Barcoding Type Specimens

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?

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

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

Factors affecting success• Taxonomy

o Tissue sizeo Primer binding efficiency

• Age• Killing method• Handling• Storage

Does age affect success?

Does size affect success?

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:

Barcoding Type Specimens

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