Post on 06-Jan-2016
description
DNA Sequencing Technology and its Applications in Evolution Research
Julie Urban, Ph.D.Assistant Director, Genomics & Microbiology Laboratory
NC Museum of Natural Sciences
Planthopper Evolution
Planthopper Evolution1953
15 Feb, 2001
Planthopper Evolution(2001)
Planthopper Evolution(2001) (2008)
XX
Today
Ion Proton ($149,000)human genome for < $5,000, ~1 day (?)
MiSeq ($126,000)
Ion Torrent ($90,000)
Planthopper Evolution
Planthopper Evolution
Planthopper Evolution
Capillary sequencing
Sequencing by synthesis
Single molecule methods
Planthopper EvolutionCapillary (Sanger) Sequencing
Capillary (Sanger) Sequencing
Fluorescence Important in (most) Next Gen Sequencing
Planthopper EvolutionSteps Common to all Next Generation Sequencing Systems
1. Make Library:-- fragment DNA into unique fragments-- add custom adaptors (“linkers”)
2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
All sequence DNA in “massive parallel” fashion
Planthopper EvolutionRoche/454 Sequencing
1. Make Library:-- fragment DNA into unique fragments-- add custom adaptors (“linkers”)
Planthopper EvolutionRoche/454 Sequencing
2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Planthopper EvolutionRoche/454 Sequencing
2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Planthopper EvolutionRoche/454 Sequencing
2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Planthopper EvolutionRoche/454 Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionIllumina Sequencing
1. Make Library:-- fragment DNA into unique fragments-- add custom adaptors (“linkers”)
Planthopper EvolutionIllumina Sequencing
1. Make Library:-- fragment DNA into unique fragments-- add custom adaptors (“linkers”)
Planthopper Evolution2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Illumina Sequencing
Planthopper Evolution3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Illumina Sequencing
Planthopper Evolution3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Illumina Sequencing
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
1. Make Library:-- fragment DNA into unique fragments-- add custom adaptors (“linkers”)
Planthopper Evolution2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Life Tech/ABI Ion Torrent & Proton Sequencing
Planthopper Evolution2. Amplify Library-- make many identical copies (“clones”) of each unique fragment-- copies are made on a solid surface
Life Tech/ABI Ion Torrent & Proton Sequencing
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionLife Tech/ABI Ion Torrent & Proton Sequencing
3. Sequencing by Synthesis-- fragments are copied again (sequencing reaction)-- incorporation of each base “fires” a signal-- system detects signal coming from all clones “firing” at once for that unique fragment
Planthopper EvolutionSingle Molecule Systems: Pacific Biosciences
Planthopper EvolutionSingle Molecule Systems: Oxford Nanopore
Capillary sequencing
Sequencing by synthesis
Single molecule methods
Planthopper EvolutionApplications of Next Generation Sequencing
Metagenomics
Planthopper EvolutionApplications of Next Generation Sequencing
Microbiome
http://commonfund.nih.gov/hmp/
Human Microbiome Project
Planthopper EvolutionApplications of Next Generation Sequencing
Microbiome
Planthopper EvolutionApplications of Next Generation Sequencing
Bacterial Genome Sequencing
Bacteriome = insect organ made of bacteriocytes
Planthoppers
Planthopper EvolutionApplications of Next Generation Sequencing
Ancient (=degraded) DNA Sequencing
Planthopper Evolution
Planthopper Evolution
Annual Review of Genomics and Human Genetics, 2008. 9: 387-402.
Youtube: Current Topics in Genome Analysis 2012; NHGRI Lecture Series.
-- Elaine Mardis-- Jonathan Eisen-- Julie Segre (Microbiome)
Collaborators and Funding
Megan EhlersNCSU undergrad
Greg PahelNCMNS
Rob DunnNCSU
Holly MenningerNCSU
Dan FergusNCMNS
Megan ThoemmesNCSU undergrad
Jason CryanNCMNS
Julie HorvatnNCMNS