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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
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
8 10 12 14 16 18 20
Length of K-mer Reads (bp)
% o
f P
aire
d K
-mer
s w
ith
Un
iqu
ely
Ass
ign
able
Lo
cati
on
E.COLI
HUMAN
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?
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