Download 슬라이드 1 - Extraordinary Everyday!

Introduction
Fish
-Nearly half of all vertebrates species
- 15700: marine, 13700 freshwater species (Fish base)
FISH-BOL(Fish Barcode of Life Initiative)
-Establish a reference library of DNA barcodes for all fish species
- fast, accurate, cost-effective system for molecular identification
•Facilitating species identification
•Flagging potentially previously unrecognized species
•Enabling identifications where traditional methods are not applicable
(immature stages or body fragments)
•Provide powerful tool for enhanced understanding
of the natural history and ecological interactions
High-quality sequence records
-10 specimens per species: more than 0.5 million specimen
Ward(2005): proof-of-principle study
- 200 species of Australian marine fishes
- 5000 barcodes from over 2000 species
-Effort
- Efficient polymerase chain reaction of barcode region
- Deliver high quality sequence data
- Ward(2005)
-Use 2 forward and 2 reverse primers (4 combinations)
-=> complex
2 solution
1. A single primer set with degenerate sites (COI-1)
2. A Cocktail whose component primers are tailed with M13 (COI-2, COI-3)
to facilitate high throughput sequencing
=> Test their effectiveness on a single species(94 different fish families)
Materials and methods
primer design
1. 159 fish mitogenome
2. align: Bioedit
3. anlayzing: CODEHOP
4. M13 Tagging
DNA quality test
Sequencing primer
mixture
Materials and methods
PCR amplification and sequencing
1. 94 SPECIES, 2 negative controls
( Myxini, Cephalaspidomorphi, Holocephali, Elasmobranchii, Actinopterygii)
2. 16S rRNA gene: positive controls for DNA extraction
Results
A
Acipenser fulvescens
B
C
D
Goniistius zonatus
E
F
G
Non-specific band
H
Amplifying target region:
96.8%
Average sequencing success
COI-3: 95.2%
COI-2: 93%
COI-1: 86.0%
Average scores and read lengths
COI-3: 41,631bp
COI-2: 39,645bp
COI-1: 38,608bp
COI-3: Myxini, holocephalid amplify
COI-2 & COI-3: all 93 species
Discussion
5’ region of the COI gene: the basis for a DNA barcoding system
=> The availability of primers aiding from a broad range taxa
=> Enough variation
=> Require multiple primer combination
Primer sets in this study
Amplify the barcode region form most taxa
COI-2 cocktail(for mammals): performs very well for fish
COI-3 cocktail(for fishes): performs very well for other taxa
Amplified the barcode for > 3000 species
Discussion
Amplification
* 16S primers: a simple test for DNA quality as a positive control
 COI and 16S fail: DNA degradation or the presence of PCR
inhibitors
*Adding a new primer to the existing cocktails
 same M13 tag and same amplicon
 considering very large number of complete mitogenome
 Employing degenerate or inosine-containing primers to overcome
3’ end mismatches
-> increase the chance of co-amplifying other gene regions or
NUMTs (rarely problematic in fishes)
Discussion
Sequencing
•Some reads : obscured with background signal (5’-terminus)
•COI-2, COI-3 read averaging 30bp longer than COI-1(untailed)
-> conventional primers: prone to form dimers
-> without clean up, incorporated into sequencing (5’ end)
*M13 tags: more overlap in the bidirectional reads
More overlap: more reliable and longer sequence records in the
reference database, important for automating steps
Summary
•Primer cocktails: high effective in generating amplicons for
diverse fish taxa and other groups of vertebrates