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Barcoding Type Specimens

This document outlines the ambitious project aimed at barcoding ancient DNA specimens, defined as over 100 years old. We explore the project's goals—developing efficient protocols for various Lepidoptera families—while addressing significant obstacles like small tissue sizes and contamination. Current progress indicates a 65% success rate in sample processing. With solutions such as universal primers and sterile practices, we aim to enhance taxonomic clarity and resolve cryptic species complexes. Preliminary findings provide insight into the future of ancient DNA research.

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Barcoding Type Specimens

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  1. Barcoding Type Specimens Goals, Obstacles, and Current Progress

  2. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

  3. A primer on ancient DNA • >100 years old is considered "ancient” Quality (%) Age(yrs)

  4. How old can we go?

  5. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

  6. Why barcode type specimens? • Barcode database construction • Helps put names to faces • Establish links to modern specimens • Taxonomic clarification • Help resolve cryptic species complexes • Help resolve unnecessary splits

  7. Primary goals • Develop type specimen protocol • Effective, cheap, high-throughput • Apply protocol to: • ~3000 geometrids • ~300 xyloryctids • Other Lepidoptera families • Other orders

  8. Oldest legitimate DNA? 130,000 years old 45,000 years old 700,000 years old

  9. Obstacles • Small tissue size = less DNA • Some legs <10 ug • 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

  10. Solutions: Tissue size • Abdominal lysates • Alternative to legs • Made prior to dissections • More DNA than a single leg • Concentrate DNA extract • Increase PCR cycles

  11. Additional obstacles • Project specific • Hundreds of species • Universal primers • Contaminants • Universal primers may also amplify contaminants • Cannot wash or take "core" sample from tissue • High-throughput • Must be cost effective • Cannot extensively focus on any one sample

  12. Solutions: Many species • Universal primers • Degenerate primers • Primer cocktail • Target conserved yet hypervariable region • 164 bp region of COI • Second attempt for specimens that failed first pass • 94 bp region of COI 164 bp 94 bp 658 bp BARCODE

  13. Solutions: Contamination • Dedicated room, equipment, reagents, and workstations • Sterile practices • Full body suit, hood, mask, gloves • Frequent glove changes • Avoid working over samples • Avoid generating aerosols

  14. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

  15. Type Specimen Protocol EXTRACTION LYSIS DNA (94 bp) (non-destructive) (single column) PCR SEQUENCING EDITING (164 bp) (658 bp)

  16. Current Progress • Processed: • Geometridae: 948/3000 (32% complete) • Xyloryctidae: 224/300 (75% complete) • Other: 383/383 (100% complete) • Total:1555/3683 (42% complete) • Success: • Geometridae: 629/948 = 66% • Xyloryctidae: 103/224 = 46% • Other: 278/383 = 73% • Total: 1010/1555 = 65%

  17. Quality of Data

  18. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

  19. Factors affecting success • Taxonomy • Tissue size • Primer binding efficiency • Age • Killing method • Handling • Storage

  20. Does age affect success?

  21. Does size affect success?

  22. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

  23. Destructive processing • Pre-lysis tissue grinding • Applicable to dry legs • Hypothesis: Grinding tissue prior to lysis will increase accessibility of DNA

  24. Real-time PCR • Monitor amplification in real-time • Sequence ONLY true positives Human Contaminants REAL Primer Dimers

  25. Further Experiments • Samples: Ancient lep specimens we can destroy • Allows us to measure one variable while controlling all others • Test: • Pre-lysis tissue treatments • Tissue types • Size (i.e. mass) • Primer binding efficiency

  26. Acknowledgments Funding provided by:

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