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

1953

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

Capillary sequencing Sequencing by synthesis Single molecule methods

Planthopper Evolution Capillary (Sanger) Sequencing

Fluorescence Important in (most) Next Gen Sequencing

Planthopper Evolution Steps 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 Evolution Roche/454 Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

Planthopper Evolution Roche/454 Sequencing 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface

Planthopper Evolution Roche/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 Evolution Illumina Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

Planthopper Evolution Illumina Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

Planthopper Evolution 2. Amplify Library -- make many identical copies (“clones”) of each unique fragment -- copies are made on a solid surface Illumina Sequencing

Planthopper Evolution 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 Illumina Sequencing

Planthopper Evolution 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 Illumina Sequencing

Planthopper Evolution Life Tech/ABI Ion Torrent & Proton Sequencing 1. Make Library: -- fragment DNA into unique fragments -- add custom adaptors (“linkers”)

Planthopper Evolution 2. 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 Evolution 2. 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 Evolution Life 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 Evolution Life 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 Evolution Life 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 Evolution Life 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 Evolution Life 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 Evolution Single Molecule Systems: Pacific Biosciences

Planthopper Evolution Single Molecule Systems: Oxford Nanopore

Capillary sequencing Sequencing by synthesis Single molecule methods

Planthopper Evolution Applications of Next Generation Sequencing Metagenomics

Planthopper Evolution Applications of Next Generation Sequencing Microbiome Human Microbiome Project

Planthopper Evolution Applications of Next Generation Sequencing Microbiome

Planthopper Evolution Applications of Next Generation Sequencing Bacterial Genome Sequencing Bacteriome = insect organ made of bacteriocytes Planthoppers

Planthopper Evolution Applications of Next Generation Sequencing Ancient (=degraded) DNA Sequencing

Planthopper Evolution

Annual Review of Genomics and Human Genetics, : Youtube: Current Topics in Genome Analysis 2012; NHGRI Lecture Series. -- Elaine Mardis -- Jonathan Eisen -- Julie Segre (Microbiome)

Collaborators and Funding Megan Ehlers NCSU undergrad Greg Pahel NCMNS Rob Dunn NCSU Holly Menninger NCSU Dan Fergus NCMNS Megan Thoemmes NCSU undergrad Jason Cryan NCMNS Julie Horvatn NCMNS