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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
och
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).