Barcoding Type SpecimensGoals, Obstacles, and Current Progress

Outline1. Ancient DNA2. Project overview

a. Purposeb. Primary goalsc. Obstacles and solutions

3. Current progress4. Preliminary findings5. Future prospects

A primer on ancient DNA• >100 years old is considered "ancient”

Qua

lity

(%)

Age(yrs)

How old can we go?

Outline1. Ancient DNA2. Project overview

a. Purposeb. Primary goalsc. Obstacles and solutions

3. Current progress4. Preliminary findings5. 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 protocol

o 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 DNA

o 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 lysates

o Alternative to legso Made prior to dissectionso More DNA than a single leg

• Concentrate DNA extract

• Increase PCR cycles

Additional obstacles• Project specific

o 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 primers

o 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 DNA2. Project overview

a. Purposeb. Primary goalsc. Obstacles and solutions

3. Current progress4. Preliminary findings5. Future prospects

Type Specimen ProtocolLYSIS

EXTRACTION 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 DNA2. Project overview

a. Purposeb. Primary goalsc. Obstacles and solutions

3. Current progress4. Preliminary findings5. 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 DNA2. Project overview

a. Purposeb. Primary goalsc. Obstacles and solutions

3. Current progress4. Preliminary findings5. 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: