Post on 04-Jan-2016
Molecular Biology
Dr. Chaim WachtelMay 28, 2015
Topics
• Microarray• Deep Sequencing• PCR
– Real-Time– Digital– NanostringGENE EXPRESSION
Enzymes: Reverse Transcriptase, DNA polymerase, RNA polymerase
Microarray in theory
Microarray in practice
Common Applications
•Gene expression profiling•Chromatin
immunoprecipitation on Chip (ChIP)
•SNP detection•Alternative splicing detection
Microarray
• Affymetrix• Agilent
– Same idea different implementation– Pros and cons to each method
The ChipsAgilent Affymetrix
Printing of Affymetrix Chips
Printing of Agilent Chips
Printing of Agilent Chips
Probes are 60 nucleotides in length vs 25 for Affymetrix
Affymetrix workflow
Agilent workflow
• Feature Extraction• Bioinformatics
Analysis“home-made”=low-density
Agilent array
Pros and Cons
• Cost• Dual color vs single• Specificity
Deep Sequencing
Next Generation Sequencing (NGS)
High Throughput Sequencing (HTS)
Timeline and Comparison of Commercial HTS Instruments
High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder
Overview of Selected HTS Applications
High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder
In 1958, he was awarded a Nobel Prize in chemistry "for his work on the structure of proteins, especially that of insulin". In 1980, he shared with Walter Gilbert half of the chemistry prize "for their contributions concerning the determination of base sequences in nucleic acids". The other half was awarded to Paul Berg "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA"
Who is this? And what is he holding???
Sanger Sequencing
Sanger program
Capillary Sequencing
Machines have 1-96 capillaries
Multiple Platforms
• Illumina• 454• Solid• Nanopore/Oxford• Ion Torrent• Pacific Biosciences
Different companies different methods
Workflow for Sequencing
Workflow for Sequencing
• Depends on project- RNA or DNA• Protocols are ~90% the same for all systems
Illumina
Workflow for Sequencing
• Bridge PCR• Emulsion PCR
Sequencing with Illumina
Ilumina Machines
Ilumina Machines
Sequencing with Solid
The Ion Torrent
Scalability
The Chip
PacBio and Nanopore
High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder
Nanopore Technology
Movie time
• https://vimeo.com/127689053?from=outro-embed
• https://nanoporetech.com/products-services/minion1
How does it work?
What’s the big deal??
• GridION, consists of an array of sequencers, each containing 2,000 nanopores.
• That is expected to increase to 8,000 nanopores in the next-generation device. Twenty of these, running simultaneously, could sequence a full human genome in 15 minutes, the company said, describing both devices as "plug-and-play."
• MinION, with 512 nanopores• will be capable of sequencing 150,000 DNA bases per hour• the 48,490-bp genome of lambda DNA was sequenced as a
complete fragment—in one read. This read length is three orders of magnitude larger than the typical length of DNA fragments that are read in current sequencing approaches using fluorescent nucleotide labeling3.
• $$$$- MinION will cost $900
Pacific Biosystems
• https://www.youtube.com/watch?v=NHCJ8PtYCFc&list=UU2y78sjVOumGc2da1tN629g