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