Greg Phillips Veterinary Microbiology gregory@iastate.edu

Greg PhillipsVeterinary Microbiologygregory@iastate.edu

Next Generation DNA Sequencing

Review of DNA sequencing technology

3 Next Gen platforms-technology (and advertising)

New applications of Next Gen technology

Next Next Gen technology• $1,000 genome

The First Generation-Sanger DNA Sequencing

Sanger DNA Sequencing by Capillary Electrophoresis & Fluorescent Detection

Long read length >1000 bp)

Traditional Applications of DNA Sequencing

Gene sequencing• New gene discovery• Mutations (natural variants & synthetic)

Genome sequencing• New genomes (microbial to human)• Comparative genomics

Metagenomics • Environmental samples of mixed populations

Shotgun Sequencing Approach

Next-Generation DNA Sequencing:3 Platforms

454 (Roche)• Pyrosequencing• Medium read length (~500 bp)

Illumina (Solexa)• Sequencing by synthesis• Short read sequencing (up to 100 bp)

SOLiD (Applied Biosystems)• Sequencing by ligations• Short read sequencing (~35 bp)

Next-Generation DNA SequencingAdvantages/Disadvantages

Depth of coverage of genome sequencing• Sanger: 4-5x • 454: >20x• Illumina, SOLiD: up to 100x

Library preparation less laborious• Methods unique to each platform

Lower cost

VS.Shorter read lengths

• De novo sequence assembly difficultLower accuracy in homopolymer tractsLarge amount of data generated

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

454 Sequencing TechnologyPyrosequencing

Library Preparation

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

454 Sequencing TechnologyAmplification

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

454 Sequencing TechnologySequencing Reaction

454 Sequencing TechnologyDetection

Roche/454 DNA SequencingPyrosequencing

Illumina Sequencing TechnologySequencing by Synthesis

Library Preparation

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

Illumina Sequencing TechnologyBridge Amplification

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

Illumina Sequencing TechnologyDetection

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

Illumina Sequencing Technology

ABI/SOLiD DNA Sequencing TechnologySequencing by ligation

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

ABI/SOLiD DNA Sequencing TechnologySequencing by ligation

Mardis. 2008. Annu. Rev. Genomics Human Genet. 9:387.

ABI/SOLiD DNA Sequencing Technology

Characteristics of New DNA Sequencing Technology

Rothberg &Leamon. 2008. Nat. Biotechnol. 26:1117

New DNA Sequencing Technology is Well-Suited for Microbial Genomes

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Next-Generation DNA Sequencing-Applications

Genome sequencing• Short read de novo assembly requires new

bioinformatic tools• Genome resequencing of mutants

Metagenomics • …the application of modern genomics techniques to

the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species -Chen, K; Pachter L (2005). . PLoS Comp Biol 1: 24.

Next Gen Sequencing & Metagenomics

• Microbial communities in a natural environment

Seawater Soil

• Host-associated microbial communities

• Human microbiome• Gut• Oral cavity

• Microbial communities in a “managed-environment”

Sewage treatment plant Aquaculture facility

Next-Generation DNA Sequencing-More Applications

RNA-seq• Transcriptional profiling• Consensus sequencing

ChIP sequencing• Chromatin immunoprecipitation• Identification of protein binding sites

Small RNA-seqEpigenetics

Marguerat, et al. 2008. Biochem. Soc. Trans. 36: 1091–1096.

Next-Generation DNA SequencingRNA-seq

Next-Generation DNA SequencingChIP-seq

Chromatin immunoprecipitation

Next-Generation DNA SequencingSmall RNA-seq

Animals/Plants/Bacteria encode small, non-coding RNAs that function in:

Gene regulationAntiviral defense mechanisms

DevelopmentUnknown functions

Next-Generation DNA SequencingEpigenetics

The Next-Next Generation-Helicos Single Molecule DNA Sequencing

The Next-Next Generation-Helicos Single Molecule DNA Sequencing

Single Molecule DNA Sequencing-Pacific Biosciences

http://www.pacificbiosciences.com/video_lg.html

Eid et al. 2009 Science

Nanopore DNA Sequencing

http://www.nanoporetech.com/sequences/index/34

What do YOU think?