Elements Not PCR-based For non-specific DNA expansion Bacteriophage Φ29 DNA polymerase Random exonuclease-resistant primers Advantages Amount of starting template Length of products Less bias and coverage Promising, robust, and reliable
Sufficient amount, length and coverage
α-Proteobacteria Less than products from whole cells
Library
Constructing Vector transformation Verifying functional screening sequence-based screening substrate induced gene expression screening (SIGEX) Other screening methods
!! Expression of the metagenomics library.
Sequencing DNA extraction clone library (plasmid) shotgun sequencing Assembly poisson distribution manual curation evaluation Sort into organisms Results contigs, scaffolds, unassembled singletons diversity some cannot be readily separated.
Environmental genomics
By: Shang, Lei School of Life Sciences 2010-4-22
Outline
1
Definition
2
Main techniques & examples ★
3
Applications
4ConcBiblioteka usionDefinition
DNA extraction
Sequencing (Shotgun)
assembly
【Whole Genome Amplificatio n (WGA)】
Metagenomic library construction
Verification
WGA
- Multiple displacement amplification (MDA)
Conclusion
A promising method
Future work
metagenomic library construction and expression single-cell genomics functional genes and pathways protocol improvement new bioactive compounds
Little difference
Quantity
DOP-PCR > PEP
DNA polymerase activity Increased annealing and extension time
Develo pment
Applications
Academic microbe resources – classification & diversity mechanism and evolution structure and function of microorganism community relationship between microbe and environment Practical active materials pollution control [water and soil] Preimplantation Genetic Diagnosis & Prenatal Diagnosis
DOP-PCR
Exponential amplification
No
Detect variance of different loci
Yes
The starting template has no effect on product
Merits & Pitfalls
Coverage of genome
WGA from single cells
MDA Primer extension preamplification (PEP) Degenerate oligonucleotide primed PCR (DOP-PCR) Tagged random primers (T-PCR) Linker-adaptor PCR
This is the first article on the applicability of MDA to metagenome application from scleractinian coral.
Aims:（1） evaluate the coverage and biases （2） amplify metagenomes
1991 Pace
“ environmental genomics” first bacteriophage library
1998 EGP
(Environmental Genome Project)
1998 Handelsman
“ metagenome” the genome of total microbiota found in nature
Broad sense Narrow sense
total biota
all bacteria and fungi
Techniques
- For uncultured microbes(★)
Ⅰ total DNA extraction Environmental samples Gene discovery [PCR\ORF] Function analysis
Ⅱ single-cell genomics
Problems & Solutions
(Hutchison and Venter, 2006)
Problems & Solutions
background synthesis remove sources of contaminating DNA template. reduce the reaction volume.
(Venter et al., 2004)
ORF
- Subtractive hybridization magnetic bead capture
Merits multi gene targets high specificity
Pitfalls requires library not always fulllength ORF
bias [“plones”]
chimeric clones in sequencing libraries enzymatic treatment before cloning into vector computational methods 454 technology
breakage of large chromosomal DNA several copies of chromosome
Efficiency/ADO Short products
Most complete (not all)
With each additional cycle Shorter PEP protocol Improved PEP PCR
Most complete (1/3)
500 bp in average
Anammox
Nitrogen cycle wastewater treatment
K.stuttgartiensis
> 200 genes metal-respiration Nirs NO acetyl-CoA pathway
(Strous et al., 2006)
Thanks!
Phylum Planctomycetes
deepest branching normal phylum related to the Chlamudiae
Phylogenetic analysis Orthologous groups
gene transfer superphylum
(Peng et al., 2006)
Comparison
PEP
Princip le
Sequence-independent Primer Temperature (T) Random mixture 92℃-37 ℃-55 ℃ Yes Partially degenerate Annealing T increase