Barcoding Type Specimens Goals, Obstacles, and Current Progress.
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Transcript of Barcoding Type Specimens Goals, Obstacles, and Current Progress.
Barcoding Type Specimens
Goals, Obstacles, and Current Progress
Outline1. Ancient DNA
2. Project overviewa. Purpose
b. Primary goals
c. Obstacles and solutions
3. Current progress
4. Preliminary findings
5. Future prospects
A primer on ancient DNA
• >100 years old is considered "ancient”
Qua
lity
(%)
Age(yrs)
How old can we go?
Outline1. Ancient DNA
2. Project overviewa. Purpose
b. Primary goals
c. Obstacles and solutions
3. Current progress
4. Preliminary findings
5. Future prospects
Why barcode type specimens?
• Barcode database constructiono Helps put names to faces
• Establish links to modern specimenso Taxonomic clarificationo Help resolve cryptic species complexeso Help resolve unnecessary splits
Primary goals
• Develop type specimen protocolo Effective, cheap, high-throughput
• Apply protocol to:o ~3000 geometridso ~300 xyloryctidso Other Lepidoptera familieso Other orders
Oldest legitimate DNA?
130,000 years old
45,000 years old
700,000 years old
Obstacles
• Small tissue size = less DNAo Some legs <10 ugo 100,000 fold less template than standard ancient
bone samples
• Variable killing and storage conditions
• Variable handling over the decades
400 mg 0.04 mg
Solutions: Tissue size
• Abdominal lysateso Alternative to legso Made prior to dissectionso More DNA than a single leg
• Concentrate DNA extract
• Increase PCR cycles
Additional obstacles
• Project specifico Hundreds of species
Universal primers
o Contaminants Universal primers may also amplify contaminants Cannot wash or take "core" sample from tissue
o High-throughput Must be cost effective Cannot extensively focus on any one sample
Solutions: Many species
• Universal primerso Degenerate primerso Primer cocktail
• Target conserved yet hypervariable regiono 164 bp region of COI
• Second attempt for specimens that failed first pass o 94 bp region of COI
658 bp BARCODE
164 bp
94 bp
Solutions: Contamination
• Dedicated room, equipment, reagents, and workstations
• Sterile practiceso Full body suit, hood, mask,
gloveso Frequent glove changeso Avoid working over sampleso Avoid generating aerosols
Outline1. Ancient DNA
2. Project overviewa. Purpose
b. Primary goals
c. Obstacles and solutions
3. Current progress
4. Preliminary findings
5. Future prospects
Type Specimen Protocol
LYSISEXTRACTION DNA
(non-destructive) (single column)
PCRSEQUENCINGEDITING
(164 bp)
(94 bp)
(658 bp)
Current Progress
• Processed:o Geometridae: 948/3000 (32% complete)o Xyloryctidae: 224/300 (75% complete)o Other: 383/383 (100% complete)o Total:1555/3683 (42% complete)
• Success:o Geometridae: 629/948 = 66%o Xyloryctidae: 103/224 = 46%o Other: 278/383 = 73%o Total: 1010/1555 = 65%
Quality of Data
Outline1. Ancient DNA
2. Project overviewa. Purpose
b. Primary goals
c. Obstacles and solutions
3. Current progress
4. Preliminary findings
5. Future prospects
Factors affecting success• Taxonomy
o Tissue sizeo Primer binding efficiency
• Age
• Killing method
• Handling
• Storage
Does age affect success?
Does size affect success?
Outline1. Ancient DNA
2. Project overviewa. Purpose
b. Primary goals
c. Obstacles and solutions
3. Current progress
4. Preliminary findings
5. Future prospects
Destructive processing
• Pre-lysis tissue grinding
• Applicable to dry legs
• Hypothesis: Grinding tissue prior to lysis will increase accessibility of DNA
Real-time PCR
• Monitor amplification in real-time
• Sequence ONLY true positives
REAL
Primer Dimers
Human Contaminants
Further Experiments• Samples: Ancient lep specimens we can
destroy
• Allows us to measure one variable while controlling all others
• Test:o Pre-lysis tissue treatmentso Tissue typeso Size (i.e. mass)o Primer binding efficiency
AcknowledgmentsFunding provided by: