

• Pre-Hybrid Selection and post-Hybrid Selection amplification PCR.

A- Pre-(to increase the pond of fragment DNA)

B- Post- (to increase the DNA that was captured)

Shear

Modify ends/Adaptors Selection

AmplificationAmplify and or Attach

Sequence & Detection

Overall Steps

Library Preparation

• EMULSION PCR orCLUSTER AMPLIFICATION

STEP C: Clonal amplification to increase signal detection

454/RocheSolid/LifeHelicos

Illumina

Amplification: where the technologies differ?

Amplified Materials deposited in picotiter plate or on slide via 3’ modification of the 3’end

SOLID; 454; ION TORRENT

Emulsion PCR

ILLUMINA

Amplification on Slide and Cluster Generation

ILLUMINA

Illumina

Common Steps

• Library Preparation

• Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Shear

Modify ends/Adaptors Selection

AmplificationAmplify and or Attach

Sequence & Detection

Overall Steps

Library Preparation

ILLUMINA

Helicos

454 & Solid

Amplified Materials deposited in picotiterplate or on slide via 3’ modification of the 3’end

45

4/R

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e

Solid

Common Steps

• Library Preparation

• Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Shear

Modify ends/Adaptors Selection

AmplificationAmplify and or Attach

Sequence & Detection

Overall Steps

Library Preparation

Illumina

Illumina

Helicos

Helicos

454The addition of one of the four deoxynucleotide triphosphates (dNTPs)(in the case of dATP we add dATPαS which is not a substrate for a luciferase) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.

Solid

Common Steps

• Library Preparation

• Amplification Steps

• Attachment to a matrix (FlowCell)

• Sequencing & Detection

• Interpretation

Alignment et Sequence Reconstruction

All fragments put together and align to region of interest

Utilities• •Whole Genome re-sequencing

• –Bacterial genomes to identify SNPs that confer drug resistance

• •Targeted Re-sequencing (Cardio Gen Scan)

• –Sure Select

• –Regular PCR & Long Range PCR (small panels/ Patch Assay)

• •Coding exons (Whole Exome and Clinical Exomes)

• •Detect Rare variants

• –Can detect a 1/20 event (1 het among 10 samples)

• –Somatic mutations in cancer samples

Other emergent platforms

• Ion Torrent

• PacBio

• Complete Genomics

Ion Torrent

Sequence multiple time same fragment

Pacific BioSciences

NanoBall (Complete Genomics)

Conclusions

• Several new platforms are emerging (variation on a same theme) that will increase throughput and reduce cost.

• The Next Gen Sequencing approaches are really the Now Gen Sequencing approaches and they are making a real impact in life sciences and soon in clinical diagnostics (starting with our own CGS test).