BACS/Fosmid Libraries Whole Genome Shotgun Sequencing

35 Pages·2005·1.67 MB·English

Metagenomics Tools:
BACS/Fosmid Libraries
Whole Genome Shotgun
Sequencing
Amy Apprill
OCN 750: Molecular Methods in Biological Oceanography
November 17, 2005
Why Metagenomics?
- Limited physiology and functional role information
known about microbes from cultures
-Phylotypes of noncultured microbes derived from
rRNA genes only provide phylogenetic info, no
information about physiology, biochemistry, or
ecological function; subject to PCR-based biases
- Metagenomics allows isolation of large portions
of genomes which provide access to genes for
protein-coding for biochemical pathways
→ insight into specific physiological and
ecological functions, metabolic
variability of an environment
History of Marine Metagenomics
1991: Lambda phage used as a vector to create 10-20 kb insert shotgun library of
picoplankton rRNA gene sequences, but also revealed other genes of interest
(Schmidt TM, DeLong EF, Pace NR, 1991)
1992: Introduction of BAC & Fosmid cloning vectors from E. coli improved cloning
efforts by controlling copy numbers
- BAC vectors replicate >300kb & display few chimeras (Shizuya et al. 1992)
1996: First environmental fosmid library with environmental samples from Oregon
coast (Stein et al. 1996)
2000: First BAC library from marine environment (Beja et al. 2000);
proteorhodopsin discovered from Monterey Bay BAC (Beja et al. 2000)
: (
2002 AAnP diversity uncovered from Monterey Bay BAC Beja et al. 2002)
2005: Whole genome shotgun sequencing approach used on first marine
environmental samples from the Sargasso Sea (Venter et al. 2005)
BAC: bacterial artificial chromosome
A modified plasmid that contains an origin of replication derived from
the E. coli F factor frequently used for large insert cloning
experiments; exists within the cell very much like a cellular
chromosome.
Specifics:
- 100- 300kb (even 600kp!) inserts; 1 insert
~10-15% bacterial genome
- Requires large amounts DNA (800-2000
L seawater)
- Useful for screening specific protein-
coding genes and genes of uncultivated
microbes
- Used to discover proteorhodopsin in
several phylotypes, genes for anoxygenic
photosynthesis
Marine
bacteria
BAC/ fosmid
construction
- general
DeLong, 2005
How to create a BAC from seawater:
1. Collect ~1000 L seawater
2. Pre-filter, use TFF to pellet cells
3. Agarose embed cell pellet
4. Lyse agarose embedded cells
5. Prepare large DNA fragments by
HindIII digestion of agarose slices
- Run PFGE
- Excise 150-400 kbp
regions
- Extract gel-embedded DNA
(Beja et al 2000, Fig. 1)
How to create a BAC, cont.
6. Ligate DNA into
vector (previously
removed from cells)
7. Transform
vector into cells
using
electrophoration
8. Screen for
phylogenetic info,
http://www.ptf.okstate.edu/pulser.html
purify & sequence
Pulse Field Gel Electrophoresis of BAC clones
digested with NotI describes size of inserts
plasmid
(Beja et al 2000, Fig. 2A)
BAC Screening: rRNA Gene Surveys using
Multiplex PCR
- Digest BAC/fosmid DNA to remove E. coli chromosome
- Screen fragments for rRNA gene from clones using 3 bacterial
primer sets (SSU & LSU) and Archaea-specific
- Excise amplicons form gel, purify
- Clone & sequence purified products
Phylogenetic-informative multiplex PCR products describes
phylogenetic groupings
(Beja et al 2000, Fig. 5)
BAC Screening: ITS-LH-PCR
Uses natural length variations in ITS, and location of tRNA-alanine
gene within the ITS, to ID unique gene fragments corresponding to
phylogenetic groupings
1. Pool plasmid-safe treated
DNA and PCR with
fluorescent labeled SSU &
LSU primers to amp ITS &
tRNA genes
2. Capillary electrophoresis
compares size stds to
fragment lengths
3. Sequence unknown
fragments w/ ITS primers
Figure 4. Suzuki et al. 2004
and 16S primers